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HIV-1 Structural Proteins Serve as PAMPs for TLR2 Heterodimers Significantly Increasing Infection and Innate Immune Activation

Immune activation is critical to HIV infection and pathogenesis; however, our understanding of HIV innate immune activation remains incomplete. Recently we demonstrated that soluble TLR2 (sTLR2) physically inhibited HIV-induced NFκB activation and inflammation, as well as HIV-1 infection. In light of these findings, we hypothesized that HIV-1 structural proteins may serve as pathogen-associated molecular patterns (PAMPs) for cellular TLR2 heterodimers. These studies made use of primary human T cells and TZMbl cells stably transformed to express TLR2 (TZMbl-2). Our results demonstrated that cells expressing TLR2 showed significantly increased proviral DNA compared to cells lacking TLR2, and mechanistically this may be due to a TLR2-mediated increased CCR5 expression. Importantly, we show that HIV-1 structural proteins, p17, p24, and gp41, act as viral PAMPs signaling through TLR2 and its heterodimers leading to significantly increased immune activation via the NFκB signaling pathway. Using co-immunoprecipitation and a dot blot method, we demonstrated direct protein interactions between these viral PAMPs and TLR2, while only p17 and gp41 bound to TLR1. Specifically, TLR2/1 heterodimer recognized p17 and gp41, while p24 lead to immune activation through TLR2/6. These results were confirmed using TLR2/1 siRNA knock down assays which ablated p17 and gp41-induced cellular activation and through studies of HEK293 cells expressing selected TLRs. Interestingly, our results show in the absence of TLR6, p24 bound to TLR2 and blocked p17 and gp41-induced activation, thus providing a novel mechanism by which HIV-1 can manipulate innate sensing. Taken together, our results identified, for the first time, novel HIV-1 PAMPs that play a role in TLR2-mediated cellular activation and increased proviral DNA. These findings have important implications for our fundamental understanding of HIV-1 immune activation and pathogenesis, as well as HIV-1 vaccine development.

HIV-1 Proteins as TLR2 PAMPs Frontiers in Immunology | www.frontiersin.org introduction Chronic immune activation and inflammation serve as fundamental drivers of HIV-1 infection, replication, and pathogenesis (1)(2)(3)(4)(5)(6). This is supported by the classical observation that pathogenic SIV is associated with high levels of immune activation and pathogenesis leading to death in non-natural non-human primate (NHP) hosts, whereas non-pathogenic SIV infection in natural NHP hosts is associated with low levels of immune activation despite high viral loads (7).
Importantly, chronic immune activation and inflammation, the hallmark of HIV infection, eventually leads to premature co-morbidities, including cardiovascular, neurological, liver, brain, and bone pathologies (8)(9)(10)(11). This despite the use of combination antiretroviral therapy (cART), which dramatically reduces systemic viral load, and, we have previously shown, normalizes PBMC TLR expression (12), suggesting that HIV or components thereof are involved in increased and dysfunctional innate immune activation (12).
To this point, HIV-1 structural proteins and glycoproteins have been detected persistently in germinal centers of lymphoid tissue from HIV-infected individuals, including those receiving cART with undetectable viral loads (13)(14)(15). Moreover, in vitro studies have observed a broad spectrum of effects including, increased proliferation, maturation, cytokine production, cell surface marker expression and HIV-1 replication in PBMC, epithelial, and endothelial cells after exposure to HIV-1 ENV, gp120/160 or matrix protein, p17 (16)(17)(18)(19)(20). gp120 plays a role in the immunostimulatory effects related to HIV-1-associated dementia (21). gp41 has been shown to significantly enhance HIV-1 infection and replication in vitro (22). Likewise p24 and p17 have been shown to possess potent immunostimulatory properties leading to increased HIV-1 replication in PBMCs of infected individuals receiving cART (23), as well as activated PBMCs in vitro (16). p17 has been shown to hijack CXCR2 and syndecan2 by activation of Jak/STAT pathway that is responsible for local activation and recruitment of inflammatory cells in HIV/HCV co-infected or HIV-1 mono-infected patients (24). Furthermore, HIV-1 has been shown to modulate the innate immune system by activating specific pattern recognition receptors (PRRs) in order to enhance viral replication in plasmacytoid dendritic cells (25)(26)(27). Despite these fundamental findings, to date, the only well recognized HIV-1 PAMP is uridine-rich HIV-1 ssRNA, which is detected by intracellular TLR7/8 (28). The innate immune system is the first line of defense against infection and consists of innate immune cells that are able to respond to infectious pathogens by PRRs, defined as innate immune activation (29). Toll-like receptors (TLRs), a family of innate PRRs, play a critical role in the early recognition of pathogens and are largely responsible for activating innate immunity and shaping subsequent adaptive immune responses (30,31). Typically, the recognition of PAMPs via TLR engagement triggers a signaling cascade resulting in the activation of transcription factor, nuclear factor kappa B (NFκB), leading to the downstream production of anti-viral and pro-inflammatory cytokines (32). NFκB is particularly important during HIV-1 infection since its activation facilitates viral replication by binding the long terminal repeat (LTR) (33).
Therefore, identifying novel interactions between TLRs and HIV will have important implications for our fundamental understanding of HIV-mediated innate immune activation and pathogenesis.
While classically considered in the context of bacterial recognition, TLR2 is unique among the TLR family in that it can heterodimerize with co-receptors TLR1, TLR6, and TLR10 (34,35), thus profoundly increasing the diversity of PAMPs recognized. Of particular interest, a number of viral proteins have been identified as novel PAMPs for TLR2 including cytomegalovirus (CMV) glycoprotein B (36), herpes simplex virus (HSV) gH/gL and gB (37), hepatitis C virus (HCV) core protein (38), and measles virus hemagglutinin A glycoprotein (39). Specifically regarding HIV, Thibault et al. (40) described that detection of pathogen-derived products through TLR2 induced an effector phenotype in naïve and memory CD4+ T cells and increased HIV replication (40). Conversely, the extracellular portion of TLR2, which is found systemically and in mucosal fluids, significantly inhibits proinflammatory cytokine production (41)(42)(43) and directly inhibits cell-free HIV-1 infection in vitro (44,45). Moreover, we previously demonstrated that soluble TLR2 (sTLR2) directly interacts with HIV-1 p17, p24, and gp41 and inhibits viral protein-induced NFκB activation and inflammation (45). These findings led us to further hypothesize that structural proteins of HIV-1 may serve as PAMPs for cellular TLR2 heterodimers.
In this paper, we report significantly increased HIV-1 provirus in TLR2-bearing cells compared to cells that do not express TLR2. Our data also demonstrates that HIV-1 p17, p24, and gp41 directly bind to TLR2 and significantly increase cellular activation in vitro, an effect that was knocked down by TLR2-specific siRNA or anti-TLR2 antibodies. Lastly, our data identified an NFκB signaling pathway responsible for the increased proinflammatory cytokine production and significantly increased CCR5 expression observed in primary human T cells.

Materials and Methods cell lines and reagents
TZMbl (JC53-BL) cells (Dr. D. Montefiori, Duke University, North Carolina) and TZMbl-2 were cultured in DMEM without and with 0.8 mg/mL geneticin (G418; Invitrogen, Burlington, ON, Canada), respectively, as described previously. HEK293-TLR2/1 and TLR2/6 (InvivoGen, Burlington, ON, Canada) were cultured as previously described (44). HIV-1 proteins included p17 (Virogen, Mississauga, ON, Canada), p24, gp41, nef (Genway Biotech, Inc., San Diego, CA, USA), and gp120 kindly provided by NIH AIDS Research and Reference Reagent Program. TLR ligands, ssRNA40 (Mobix, McMaster University), LPS, Pam3CSK4 (InvivoGen), and poly I:C (Sigma-Aldrich, Burlington, ON, Canada) were diluted in phosphate-buffered saline (PBS). establishment of a stable Tlr2 Transfected TZMbl-2 cell line Human TLR2 cDNA was generated by RT-PCR from PBMC and cloned into pcDNA3.1(+)-002 (kindly provided by Dr. Jonathan Bramson, McMaster University). The construct was used as a template for PCR-amplification of the TLR2 gene and inserted into pIRES2-ZsGreen1 vector (Clontech, Burlington, ON, Canada), named pIhT2G. DNA sequencing confirmed its structure, and TLR2 protein expression was detected using western blot analysis. The plasmid was transfected into TZMbl cells with Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) according to the manufacturer's instructions. Three rounds of selection for single transfected cells were completed with medium containing 0.8 mg/mL G418. Long-term expression of TLR2 was confirmed using RT-PCR and western blot analyses up to passage 15. All experiments were completed in cells before this mark. Importantly, the confirmatory use of TZMbl-2 was a rationale choice given its stable expression of canonical HIV-1 receptors and Tat-regulated reporter genes, which allowed for sensitive and reproducible quantification of HIV-1 integration (46).

Viral stocks and hiV-1 integration assay
HIV-1 R5-tropic BaL was prepared and tissue culture infectious doses (TCIDs) of pooled supernatants as well as in vitro functional assays were determined using TZMbl and TZMbl-2 cells, as previously described (47). Optimal time for cell-free R5 HIV-1 proviral DNA was determined using qRT-PCR analysis of the Pol gene prior to infection studies and found to be 8 h post exposure in TZMbl cells ( Figure S2 in Supplementary Material).
TZMbl cells were plated in 24-well plates and left overnight. At approximately 80% confluence, cells were transfected for 24 h with Lipofectamine 2000 containing 0.5 μg/well of plasmid phTLR2 or phTLR3, which was generated in the same way as phTLR2, or vector pcDNA3.1(+)-002m, respectively. The cells were superinfected with 100 TCID50 HIV-1 for 48 h. Total cellular DNA was extracted for analysis of relative integration by qPCR of HIV-1 pol and18S rRNA genes. CD4+ T cells were enriched from donated human PBMC with EasySep Human Enrichment CD4+ T cell Kit (Stemcell). The CD4+ T cells were stimulated with 5 μg/mL phytohemagglutinin (PHA) -20 U/mL IL2 -RPMI20 for 3 days. The cells were then pre-treated with RPMI10 containing 5 μg/mL polybrene (Sigma) and 5 μg/mL antibody cocktails, either a mixture of non-specific antibodies (normal rabbit and goat IgG sc-2027, sc-2028) or a mixture of TLR2 antibodies (IMG-410A, sc-8689, sc-21760x, and R&D AF2616) at 37°C for 1 h, followed by infection with HIV-1 300 TCID50/well in a 96-well plate overnight. 5 μg/mL infuvirtide (T20), HIV-1 inhibitor, was used as a negative control. Media was removed and replaced with RPMI20 containing the same antibody cocktails for a total of 6 days. DNA was extracted with 0.2 mg/mL proteinase K-CLB (1.2%SDS-50 mM Tris-HCl pH8.0-4 mM EDTA-4 mM CaCl2) and phenol. Relative proviral DNA was then assessed by qPCR.
Quantitative reverse-Transcriptase real-Time Polymerase chain reaction and conventional rT-Pcr Total RNA was extracted from TRIzol samples as previously reported (48). Reverse transcription (RT) reactions and qRT-PCR of selected genes and internal controls, RPL13A or 18S rRNA gene, were completed as described previously (48). PCR primers for COX-2, TNF-α, CCR5, and HIV Pol were designed using the program, Primer 3.0 (http://frodo.wi.mit.edu), and supplied by Mobix (McMaster University, Hamilton, ON, Canada). Total RNA was harvested from all cells used in this study and mRNA levels for TLR1, 2, 3, 4, 6, 10 and β-actin were assessed using conventional RT-PCR.
immunoassays Endotoxin detection assay (kindly provided by Dr. Dawn Bowdish, McMaster University) was completed according to the manufacturer's instructions (Lonza, Rochester, NY, USA). OptiEIA was used to measure IL-8 levels in cell culture supernatants according to manufacturer's instructions (BD Biosciences).
Dot Blot assay 20 pmol of HIV proteins, test reagents (Cell Sciences), and negative controls (urea and glutathione transferase enzyme tag; Virogen) were blotted onto nitrocellulose membrane as previously described (50), and incubated with the cytosol or cell membrane fractions of either TZMbl or TZMbl-2, probed with anti-TLR1 or anti-TLR2 antibodies, and detected with Femto chemiluminescent substrates (Thermo).

co-immunoprecipitation
The plasmid, pIhT2G was subcloned by removing the TLR2 stop code and inserting an oligonucleotide containing three repeated Flag-tags and a stop code, which was PCR-amplified from p3 × Flag-CMV-7.1 (Sigma) as a template, generating TLR2-flag expression plasmid, pIhT2FG. This plasmid was transfected with Lipofectamine 2000 into TZMbl or HEK293 cells for 2 days and TLR2-Flag expression was confirmed by western blot with anti-TLR2 (R&D) and anti-Flag (Sigma) antibodies, respectively. The bulk prepared TLR2-Flag lysates and CD14 lysates, pCD14 kindly provided by Evelyn Kurt-Jones (Univ. Massachusetts Med. School, Worcester, MA, USA), with 20 mM CHAPS, 3-((3-cholamidopropyl)dimethylammoniol)-1-propanesulfonate, in a binding washing buffer (BWB) containing 8 mM NaPO4-2 mM KPO4-10 mM KCl-140 mM NaCl from HEK293 cells were used as resource of TLR2 protein in co-IP. The mixture of 50 μg cell lysates containing either TLR2-Flag or none with individual 10 μg pure HIV-1 proteins, p17, p24, or gp41, was rotated in BWB at 4°C overnight. The positive control was a reaction of cell lysates containing TLR2-Flag and CD14 in presence of 10 μg/ mL Pam3CSK4. Then anti-Flag, anti-p24 (sc-69728), anti-gp41 (GWB-22A6B9), and anti-CD14 (sc-58951) antibodies were added respectively and rotated at 4°C for an additional 2 h. The complexes were pulled down with 10 μL Dynabeads protein G (Invitrogen). The beads were washed three times with BWB on a 96-well magnet stand. Finally the beads were boiled in BWB and subjected to WB. The membranes were probed with anti-p17 (sc-69725) and anti-Flag antibodies, respectively.

statistical analysis
All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad). The Mann-Whitney U-test was used to directly compare two groups for HIV integration, immune responses, cellular activation, and CCR5 expression. All P-values are two-tailed and considered statistically significant if P < 0.05.

results characterization of Tlr expression in TZMbl cells and establishment of a Functional stably Transformed Tlr2 cell line, TZMbl-2
In order to investigate the effect cellular TLR2 expression has on host innate responses to HIV-1 proteins and infection, we initially set out to utilize the well-characterized HIV-1 luciferase reporter assay, TZMbl cell line (46). Early results showed that TZMbl cells do not endogenously express TLR2, consequently we established a stably transformed TZMbl cell line, TZMbl-2, that showed a substantial increase in TLR2 protein expression ( Figure S1A in Supplementary Material). In addition, TLR1 protein was detected in TZMbl and TZMbl-2 cytosol fractions ( Figure S1A in Supplementary Material). Importantly, TZMbl-2 exposed to TLR2 ligand, Pam3CSK4, produced significantly increased IL-8 levels in a dose-dependent manner compared to TZMbl ( Figure S1B in Supplementary Material, P < 0.05, 0.001, 0.01, respectively). Taken together, these results indicated that TZMbl-2 cells expressed functional TLR2 and TLR1, and produced a pro-inflammatory response to synthetic bacterial TLR2/1 ligand, Pam3CSK4.

cellular Tlr2 expression is associated with increased hiV-1 Proviral Dna
Given that multiple viruses induce immune activation via TLR2 as a means to facilitate entry (36,38,39), as well as our recent paper which showed that soluble TLR2 (sTLR2) directly interacted with HIV-1 structural proteins (45), we explored whether cellular expression of TLR2 increased cell-free HIV-1 integration and viral-induced inflammation.
To first test our hypothesis, we transiently transfected TLR2, TLR3, or empty plasmid, into TZMbl cells and infected the cells with cell-free R5 HIV. Results revealed significantly increased HIV-1 proviral DNA compared to empty-plasmid control, whereas no significant increases in integration were identified in empty plasmid or TLR3-transfected TZMbl cells ( Figure 1A; P = 0.0324).
Next, we infected TLR2 stably transformed cell line, TZMbl-2 and TZMbl with HIV-1 and evaluated viral DNA at 8 h post infection. These data indicated that TZMbl-2 had significantly increased proviral DNA compared to TZMbl cells at various infectious doses ( Figure 1B; P = 0.0044, P = 0.017, P = 0.0132, respectively).
To directly investigate whether TLR2-specific activation preceding exposure to virus altered HIV infection, various concentrations of Pam3CSK4 were added to TZMbl-2 and TZMbl cells for 24 h prior to the addition of HIV-1. The results indicated significant increases in HIV infection in TZMbl-2 in a Pam3CSK4 dose-dependent manner ( Figure 1C; P = 0.0041, 0.0137, 0.0051, respectively), while no significant increase was noted in TZMbl cells. Thus, TLR2 stimulation via Pam3CSK4 enhanced HIV infection.
Additionally, in primary human CD4+ T cells we tested whether neutralizing TLR2 using 1 or 5 μg/mL TLR2-specific antibodies could inhibit infection. We observed significant reduction in HIV DNA following treatment with 5 μg/mL of anti-TLR2 Ab compared to lymphocytes exposed to non-specific antibodies ( Figure 1D; P = 0.0286).
Taken together these data indicated that cellular expression of TLR2 played an important role in significantly increased HIV-1 infection compared to cells that did not express TLR2, and TLR2 effects were blocked by anti-TLR2 Ab.
hiV-1 p17 and gp41 induce Pro-inflammatory cytokines through a Tlr2-Dependent Mechanism Numerous reports have highlighted the HIV-1 glycoproteins and matrix proteins' role in inducing pro-inflammatory cytokines in both PBMC and specific epithelial cell subtypes (17)(18)(19). With this in mind, we set out to determine whether specific HIV-1 proteins induced immune activation as shown by pro-inflammatory cytokine production in cells expressing TLR2. Here TZMbl-2 and TZMbl cells were exposed to HIV-1 p17, p24, gp41, and gp120, positive control (Pam3CSK4) or negative controls (medium and protein tag glutathione transferase; GST). Results showed significantly increased IL-8 production in TZMbl-2 after exposure to p17, gp41, and Pam3CSK4 compared to medium, but not in TZMbl cells (Figure 2A, P = 0.0002, P = 0.0004, P < 0.0001, respectively). In contrast, p24 or gp120 protein exposure did not substantially increase IL-8 production in either TZMbl-2 or TZMbl cell lines (Figure 2A).
Taken together, these data indicated specific synergistic effects of the TLR2/1 heterodimer in sensing HIV proteins, p17 and gp41, and suggested that TLR2/6 heterodimer senses p24 leading to immune activation and production of pro-inflammatory cytokine. Further, the results confirm the importance of TLR2 in HIV protein-mediated activation of primary human T cells. TZMbl and TZMbl-2 cells were exposed to increased concentrations of Pam3CSK4 for 24 h prior to the addition of 100 TCID50 of virus. After 48 h, relative luciferase units (RLUs) were evaluated and showed significantly increased HIV infection in TZMbl-2 cells exposed to 5, 50, 500, and 5000 ng/mL of Pam3CSK4, prior to the addition of virus. A representative data set from three independent experiments is shown ±SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ^P < 0.0001. To understand the interaction by which HIV-1 proteins signal through TLR2 and/or its binding partner, TLR1, a series of siRNA knockdown assays were performed. Western blot analyses of total cell lysates showed substantially reduced endogenous TLR1 and TLR2 protein expression in TZMbl-2 cells treated with siRNA directed against TLR1 or TLR2 compared to non-specific control siRNA ( Figure 3A). After siRNA knockdown, TZMbl-2 cells were exposed to viral proteins overnight and IL-8 levels were assessed. Results indicated that single siRNA knockdown of TLR1 or TLR2 ablated viral p17 and gp41-induced IL-8 production, as well as significantly reduced Pam3CSK4-induced IL-8 levels ( Figure 3B; P = 0.0004, P = 0.0119, P = 0.0152; Reduction, P = 0.0051 and Figure 3C; P = 0.0004, P = 0.0119, P = 0.0074; Reduction, P = 0.0092). Little to no IL-8 response was elicited by Pam3CSK4, p17 or gp41 following treatment with a combination of TLR1/TLR2 siRNA ( Figure 3D; P = 0.0004, P = 0.0119, P = 0.0074; Reduction, P = 0.0052). These results confirmed that both TLR1 and TLR2 are involved in recognizing these specific HIV-1 structural proteins.
Collectively, these data provided strong evidence that HIV-1 proteins, p17 and gp41, act as PAMPs and have a structural signature recognized by TLR2/1 heterodimer that activated innate immune responses in host cells.

hiV-1 Proteins Bind Tlr2 from TZMbl-2 cell lysates
Two approaches were utilized to determine physical protein-toprotein interaction between TLR2 and specific HIV-1 proteins. Initially a dot blot detection method was used, as previously described (50). Recombinant HIV-1 proteins (p17, p24, gp41, gp120, and nef), positive controls CD14 and 1:20 diluted human breast milk (BM), which contains high levels of sTLR2 (41,44), as well as negative carrier controls (urea solution, PBS, GST) were blotted onto nitrocellulose membrane. After blocking, the membranes were exposed to TZMbl or TZMbl-2 cell lysates and viral protein-TLR interactions were identified using TLR2 or TLR1-specific antibodies. Results indicated that HIV-1 p17, p24, gp41, and pure CD14 directly interacted with membrane bound TLR2, whereas no interaction was detected between TLR2 and gp120, nef, or ssRNA40 ( Figure 4A). TZMbl lysate incubation did not reveal any interactions with viral proteins, or CD14 control, however the membrane control was positive, thus demonstrating that there was no non-specific antibody binding ( Figure 4B). Furthermore, TZMbl lysates probed with anti-TLR1 antibodies indicated strong interactions between TLR1 and viral proteins p17 and gp41, but not with p24 or other viral components ( Figure 4C). Co-immunoprecipitation (co-IP) assays validated our findings and demonstrated that TLR2 interacted with p17, p24, gp41, and CD14 ( Figure 4D). Taken together, these data demonstrate, for the first time, direct protein-to-protein interactions between TLRs and HIV-1 structural proteins in which TLR2 preferentially bound p17, p24, and gp41 while TLR1 only interacted with p17 and gp41.

hiV-1 Proteins activate nFκB signaling in TZMbl-2 and Primary human T cells
Recognition of PAMPs by TLRs typically induce pro-inflammatory responses via the phosphorylation of IκBα, the inhibitory subunit of NFκB, and subsequent translocation of NFκB subunits p50/p65 into the nucleus (32). Given the central role of NFκB to activation of innate immune responses, we sought to qualitatively assess the impact of HIV-1 protein exposure on the phosphorylation of IκBα in TZMbl-2 cells and primary human T cells. Following exposure to HIV-1 proteins (p17, p24, gp41, and gp120), positive control (Pam3CSK4), and negative control (medium), western blot analysis of TZMbl-2 cell lysates showed a substantial increase in phosphorylated IκBα in cells exposed to p17, gp41, and Pam3CSK4, compared to medium and gp120 ( Figure 5A). Furthermore, assessment of NFκB subunit p65 nuclear translocation in TZMbl-2 cells revealed substantially increased p65 in nuclear fractions of TZMbl-2 cells that were exposed to p17, gp41, and Pam3CSK4, but not in cells exposed to medium or p24 (Figure 5A). TLR2 from cell lysates. TLR2/HIV protein complexes were co-IP with anti-flag (α-flag), anti-p24 (α-p24), anti-gp41 (α-gp41), or anti-CD14 (α-CD14) antibodies and harvested with protein G-beads. Co-IP interactions were identified between TLR2 and HIV-1 proteins or CD14 after separation by SDS-PAGE and staining using α-p17 or α-Flag antibodies, respectively. In all panels, aliquots of cell lysate containing TLR2 or recombinant proteins were conducted in parallel for identification of specific protein bands. A representative data set of at least three independent experiments in shown. Given that HIV-1 infection induces COX-2 in multiple cell types (52,53), we examined COX-2 mRNA in TZMbl-2 cells exposed to HIV-1 proteins (p17, p24, gp1, and gp120), positive control (Pam3CSK4), and negative control (medium). Our results showed significantly increased COX-2 cDNA expression levels in TZMbl-2 cells exposed to p17, gp41, and Pam3CSK4 compared to medium (Figure 5B; P = 0.0033; P = 0.0022; P = 0.0002, respectively). As well, TNF-α is strongly implicated in HIV-1 pathogenesis (54); therefore, we examined cDNA expression using qRT-PCR and showed significantly increased TNF-α expression in TZMbl-2 cells exposed to p17 and gp41, and Pam3CSK4 compared to medium (Figure 5C; P = 0.0058, P = 0.0064, P = 0.0051, respectively).
Next, we examined primary human T cells exposed to specific HIV-1 structural proteins, p17, p24, gp41, or positive control, Pam3CSK4 and observed significantly increased phosphorylation of IκBα (p-IκBα) (Figure 6A). We then evaluated the level of IL-8, as an indicator of primary T cell activation, and observed that p17 elicited significantly increased production of the chemotactic cytokine at equivalent concentrations of TLR2 PAMP, Pam3CSK4. In contrast, p24 and gp41 activated primary T cells to produce substantial amounts of IL-8, but required 5-and 25-fold, respectively, greater concentrations compared to p17 and Pam3CSK4 (Figure 6B). Thus, activation of primary human T cells by gp41 required a significantly greater dose compared  Frontiers in Immunology | www.frontiersin.org to p17 and p24. Lastly, gp120 produced basal IL-8 levels at all doses tested. It has previously been shown that the heightened state of cellular activation of HIV-1 infected individuals is associated with increased CCR5 expression in CD4+ T cells, which may help propagate R5 viruses (55). In addition, Heggelund et al. (56) previously demonstrated that direct TLR2 stimulation significantly increased CCR5 protein expression in primary mononuclear cells. Thus, we next determined whether primary T cells exposed to viral proteins (p17, p24, gp41, and gp120) or Pam3CSK4 increased co-receptor expression, and observed that CCR5 cDNA expression was significantly elevated after 4 h of exposure to p17, p24, gp41, as well as Pam3CSK4 compared to medium (Figure 6C; P = 0.0014, 0.0256, 0.0027, respectively).
Together, these data indicate that specific HIV-1 structural proteins (p17, p24, and gp41) induce significant cellular activation and increased HIV-1 co-receptor expression in primary human T cells. HIV-1 Proteins as TLR2 PAMPs Frontiers in Immunology | www.frontiersin.org p24 Blocks p17 and gp41-induced Production of Pro-inflammatory cytokines Data shown above (Figures 4A,B) indicated a strong proteinto-protein interaction between p24 and TLR2, yet did not induce a pro-inflammatory response in TZMbl-2 cells that lack expression of TLR6. Therefore, we sought to determine whether p24 was interacting with TLR2 in a manner that impacted TLR2-dependent pro-inflammatory activation induced by p17 and gp41.
Collectively, these data suggest that p24 can bind to TLR2 and block activation by p17 and gp41 HIV structural proteins, but not Pam3CSK4. Thus providing a novel mechanism capable of manipulating innate immune signaling through a TLR2dependent mechanism.

Discussion
Chronic immune activation is a central driver of HIV-1 infection and progression to AIDS (1,2,(4)(5)(6), and can be partially attributed to the rapid and massive depletion of gastrointestinal CCR5+CD4+ T cells following infection (57,58), which compromises the integrity of the mucosal barrier and facilitates translocation of bacteria from the gut (1, 2, 59). Indeed, increased lipopolysaccharide (LPS) levels in sera, a result of microbial translocation through the gut-associated lymphoid tissue (GALT), has been documented to strongly correlate with immune activation in chronically HIV-1-infected individuals (59). However, we previously demonstrated that following cART and subsequent decrease in viral load, TLR expression in previously untreated HIV-infected individuals normalized, thus suggesting that HIV itself may be involved in increased activation (12). Additionally, we recently showed that soluble TLR2 (sTLR2), which is found in human milk, other mucosal fluids, and systemically, inhibited HIV infection in vitro (44), through a direct interaction with HIV-1 structural proteins, and inhibited virally induced NFκB activation and inflammation (45). These findings led us to ask a rather simple but important question, namely, do HIV-1 structural proteins act as PAMPs for TLR2 and its heterodimers? Here, we tested a number of HIV-1 structural proteins as PAMPS, and demonstrated that specific HIV proteins can engage and activate membrane bound TLR2 heterodimers, while others block TLR2 activation. Moreover, we demonstrated that cellular TLR2 expression significantly increased HIV-1 infection/integration in vitro. Collectively, we provide an illustrated summary of our results highlighting HIV PAMPs interactions with TLR2 heterodimer signaling, leading to increased immune activation, CCR5 expression, and HIV integration (Figure 8).
The role of TLRs in HIV pathogenesis has not been fully elucidated; however, multiple manuscripts have described the importance of HIV-1 specific cellular activation through TLRs that facilitates viral replication in pDC and T cells (25-27, 60, 61). Specifically, activation of TLR2 has been shown to increase viral replication and the production of pro-inflammatory cytokines in naïve and memory T cells (40,61,62). To investigate the role of TLR2 directly sensing HIV-1 structural proteins and contributing to increased infection and pro-inflammatory cytokine production, we tested primary human T cells and a TLR2 stably transformed TZMbl cell line (TZMbl-2). Our data demonstrate that expression of TLR2 significantly increases HIV integration and production of pro-inflammatory cytokines. These data support earlier investigations that described increased TLR2 expression on primary cells from HIV-infected patients that when activated through this specific TLR led to increased viral replication and TNF-α production (56), which suggests an important role for TLR2 in innate immune activation. Indeed, cellular exposure to specific HIV-1 structural proteins, namely p17, p24, and gp41 significantly increased NFκB-induced cellular activation and inflammation, which could be knocked down following pretreatment with anti-TLR2 antibodies or TLR2/1 specific siRNA. Interestingly, not all the HIV-1 structural proteins tested triggered cellular activation through the same heterodimer pair. We demonstrated that cells expressing TLR2/1 responded to p17, gp41, and Pam3CSK4, but not p24 or gp120, while TLR2/6 heterodimer led to production of pro-inflammatory cytokines in the presence of p24. Previous publications have provided evidence that multiple HIV-1 proteins play key roles in altering cellular activity. Indeed, p17 has previously been shown to induce the production of pro-inflammatory cytokines in vitro and in vivo (17,63), and act as an adjuvant in vaccine strategies tested in animal models (64). Furthermore, CXCR1 has been identified as a p17 receptor that triggered adhesion and chemotactic-like migration in monocytes (65). Given that CXCR1 is not expressed on many epithelial cell types, including HeLa (66), the data provided here revealed TLR2 as a novel innate HIV structural protein PRR. Importantly, we previously published on the fact that our protein preparations showed little to no endotoxin contamination (45), and that viral proteins were sensitive to proteinase K digestion, thus further supporting that TLR2 stimulation by HIV-1 PAMPs was not due to LPS contamination. Moreover, siRNA knockdown of TLR2 and/or TLR1 but not TLR4 ablated the production of pro-inflammatory cytokines after HIV-1 PAMP exposure, and HIV-1 PAMPs did not induce production of IL-8 in TZMbl cells which endogenously express TLR4 but not TLR2. Furthermore, TLR2 antibodies abrogated IL-8 production in primary T cells, and HEK293 cell lines do not express TLR4, yet responded to specific HIV proteins in a TLR2-dependent manner. These results indicate a novel mechanism in which HIV-1 structural proteins induce immune activation through TLR2 heterodimers that was not due to endotoxin contamination.
Recognition of viral proteins by TLR2 heterodimers is not unique to HIV-1. A number of reports highlight the importance of TLR2 expression in sensing multiple viral proteins that led to increased cellular activation and facilitated viral entry (36)(37)(38)(39). However, previous publications demonstrated a TLR2mediated enhancement of HIV integration in resting T cells (67). Interestingly, our evaluation of PRR expression in a population of highly exposed seronegative (HESN) female commercial sex workers (CSWs) showed significantly decreased expression of TLR2 in cervical epithelial and cervical mononuclear cells compared to HIV-uninfected and HIV-infected CSWs from the same cohort (48). Here, we showed that expression of TLR2 significantly increased HIV integration compared to cells that did not express this particular PRR in vitro. Interestingly, in a recent study evaluating TLR polymorphisms associated with HIV outcomes in Sub-Saharan Africans, it was shown that a single nucleotide polymorphism (SNP) in TLR2 was associated with HIV-1 set-point (68). Together indicating that TLR2 may play an important role in viral infection, particularly HIV, and needs to be investigated further.
For us, the question remained, what was the mechanism by which HIV-1 induces cellular activation leading to increased viral integration through TLR2? Using two approaches, a dot blot detection method and co-immunoprecipitation, we examined the direct protein-to-protein interactions between TLRs and HIV-1 structural proteins and showed that they physically associated. Specifically, structural proteins, p17, p24, and gp41, bound directly to TLR2, while only p17 and gp41 interacted with TLR1, indicating that one of the functional sensors for HIV-1 is a TLR2/1 heterdimeric complex. Indeed, this would help explain why primary cells of HIV-1 infected individuals have significantly increased TLR2 expression compared to uninfected individuals (12,56).
Following the physical interaction between p17, p24, or gp41 and TLR2, there was a substantial increase in phosphorylated IκBα in primary and transformed cell lines compared to medium, which led to nuclear translocation of NFκB subunit p65. This is an important step not only in the TLR2 signaling pathway but is also required for induction of HIV-1 gene expression via viral LTR binding (69). Moreover, primary human T cells showed significantly increased COX-2 and TNF-α expression in a TLR2-dependent manner, and importantly a TLR2-dependant increase in HIV co-receptor, CCR5 expression in primary T cells. These observations are in line with previous accounts of a TLR2-dependent increase of CCR5 expression in permissive cells (56,67). These data may provide a key mechanism for increased susceptibility to HIV infection, and help explain why expression of TLR2 significantly increased HIV integration in permissive cells while providing important information about the innate immune mechanism by which HIV-1 proteins can promote infection through increased CCR5 expression on target cells.
Our data showing that in the absence of TLR6, p24 blocked p17-and gp41-induced pro-inflammatory cytokine production in a dose-dependent manner was an unexpected finding, but are particularly intriguing since they provide evidence for a mechanism by which HIV-1 can manipulate the innate immune balance between viral protein-induced immune activation and inhibition in a TLR2-dependent manner. p24 was unable to block Pam3CSK4-induced immune activation, which is also intriguing since it is well documented that Pam3CSK4 binds in the crevice of the m-shaped heterodimer produced through the interaction of TLR2 and TLR1 (70,71). Therefore, we speculate that HIV proteins bind to alternate conformations or in different regions of this heterodimer than previously described for Pam3CSK4 (41). Another unexpected outcome was that gp120 had little to no effect on cytokine production or IκBα phosphorylation, which was unexpected given recent publications showing the induction of pro-inflammatory cytokines in genital epithelial cells (72). This suggests that cellular responses to innate activation by HIV may be contextual and thus requires further investigation.
Previous publications have shown that gp41 activated NFκB in exposed lymphocytes and gp120 altered inflammatory responses in vitro (73,74). Importantly, it was shown by Popovic et al. (15) that HIV structural proteins and glycoproteins were present and persisted in germinal centers of lymph nodes in abundant amounts both before and during cART (15). Furthermore, numerous studies have shown the presence of extracellular or virus-free HIV-1 proteins present in body fluids (75,76), e.g., extracellular Vpr was detected in serum of HIV-infected individuals at levels of 5-10 ng/mL (77). Taken together with data indicating HIV-1 infected cells continually shed large amounts of viral proteins as well as defective viral particles (78), it seems critical for future experiments to determine the role this viral milieu provides in driving innate activation and HIV-related pathogenesis in HIVinfected individuals.
In conclusion, the present investigation extends our current understanding of innate sensing of HIV-1 and for the first time reveals novel HIV-1 PAMPs, including p17, p24, and gp41, that can manipulate innate sensing and cellular activation. Further, our finding that TLR2 expression significantly increased HIV integration via increased CCR5 expression provides a mechanism by which HIV-1 can regulate host infection and persistence. Perhaps by manipulating TLR2-related innate activation, one can significantly decrease the reservoir of latently HIV-infected cells. Additionally, the data shown here indicated that TZMbl-2 cells are more highly activated after exposure to HIV-1 structural proteins and have significantly increased HIV integration rates compared to TZMbl cells. Therefore, TZMbl-2 cells might be more suitable compared to TZMbl cells for assessing HIV-1 infectivity, integration and, measurement of effectiveness of neutralizing antibodies since TZMbl cells may underestimate the level of viral infection and integration. Lastly, identification of these novel HIV PAMPs has potential implications for the development of HIV vaccines. Indeed, HIV proteins that serve as innate PAMPs have auto-adjuvant activity and thus may prove to be a more effective antigen delivery systems to promote immunogenicity of HIV vaccines. Together, these results have important implications for our fundamental understanding of innate immune activation by HIV-1 and may provide insight into the design of novel vaccine strategies, as well as targeting latently infected cells to bring us one step closer to HIV cure.

Funding
This work was supported by a Large Team grant from the Canadian Institutes of Health Research (CIHR) to KR (PI) as part of the Canadian HIV Vaccine Initiative (CHVI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


extfieldsofstudy: Biology Medicine

text:
Check'-ing DLBCL

MYC is a potent oncogene that encodes a transcription factor which regulates a plethora of target genes related to cell growth and cell cycle. Not surprisingly, a vast majority of human cancers are characterized by high constitutive Myc levels promoting oncogenesis. In particular, in mature B-cell neoplasms Myc overexpression is often associated with an aggressive clinical behavior. Interestingly, a high degree of Myc overexpression can be sensed as an oncogenic stress, and, as a protective mechanism, the cell can experience senescence or apoptosis. Myc not only causes replication stress but also activates the DNA damage response (DDR) through ATM and ATR kinases, which, in response to DNA breaks switch on a transduction pathway that activates p53. This cascade activates Chk1, which, in turn, inactivates Cdc25 phosphatase and other proteins of the replication fork and spindle checkpoint. All these modifications promote G2/M cell cycle arrest, in order to facilitate DNA repair and thus, prevent the cell from a premature and catastrophic mitosis in cells harboring extensive DNA damage.

Most human tumors are characterized by genomic instability, some of its manifestations are lagging chromosomes and chromatin bridges, and multipolar spindles caused by supernumerary centrosomes that cause anomalous chromosome segregation. Genomic instability may be “tolerated” by a low level of checkpoint bypass. In contrast, an extremely high level of genomic instability -due to Chk1 inhibition, for example- can be used as a therapeutic strategy, since tumor cells treated with chemo or radiotherapy along with Chk1 inhibitors are more sensitive than normal cells. This link between Myc and Chk1 was first demonstrated by Hoglund et al [1] in 2011 when they treated mouse models of Myc-driven B-cell lymphomas with Chk1 inhibition and they observed marked caspase-dependent apoptosis. They suggested that not only Myc-driven B-cell lymphomas would benefit from this type of therapy, but also neuroblastoma, and breast and lung cancers, all of them characterized by high levels of Myc [1,2]. The rationale behind this observation is that tumors with high levels of Myc become dependent of Chk1 for maintaining genomic integrity, and by adding Chk1 inhibitors to the standard therapy regimens the cells increase its genomic instability to “untolerated” levels and become more sensitive to treatment (Figure (Figure1).1). This phenomenon is termed “synthetic letal”, and requires deregulation of both genes, Myc and Chk1 simultaneously. Thus, Myc-driven tumors are perfect candidates for Chk1-inhibition as a therapeutic strategy specially cases with mutated p53, proven to be insensitive to other types of therapies. Moreover, Ferrao et al. [3] demonstrated the benefits and efficacy of treating Myc-driven lymphomas with single agent Chk inhibitor and showed that the dual Chk1-Chk2 inhibitor was more powerful in p53 deficient cells than the Chk1 inhibitor alone.

Figure

Targeting MYC-induced genomic instability in DLBCL with CHK inhibitors

In a recent issue of Oncotarget, Derenzini et al [4] point to the inhibition of DDR, via Chk inhibition, as a potential therapeutic strategy in diffuse large B-cell lymphoma (DLBCL). Cells with Myc-driven replicative stress and genomic instability after Chk inhibition were driven to mitotic catastrophe due to high levels of DNA damage and with “untolerated” genomic instability that eventually ended in cell death (Figure (Figure1).1). They studied the expression of Myc, phosporylated Chk1/2 and Cdc25c proteins, and phosporylated histone γH2AX, as a bona fide marker of inherent DNA damage. Both overexpression of Myc and constitutive expression of γH2AX were tightly related and associated with poor outcome of patients after standard chemotherapy regimens. The authors demonstrated a reduction of proliferation through inhibition of the DDR pathway using Chk inhibition. Overall, these results and previous observations suggest that pharmacological inhibition of Chk1 could represent a novel and efficient therapeutic strategy not only for DLBCL with Myc overexpresssion, but also for other malignant B-cell lymphomas characterized by high Myc levels and high degree of genomic instability, like mantle cell lymphomas, even in cases with inactive p53. Interestingly, chromosomal missegregation defects in DLBCL visualized under the microscope are much more frequent in patients with short overall survival and inferior response to therapy [5].

Some concerns before including this strategy in clinical trials are the difficulties in standardizing the quantitative evaluation of MYC expression in tumors. Similarly, it is not clear what could be the most appropriate surrogate marker for genomic instability, γH2AX phosporylation, or directly study the chromosomes or copy number arrays?

Recently, targeted sequencing of key DNA repair genes in DLBCL has found mutations in several genes, including 8% mutations of CHK2 [6], some of them of germline origin. Occasional mutations and loss of Chk2 protein expression in aggressive lymphomas, such as mantle cell lymphomas, had also been previously associated with high levels of chromosomal instability [7]. Overall, all these studies highlight the importance of the DDR pathway in aggressive lymphoma pathogenesis, but the functional consequences and also their direct link with chromosomal instability have still to be elucidated. Moreover, it should be reasonable to try the strategy of Chk inhibition in combination with chemotherapy in these cases with mutations in DNA repair genes to test if it is still efficient. It should also be taken into consideration that the different genetic background of each tumor may determine the degree of efficacy of Chk inhibitors. In summary, Chk inhibition in MYC-driven tumors should be considered in the current era of molecular targeted therapies and genome/exome sequencing.

MYC is a potent oncogene that encodes a transcription factor which regulates a plethora of target genes related to cell growth and cell cycle. Not surprisingly, a vast majority of human cancers are characterized by high constitutive Myc levels promoting oncogenesis. In particular, in mature B-cell neoplasms Myc overexpression is often associated with an aggressive clinical behavior. Interestingly, a high degree of Myc overexpression can be sensed as an oncogenic stress, and, as a protective mechanism, the cell can experience senescence or apoptosis. Myc not only causes replication stress but also activates the DNA damage response (DDR) through ATM and ATR kinases, which, in response to DNA breaks switch on a transduction pathway that activates p53. This cascade activates Chk1, which, in turn, inactivates Cdc25 phosphatase and other proteins of the replication fork and spindle checkpoint. All these modifications promote G2/M cell cycle arrest, in order to facilitate DNA repair and thus, prevent the cell from a premature and catastrophic mitosis in cells harboring extensive DNA damage.
Most human tumors are characterized by genomic instability, some of its manifestations are lagging chromosomes and chromatin bridges, and multipolar spindles caused by supernumerary centrosomes that cause anomalous chromosome segregation. Genomic instability may be "tolerated" by a low level of checkpoint bypass. In contrast, an extremely high level of genomic instability -due to Chk1 inhibition, for example-can be used as a therapeutic strategy, since tumor cells treated with chemo or radiotherapy along with Chk1 inhibitors are more sensitive than normal cells. This link between Myc and Chk1 was first demonstrated by Höglund et al [1] in 2011 when they treated mouse models of Myc-driven B-cell lymphomas with Chk1 inhibition and they observed marked caspase-dependent apoptosis. They suggested that not only Myc-driven B-cell lymphomas would benefit from this type of therapy, but also neuroblastoma, and breast and lung cancers, all of them characterized by high levels of Myc [1,2]. The rationale behind this observation is that tumors with high levels of Myc become dependent of Chk1 for maintaining genomic integrity, and by adding Chk1 inhibitors to the standard therapy regimens the cells increase its genomic instability to "untolerated" levels and become more sensitive to treatment (Figure 1). This phenomenon is termed "synthetic letal", and requires deregulation of both genes, Myc and Chk1 simultaneously. Thus, Myc-driven tumors are perfect candidates for Chk1inhibition as a therapeutic strategy specially cases with mutated p53, proven to be insensitive to other types of therapies. Moreover, Ferrao et al. [3] demonstrated the benefits and efficacy of treating Myc-driven lymphomas with single agent Chk inhibitor and showed that the dual Chk1-Chk2 inhibitor was more powerful in p53 deficient cells than the Chk1 inhibitor alone.
In a recent issue of Oncotarget, Derenzini et al [4] point to the inhibition of DDR, via Chk inhibition, as a potential therapeutic strategy in diffuse large B-cell lymphoma (DLBCL). Cells with Myc-driven replicative stress and genomic instability after Chk inhibition were driven to mitotic catastrophe due to high levels of DNA damage and with "untolerated" genomic instability that eventually ended in cell death (Figure 1). They studied the expression of Myc, phosporylated Chk1/2 and Cdc25c proteins, and phosporylated histone γH2AX, as a bona fide marker of inherent DNA damage. Both overexpression of Myc and constitutive expression of γH2AX were tightly related and associated with poor outcome of patients after standard chemotherapy regimens. The authors demonstrated a reduction of proliferation through inhibition of the DDR pathway using Chk inhibition. Overall, these results and previous observations suggest that pharmacological inhibition of Chk1 could represent

Figure: Targeting MYC-induced genomic instability in DLBCL with CHK inhibitors.
a novel and efficient therapeutic strategy not only for DLBCL with Myc overexpresssion, but also for other malignant B-cell lymphomas characterized by high Myc levels and high degree of genomic instability, like mantle cell lymphomas, even in cases with inactive p53. Interestingly, chromosomal missegregation defects in DLBCL visualized under the microscope are much more frequent in patients with short overall survival and inferior response to therapy [5].
Some concerns before including this strategy in clinical trials are the difficulties in standardizing the quantitative evaluation of MYC expression in tumors. Similarly, it is not clear what could be the most appropriate surrogate marker for genomic instability, γH2AX phosporylation, or directly study the chromosomes or copy number arrays?
Recently, targeted sequencing of key DNA repair genes in DLBCL has found mutations in several genes, including 8% mutations of CHK2 [6], some of them of germline origin. Occasional mutations and loss of Chk2 protein expression in aggressive lymphomas, such as mantle cell lymphomas, had also been previously associated with high levels of chromosomal instability [7]. Overall, all these studies highlight the importance of the DDR pathway in aggressive lymphoma pathogenesis, but the functional consequences and also their direct link with chromosomal instability have still to be elucidated. Moreover, it should be reasonable to try the strategy of Chk inhibition in combination with chemotherapy in these cases with mutations in DNA repair genes to test if it is still efficient. It should also be taken into consideration that the different genetic background of each tumor may determine the degree of efficacy of Chk inhibitors. In summary, Chk inhibition in MYC-driven tumors should be considered in the current era of molecular targeted therapies and genome/exome sequencing.


text:
[PSI+] turns 50

abstract The year 2015 sees the fiftieth anniversary of the publication of a research paper that underpins much of our understanding of fungal prion biology, namely “ψ, a cytoplasmic suppressor of super-suppressor in yeast” by Brian Cox. Here we show how our understanding of the molecular nature of the [PSI+] determinant evolved from an ‘occult’ determinant to a transmissible amyloid form of a translation termination factor. We also consider the impact studies on [PSI] have had – and continue to have - on prion research. To demonstrate this, leading investigators in the yeast prion field who have made extensive use of the [PSI+] trait in their research, provide their own commentaries on the discovery and significance of [PSI].

IN THE BEGINNING. . . Fifty years ago this year Brian Cox, then at the University of Liverpool, published an article in the journal Heredity on c (psi), an unusual genetic trait he had observed in the yeast Saccharomyces cerevisiae 1 (Fig. 1). The 'mutant' he identified abolished nonsense suppression of the ade2-1 ochre (UAA) mutation. Remarkably, in mutant by wild-type back-crosses, the underlying mutation was not inherited since the suppression of the ade2-1 mutation was recovered in the diploids and in all of their meiotic progeny. Such a non-Mendelian pattern of inheritance was diagnostic of a cytoplasmically-inherited trait. Further crosses with strains obtained from Don Hawthorne showed that the suppression was what was then sometimes called "supersuppression," and suppressed a suite of ochre mutations in addition to ade2-1. The genetics also showed that suppression depended on two factors, one being a Mendelian gene segregating 2:2 in tetrads (SUQ5 now SUP16, coding for a mutant serine-inserting tRNA) and the other inherited in a non-Mendelian fashion: both suppressors were dominant. The non-Mendelian determinant was given a Greek letter, c, as was the convention at the time for cytoplasmic traits in yeast. Naming the determinant c was not a reference to any paranormal nature, but rather was inspired by a catch phrase used to end the, then highly popular, BBC radio comedy show 'The Goons', namely "It's all in the mind, you know. " Cox assigned c C as the dominant enhanced suppression state and c ¡ the "normal" state allowing efficient polypeptide chain termination. Efficient chain termination in c ¡ cells causes the translation of the ade2-1 mRNA to terminate at the mutant UAA codon, and causes a precursor (P-ribosylaminoimidazole or AIR) of the adenine biosynthesis pathway to accumulate. This precursor oxidises to form a red pigment and the resultant red colony phenotype, as well as adenine requirement, have been used as a marker for most genetic studies of c ever since. Colonies of well-suppressed strains are white and adenine independent.
c was not the first non-Mendelian trait to be described in yeast, for example the petite mutant described 16 years earlier and designated r (rho) 2,3 is caused by a defect in or deficiency of mitochondrial DNA. A few years later came [URE3], which modifies the way yeast takes up ureidosuccinic acid, a precursor in the pathway synthesising uracil. 4,5 A further example is the killer phenotype that is determined by a dsRNA containing virus-like particle (VLP) that infest many strains of yeast. 6,7 Neither the c C nor [URE3] determinants could be linked with mitochondrial DNA, 8 or with cytoplasmically transferred nucleic acids such as dsRNA or the 2mm circle dsDNA. 9 All of these discoveries were serendipitous, and quite independent of previous publications: the question of priority is quite meaningless. The genetic behavior of these factors was, and still is, variously described as "cytoplasmic," "extrachromosomal" or "non-Mendelian." The 2mm circle has not been associated with any easily recognized phenotype and such plasmids are sometimes described as"cryptic," implying you can see them, but have no idea what they do except replicate themselves. For some 30 years we tried to promote the epithet 'occult' for c and [URE3] for something you know is there but cannot see -but it never caught on.
For nearly 30 years the c determinant remained a mystery, baffling numerous graduate students and postdocs in the Cox laboratory, which by now had moved to the Botany School, University of Oxford. During this time, all speculation about the nature of c was done in the overwhelmingly dominant paradigm of inheritance, as introduced by Watson and Crick. Disease, likewise, was expected always to be traceable to changes in native DNA or invasion by parasites introducing foreign DNA or RNA. Then in 1994 Reed Wickner proposed that the non-Mendelian [URE3] determinant in yeast was a heritable conformational variant of the cellular protein Ure2 i.e. the [URE3] determinant was a prion. 10 Furthermore, Wickner proposed that c most likely also had a prionlike determinant and concluded, from the then limited available data, that Sup35 was the strongest candidate. As is now well documented, his conjecture on c was correct; c C is the prion form of the translation termination factor Sup35 (eRF3) encoded by the SUP35 gene. In keeping with the accepted modern nomenclature, the c C trait is now referred to as [PSI C ] with cells lacking the determinant being designated [psi ¡ ].
The 'protein only' prion concept, adding a novel paradigm, was first proposed by Prusiner in 1982 as an explanation for the transmi-ssion of scrapie in sheep and goats 11 and then eventually for the human analogs kuru, Creutzfeldt-Jakob disease (CJD), fatal familial insomnia and Gerstmann-Straussler-Scheinker syndrome; all pathologies associated with the same PrP protein. 11,12 By the time of Wickner's publication in 1994 there was considerable evidence for the prion hypothesis for transmissible spongiform encephalopathies. The prion protein had been identified, the genes determining it in man, in mice, in hamsters and in sheep had been sequenced, and the existence of 2 conformations of the PrP protein in prion and normal forms had been demonstrated, as had some critical details of the molecular structure. It is undeniable that Prusiner introduced a new paradigm into the understanding of disease and heredity. Equally, one cannot deny that the acceptance of the 'protein only' prion concept was both slow and grudging, even after Charles Weissmann's laboratory in Zurich reported that mice without the PrP gene were totally resistant to scrapie, and that the species barrier to transmission between mice and hamsters disappeared if the recipient was transgenic with the PrP gene of the donor. 13 That the discovery that two phenotypically distinct heritable traits in a completely different and only remotely-related organism could be explained by the prion hypothesis, was one of the factors that promoted the acceptance of the 'protein only' phenomenon. Another factor of course, was the emergence in Britain in 1995 of "mad cow disease" in man. This variant of CJD (vCJD) was apparently caught from cows and the possible involvement of the bovine equivalent of PrP caught the attention not only of the public, but of the medical establishment. It is unforgivable that even in 1988 we published a review of c 14 that discussed in detail various models we thought might account for the c phenomenon without even considering prions, in spite of the growing body of evidence for them from the Prusiner laboratory.
In this short article we illustrate how our understanding of the molecular nature of the [ ] was shown to suppress all 3 classes of nonsense mutation, in some cases in the absence of a known suppressor tRNA. 15 As we now know, and consistent with these early observations and inferences, the efficiency of translation termination is significantly impaired in a [PSI C ] strain. On the biochemical side, Tuite and McLaughlin established an in vitro translation termination assay, and using yeast cell-free extracts and globin mRNAs, found that the extracts from [PSI C ] yeast promoted read-through of nonsense codons. This readthrough was poisoned by adding extracts from [psi ¡ ] cells 16,17 leading to the conclusion that [psi ¡ ] cells contained an unidentified"fidelity factor" that enhanced chain termination. It was not until much later, with the publication of papers by Doel et al. 18 and Ter-Avanesyan et al. 19 that Sup35 (eRF3) was implicated as that fidelity factor, and by extension as the prion-forming protein, 20 as Wickner had suggested. 10 We wish to acknowledge that even in 1994 we were unable to discuss the role of Sup35 and [PSI] in terms of the prion hypothesis. And so it was that, leading up to 1994, much information about the genetics of [PSI C ] and how it affected the biochemistry of translation termination in yeast had accumulated, from at most 3 or 4 laboratories around the world, without any understanding of who the players were or how they worked.
In 1994, with the emergence of Reed Wickner's seminal paper, 10 and followed by publications two years later from the Lindquist 21 and Ter-Avanesyan 22 laboratories demonstrating the alternative states of the Sup35 (eRF3) protein in [PSI C ] and [psi ¡ ] strains, the curtain was drawn and all was revealed. It was one of science's rare moments and it constituted a paradigm shift if only in the parochial field of yeast genetics. In the preceding moment, all explanations of a set of phenomena were based on one perception of how things worked, and then a new window opened, and with it a new set of information became relevant, while the existing data all acquired different weights, without necessarily losing their truth.
The effect of this paradigm shift is illustrated in our review published six years before Wickner's paper. 14,23 In this review we summarized the known properties of [PSI] and discussed various models for how it might work,

Box 1: Reed Wickner
The secret handshake: After learning about Brian Cox's discovery of the non-chromosomal genetic element [PSI] when I took the Cold Spring Harbor Yeast Course in 1972, I was elated to meet him at various Yeast Genetics meetings in the '70s and '80s. I was working on the double-stranded RNA viruses determining the 'killer character' of yeast, another nonchromosomal system, so I was always interested in [PSI]. Brian is always the gentleman, but with a great sense of humor, so I always think of him smiling.
Brian's discovery of the [PSI C ] non-chromosomal genetic element started the yeast prion story, and his careful characterization of [PSI C ], and particularly its genetic properties, made our later work possible. The fact that curing of [PSI C ] was reversible 70 and that mutation of the SUP35 gene could lead to loss of [PSI C ] 18 were critical facts leading to our explanation of [PSI C ] as a prion of Sup35 10 . At the end of Brian's chapter in"The Early Days of Yeast Genetics" 71 , he wrote, "The club of yeast geneticists is still in existence but has suffered a degree of dispersion and lost its exclusivity. However, a number of clubs within the club have formed informally, like eddies in a tide pool, and one of the most esoteric is the little coterie interested in cytoplasmic or extrachromosomal inheritance, and the more obscure the particle, the greater the bond. We do not have a handshake, but sometimes an eyebrow is raised. One of the pleasures that endures at the huge modern conventions on yeast genetics is meeting each other, especially those who did and still do genetics with their phenotypes, to exchange details of the latest rococo experiments. Here is to the killer people, especially Reed Wickner and Alan Bevan, to the mitochondrial people and their doyen, Piotr, to Jim Haber for giving us 20S and congratulations to them all on their graduation into molecular respectability. C and URE3, I am sure Francois Lacroute and Michel Aigle would agree, te salute cumque gratias." I was pleased at the time to be included in Brian's club along with Francois Lacroute. And it is true that those interested in non-chromosomal inheritance had common interests and approaches that made me look forward to developments in [PSI C ], [URE3], 2 micron DNA, the killer factor and the various RNA viruses that were our main interest before prions. The orphan non-Mendelian genetic elements were particularly exciting and mysterious. Brian always had interesting new data that stimulated our thinking.
Brian has continued to work on [PSI C ], the prion, and has made several important recent contributions, such as his characterization of prion 'seeds', defining them by a direct and simple assay method based on his discovery (with Mick Tuite) of the curing of [PSI C ] by millimolar concentrations of guanidine. without presuming to identify the actual molecular factor. The purported models were limited to pre-existing and well-understood examples of irregular genetics based on DNA organization or regulation. Those we favored were more or less elaborate episomal theories or positive feed-back regulation systems, similar to that proposed by Novick for example, to explain an environmentally-triggered self-sustaining inherited 'ON' state of the lac operon in E. coli 23 or to the inheritance of methylated sites in DNA, like those determining restriction-modification systems in bacteria. 24,25 The most elegant and economical model came from Jeff Strathern at one of the annual Cold Spring Harbor courses on Yeast Genetics (Fig. 2). The gene is hypothetical of course and the model does not work Box 2: Susan Liebman I first learned of the c C mystery in 1971, when I entered Fred Sherman's laboratory as a graduate student at the University of Rochester. I was to determine the efficiency of"supersuppressor" read-through and the amino acids inserted at nonsense codons, in c C and c ¡ cells. At that time posters and talks on c C were largely ignored in favor of work on promoters. Luckily for me Brian Cox became a frequent visitor to Rochester serving as a de facto second advisor. Discussions of what c C might be consumed by us and a small core of scientists whenever we met. Due to Brian's influence, my 1973 PhD thesis states, ". . . according to this hypothesis yeasts having identical genotypes may exist in the c C or c ¡ state depending upon whether or not they have been exposed to a particular substance which induces a series of chain events resulting in the continued production of that substance." One focus in my laboratory was super-suppressors and their genetic modifiers. By 1989 this led me to seek funding for a structure function analysis of Sup35, whose role as a translational release factor and c C was still unknown. After three such proposals failed I instead studied rRNA's role in translational fidelity. This enabled me to recruit Yury Chernoff who brought a side project that eventually showed deletion or overexpression of the Hsp104 chaperone cured cells of c C . This initially esoteric topic became of broad interest when Reed Wickner, combining his data on [URE3] with that of scientists in Russia that had continued to study Sup35, convincingly proposed that c C and [URE3] were, respectively, the prion forms of the SUP35 and URE2 gene products. Thereafter, together with Irina Derkatch we showed the existence of different heritable variants of [PSI C ] and that another prion, [PIN C ], affected the appearance and maintenance of [PSI C ]. Biochemical and transfection data have now proven that [PSI C ] is a self-perpetuating amyloid form of Sup35. Self-propagation of [PSI C ] requires chaperones to break prion fibers continually providing ends where soluble Sup35 molecules join and convert to prion. Other yeast proteins also exist in prion forms. These lessons are now being used to characterize human proteins with prion-like properties. Since many of these proteins are associated with neurodegenerative disease, the legacy of the 1965 paper continues. inheritance. Fortuitous or induced read-through of the UAA codon generates a protein (hypothetical) that sustains itself by promoting reading through its own internal stop codon and also, incidentally, any other codon mutations to UAA in other genes. The suppression ('[PSI C ]') phenotype so generated would be dominant and inherited in non-Mendelian fashion since it does not depend on any change in DNA sequence, only in its activity.
for Sup35, which at the time was not one of the suspects, but it is very clever.
The Cox et al. 14  My favourite part of the Brian Cox story, an inextricable piece of what I believe will prove a profoundly important scientific legacy, is the object lesson it provides on the very nature of scientific inquiry. I remember hearing a lecture by Susan Liebman (then University of Illinois) on the non-chromosomal inheritance of an omnipotent nonsense suppressor, when I was a postdoc at the University of Chicago. I always loved to think about compelling puzzles that needed tackling. I walked out of Sue's lecture realizing I had no way to tackle that one, but having a sense of real excitement from the sheer craziness of the story. A heritable change in translation termination -one of the key elements of the central dogma -didn't obey the rules of DNAbased inheritance. Wow! But as I was walking back to the lab with 2 fellow postdocs, they surprised me by complaining about how ridiculous it was to study something so odd and uninteresting. So I briefly delved into Brian's papers (which laid the entire foundation!), and that work, a brilliant investigation of an odd chance observation, was creatively and rigorously laid. I asked a friend who was attending a yeast meeting where Brian was speaking to check it out. My friend was not happy about the waste of time. I forgot about it.
Fast forward many years later. My lab was deep in the throes of deciphering the problems of protein misfolding. We had reported that Hsp104 saves cells from extreme stresses. No problem. But when we discovered that it did so by using its 12 ATPase domains to disaggregate the aggregates of other proteins, the reaction was much the same as Brian had gotten: can't be right; this is a stupid idea. While we were struggling to get the story published, I got a call from Yury Chernoff, who was working with Sue Liebman. He'd found that Hsp104 overexpression cures cells of this mysterious heritable suppressor element. Did I have any idea what Hsp104 might be doing? Time to reread Brian's papers! Prions break previous dogmas, expand our paradigms for inheritance, and provide entirely new understandings of self-perpetuating states in biology. As interesting and as vitally important as their roles in disease may be, I think it will be their roles in normal biology that will have the biggest impact. It's been 50 y since Brian's first work on this system and, believe me, we have only seen the tip of the iceberg. In another 50 y (or maybe just 10?), Brian's astonishing story of tackling the inexplicable with beautifully reasoned and rigorously controlled experiments will be in all of the textbooks. Meanwhile, it provides a number of object lessons. I ask today's anonymous manuscript reviewers with their demands for mechanistic understandings prior to publication, I ask students and postdocs who want only to publish in the top journals, and I ask granting agencies who want to fund only work that is important for human disease: where would be we if Brian hadn't published those papers? assays of stop codon readthrough in [psi ¡ ] lysates 16 (Fig. 2), being nonsense mutants which promote their own readthrough. 34 3. Observations not enlightened by the theory, but which were thought to

Box 4: Michael Ter-Avanesyan
To be honest, Brian Cox's 1965 publication describing [PSI C ], a novel non-chromosomallyinherited determinant in Saccharomyces cerevisiae, passed by me unnoticed. Indeed, though at that time I had already decided to link my life with genetics, I did not think that yeast would be the focus of my future work. Even later, when I had already become a yeast geneticist, I did not plan to study [PSI C ]. Of course, studying translation termination in Sergey Inge-Vechtomov's laboratory, I knew that such a determinant existed, but this phenomenon seemed too elusive and formally-genetic, while at that time I was attracted by things which had molecular interpretations. This is why [PSI C ] and I peacefully co-existed without touching each other until the late 1980s. However, about twenty years after its discovery, [PSI C ] began to intervene into my life, sometimes causing significant trouble! The first such intervention happened around 1987 -we could not disrupt the SUP45 gene and only after significant efforts established that this was due to decreased viability of [PSI C ]-bearing cells heterozygous for SUP45 disruption. The second instance was connected to our observation published in 1994 19 that 5 0deletions of the SUP35 gene caused disappearance of [PSI C ]. Thus, without any desire, our lab started getting involved in studies of [PSI C ]. Some of my colleagues, especially Vitaly Kushnirov who at that time was involved in studies of the SUP35 gene, were skeptical of these efforts, since even though [PSI C ] seemed to be linked to SUP35, we could not understand the physical nature of [PSI C ]. Fortunately, that same year Reed Wickner published a paper with the prion concept for [PSI C ] and from that moment [PSI C ] ceased to be only trouble for us and, on the contrary, became an object of our constant and undivided interest. From that moment, our lab's life had changed and had become much more interesting. So, we are very grateful to [PSI C ] and to Brian, who described and studied it. Undoubtedly, we also are grateful to our other colleagues, whose work in this area constantly surprised and inspired us. To conclude, I would like to stress the magic of numbers. While the study of the role of chromosomal genes (i.e., DNA) in determination and inheritance of phenotypic traits started with the work of Gregor Mendel published in 1865, the study of proteins as genetic material was initiated by Brian's work published exactly one hundred years later. deserve further investigation, or were otherwise deemed irrelevant: i. Other mutations of Sup35 that apparently affected cell cycle progression (gst1). 29 or frameshift suppression (SUF2) 28 [C]. He replied simply by sighing, and one need only re-read his landmark paper, "C, A Cytoplasmic Suppressor of Super-Suppressors in Yeast," to immediately understand.
In the course of characterizing a suppressor of multiple yeast auxotrophies, Brian noted a rare phenotypic instability that his curiosity led him to pursue. The underlying complexity turned out to be astounding: a diploid strain carrying one suppressible and one non-suppressible ade2 allele, a spontaneously arising, dominant but inefficient suppressor SUQ5, and a non-Mendelian factor [PSI C ]. Fifty years later, knowing full well that the suppressible allele is a nonsense mutation, that SUQ5 is a tRNA suppressor, and that [PSI C ] is the prion form of the release factor Sup35, the intricate logic of the crosses and their interpretations are still challenging to follow. And, then I remember that Brian designed these studies without knowledge of the molecular basis of the players. Nonetheless, he proposed the"simplest explanation" of the data, which still stands today: [PSI C ] is a cytoplasmically transmitted modulator of the SUQ5 suppressor, which he posited could be a"self-replicating particle." With the publication of this paper and the understatement,"It will obviously be of interest to establish the nature of this cytoplasmic mutation," Brian launched a new field that continues to thrive 50 y later.
Every few years I re-introduce the paper in our laboratory journal club. My senior students and post-docs have come to view it as a sign that it is time to finish up their experiments and move on if they make it to a second discussion of the paper. But, I think of it as a way to inspire the next generation to be observant, curious, and thoughtful and to appreciate that even the most impossibly complex systems can be deconstructed with persistence and simple yet elegant experiments.

CONFIRMING [PSI C ] IS A TRANSMISSIBLE AMYLOID
The original amyloid-prion identity of [PSI C ] was reinforced by subsequent prion-theory-driven studies. For example, the ability of recombinant Sup35 to form amyloid in vitro which can then be further applied in vitro to seed solutions of monomer to form more amyloid. 40,41 The amyloid products of this can then convert in vivo [psi ¡ ] protoplasts of yeast cells to [PSI C ] protoplasts to generate stable [PSI C ] clones of cells. 42,43 This latter discovery was particularly gratifying since it was a predicted property of prions that for many years had resisted attempts to confirm it using natural and recombinant forms of the mammalian PrP Sc protein. In addition, just as [PSI C ] cultures arising spontaneously comprise a set of phenotypically different "variants" (the amyloid equivalent of allelomorphs in DNA-and RNA-based heredity), so in vitro-generated Sup35 amyloid promoted the formation of such variants. Reg-ardless of in vivo or in vitro generation, the [PSI C ] variant of the source strain was promoted when [psi ¡ ] protoplasts were transfected.
An essential role for Hsp104 for the propagation of [PSI C ] in vivo was initially demonstrated through genetic inactivation of the HSP104 gene 36 and subsequently by chemical inhibition of the ATPase activity of this molecular chaperone with very low concentrations of guanidine hydrochloride. 35,[44][45][46] Likewise roles for the Hsp70 (Ssa1/2) and Hsp40 (Sis1) chaperones were subsequently uncovered. 47 Hsp104 is a requirement common to nearly all yeast prions, to the extent that it is taken to be a defining feature in establishing their prion nature. Hsp104-dependency has been used as an easily verifiable first criterion for putative prions identified by prion-associated sequences, such as poly-QN domains 48 or by their ability to mediate the de novo formation of other prions i.e. to act as [PIN C ] factors. 49,50 These properties have been used to help identify several other prions in S. cerevisiae although it is very likely that many others exist. 48,51 It is worth pointing out that, in addition to the effect of Reed Wickner's 1994 paper on the direction of [PSI C ] prion studies, it "changed the water on the beans" for prion biology in a more general way. As we note above, we think it is fair to say that acceptance of Stanley Prusiner's prion hypothesis had until then been grudging, in spite of papers from his laboratory and others illuminating the molecular basis of spongiform encephalopathies. 12,52 With the demonstration of the [URE3] and [PSI C ] prions in yeast, many more people involved in fields ranging from biomedical sciences to biophysics and particularly neurodegenerative diseases took up research in the field of amyloids and prions. The outbreak of mad cow disease in Britain in the early 1990s alerted the general public, not to mention the health industry, to the dangers of this novel form of infection and heredity. It is common for many new paradigms to languish in obscurity or odium for a long time before coming back to light: take gene linkage, gene conversion, epigenetics or Lamarkism for example? This article is therefore as much a celebration of the 21 st birthday of Wickner's paper promoting acceptance of the 'protein only' theory, as it is of [PSI C ]'s 50 th .

WHAT IS THE POINT OF [PSI C ]?
Our understanding of fungal prions leads us to predict a role for chaperones in the maintenance and propagation of amyloids, however no such intervention or requirement has yet been found. Indeed, by contrast to fungal prions, almost all mammalian amyloids and prions are pathogenic and therefore the existence of a mammalian enzyme system to maintain them would perhaps be counter-selected. This highlights the question of whether yeast and fungal prions have selective advantages. Heather True and Susan Lindquist have demonstrated that [PSI C ] is advantageous in certain stress environments (the presence of heavy metals, for example); equally, there are conditions in which being [psi ¡ ] provides a selective advantage e.g. growth at 12-15 C. 31,32 It may also be significant that several of yeast's prion-forming proteins are transcription factors, allowing the prion plus-to-minus switch (or vice versa) to be adaptive. There may be an evolutionary balance-point involved, since there are circumstances in which the prion form of Sup35 appears to be toxic. 33,53,54 There seem to be no obviously pathogenic prions in yeast, however this may be an illusion since pathogenic prions could result in cell death and therefore remain inconspicuous, unlike the disease-related prions and amyloids of humans and animals.

WHAT CAN WE-HAVE WE-LEARNED FROM STUDYING [PSI C ]?
Naturally, the question arises as to whether fungal prions have much to offer in the way of illuminating the nature, or more importantly the control, of other prions in general or mammalian prions in particular. The cellular environments of unicellular microorganisms and com-plex multicellular mammalian cells are radically different. Yeast and bacteria are rapidly proliferating, relatively undifferentiated organisms, whereas mammalian cells are mostly reproductively arrested, highly differentiated from one another, and presented with different and more varied opportunities and challenges for prion propagation than are yeast cells. Fundamentally, yeast amyloids have to be prions to survive cell division, whereas mammalian amyloids may have other options.
As we have pointed out above, nearly all prions and amyloids within mammalian cells are pathogenic, and we have argued in contrast that most have been recruited to useful functions within fungal hosts. The implication of this difference must be profound, and the differing relationships between these proteins and their host cells, in terms of maintenance or switching between one state and the other, at first sight would seem to position yeast as a poor model for better understanding mammalian prion behavior and properties. However, the underlying agents have the common molecular structure of aggregates for their pathogenic and/or sub-functional forms, as opposed to monomer or oligomers for the active "normal" form and it is the pathogenic form which is self-perpetuating. The aggregates, in most cases, are assemblies of amyloid fibers formed of parallel or anti-parallel b-sheets and protein sequences rich in hydrophilic amino acids such as Gln and Asn that drive nucleation and subsequent growth of the amyloid fiber. [55][56][57] Other sequence features of amyloidogenic proteins such as the oligopeptide repeats found in Sup35, are largely dispensable for nucleation and fiber growth, although they contribute to the mechanism of chaperone-dependent propagation. [58][59][60] It is plausible therefore that continued studies of yeast prions will result in treatments or procedures applicable to preventing the formation or propagation of prions and amyloids in general. To our benefit, yeast prions reside in an organism which has proven to be very tractable to experimentation, quick and easy to handle, with much of the technology simple, cheap and not, it seems, ethically disturbing. Genes from other organisms, including animals, are routinely expressed in yeast and mammalian prions and amyloid can be propagated as such. 61,62 One example we have recently explored for the use of yeast as a model organism which seems likely to be relevant for most amyloidand prion-forming proteins stems from the work of Ricardo Marchante. 63 His work involved the isolation of all possible alleles of PNM2-1 (i.e., Sup35-G 58 D; 18 screening for those alleles showing the 'PNM' phenotype i.e. an inability to propagate [PSI C ]. The responses of the G 58 X mutations in the formation or maintenance of [PSI C ] were documented and correlated with NMR-determined conformations of an oligopeptide of the region. The outcome was the discovery that the amyloidforming tendency of mutants correlated with high entropy (disorder) of the polypeptide studied.
Genetic crosses with a selection of the Sup35 G 58 X mutants can be used to identify the interactions of these mutants with each other or with wild-type Sup35 protein via their effects on the diploid phenotypes and patterns of inheritance in meiosis. A variety of such interactions and patterns of inheritance has already been observed, some of which have not previously been seen for Sup35 The situation described above is reminiscent of the common M129V polymorphism in the PrP gene of human populations. Although either allele can support the PrP Sc prion when in a homozygous state, when in a heterozygous state individuals are resistant to infection by many (but not all) PrP Sc variants. 64,65 Many similar interactions are seen in the PrP gene of sheep, affecting scrapie. In yeast, we see a variety of interactions between heteroallelic proteins, ranging from a total failure for one to interact with the other (frigid), through failure to reproduce (sterile), mutual modification, but separate variant forms (dating but living apart) to co-integration (happily married).
The unexpected interactions we observe between different forms of Sup35 are interesting for many reasons. Firstly, because they are single-residue alterations, they may be instructive about structural molecular reasons for amyloid formation; for example they may make it possible to classify the effects of heteroallelic situations; what interactions may promote amyloid formation and what enhance it; what interactions affect amyloid fragmentation and thus proliferation; what interactions may affect inheritance from cell to cell or what, if any, promote the enzymatic destruction of amyloid. Further study of their behavior may also prove to be a route toward designing a common strategy of therapy for the widely divergent amyloid-forming proteins in man and animals. Finally, it is worth remarking that the G58 residue is not the only residue within Sup35 which has yielded 'PNM' mutations. Angela de Pace and Jonathan Weissman described dozens of PNM mutants located in the N-terminal polyQN region of Sup35. 66 To conclude; our recent experience with the [PSI C ] prion has drawn our attention to the significance of heterozygosity. Man, in spite of the bottlenecks in his genetic history, is highly heterozygous and many of his domesticated animals are too, if rather less so, and it is apparent that heterozygosity is an important factor in the resistance to, and diagnosis and prediction of, diseases. [67][68][69] With the exception of Alzheimer's, Parkinson's and cancer, amyloidoses in man are rare and sporadic and although often highly morbid, not the subject of much pharmacological or medical attention. However, if their common molecular aetiology offers the possibility of a common approach to their control, anatomical diversity may not be a hindrance to their treatment. . The significance of prion heterozygosity: the unusual interactions between some Sup35 G 58 X mutants. In this example the suppressible mutation is ade1-14 (UGA) and there is no tRNA suppressor in any of the strains.
[PSI C ] cells form white colonies while [psi ¡ ] cells form red colonies. Shown are colonies arising from single zygote cells picked from mixes of the haploid MATa and MATa strains as indicated. The four parent haploids differed at the Gly58 residue of the protein; C is G 58 (wild type) and 'W' is a G 58 W mutant. All four parent haploids taken from a single tetrad are [PSI C ]. Unlike normal [PSI C ] x [PSI C ] crosses involving wild-type Sup35 parents, the diploid progeny heterozygous for the two sup35 alleles are clearly [psi ¡ ]. The epigenetic segregation makes its own interpretation of the underlying Mendelian ratios. 328 Tuite et al.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
No potential conflicts of interest were disclosed.


text:
Plann: A command-line application for annotating plastome sequences1

Premise of the study: Plann automates the process of annotating a plastome sequence in GenBank format for either downstream processing or for GenBank submission by annotating a new plastome based on a similar, well-annotated plastome. Methods and Results: Plann is a Perl script to be executed on the command line. Plann compares a new plastome sequence to the features annotated in a reference plastome and then shifts the intervals of any matching features to the locations in the new plastome. Plann’s output can be used in the National Center for Biotechnology Information’s tbl2asn to create a Sequin file for GenBank submission. Conclusions: Unlike Web-based annotation packages, Plann is a locally executable script that will accurately annotate a plastome sequence to a locally specified reference plastome. Because it executes from the command line, it is ready to use in other software pipelines and can be easily rerun as a draft plastome is improved.

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces
As next-generation sequencing becomes the primary source of molecular variation for phylogenetic analyses, sequencing and assembling whole plastomes is more feasible than ever. Recent papers ( Kane et al., 2012 ;Bock et al., 2013 ;Ripma et al., 2014 ) have highlighted the ease of genome skimming techniques for assembling the relatively high-coverage plastome and chondriome (mitochondrial genome) from lower-level genomic DNA short-read sequencing. De novo assembly programs can easily assemble large contiguous sequences of plastome; these sequences can be aligned to the generally syntenic and conserved gene order of the plastome, which makes wholeplastome alignments ideal for phylogenomic and phylogeographic studies ( Cronn et al., 2008 ;Parks et al., 2009Parks et al., , 2012Straub et al., 2012 ;Njuguna et al., 2013 ). Once a plastome is assembled, however, it still needs to be annotated. Generally, new plastomes are annotated in reference to known plastid genes and features, using available online tools such as DOGMA ( Wyman et al., 2004 ) or CpGAVAS ( Liu et al., 2012 ). These tools are excellent for annotating and visualizing a single plastome sequence from a single taxon, but can become tedious for annotation of plastomes for which a well-annotated sequenced relative already exists. In this situation, an interactive interface that repeatedly queries a broad range of annotated plastomes is needlessly complex.
Here we introduce Plann (Plastome Annotator), a command-line tool to automate the annotation of newly assembled plastomes based on a single well-annotated related reference plastome. Because it runs locally on the user's machine (or on a Unix server), it can be easily run independently or pipelined into existing workfl ows. Its inputs are minimal: it requires only the new plastome sequence and the GenBank-formatted fi le of the related annotated plastome to match. Its output can be immediately used in the National Center for Biotechnology Information's (NCBI) command-line tool tbl2asn to create a GenBank-ready submission. Features in the reference genome that were not matched in the new plastome are included as alignments in the report so that more attention can be paid to possible misassemblies in those regions. Plastome sequences are often used for deep phylogenetics in plants because of their highly conserved gene order and low mutation rate. In closely related taxa, the coding sequence of the genes is nearly identical, with few insertions or deletions; the variation is mostly present in the intergenic spacers. Because of these features, annotating the plastome using the template of a closely related pre-annotated plastome is a straightforward process.

METHODS AND RESULTS
The reference gene sequences to be used in the search can be obtained from a GenBank record that is already validated and has annotations describing the names and qualifi ers for all of the genes in the plastome. Plann searches for sequences in the newly assembled plastome similar to those known genes and transforms those matched genes to their corresponding genomic locations. Because it only searches one sequence against one sequence, it is a very fast process: it should only take a few seconds to run.
Plann consists of Perl scripts contained in a GitHub repository ( https:// github.com/daisieh/plann/releases/tag/v1.1 ) and licensed under a BSD opensource license. It uses two freely available command-line tools from NCBI: BLASTN and tbl2asn. The graphical user interface (GUI) application Sequin, also available from NCBI, can be used to generate the template fi le required by tbl2asn and to validate the output of tbl2asn. It has been tested on Unix and • Premise of the study: Plann automates the process of annotating a plastome sequence in GenBank format for either downstream processing or for GenBank submission by annotating a new plastome based on a similar, well-annotated plastome. • Methods and Results: Plann is a Perl script to be executed on the command line. Plann compares a new plastome sequence to the features annotated in a reference plastome and then shifts the intervals of any matching features to the locations in the new plastome. Plann's output can be used in the National Center for Biotechnology Information's tbl2asn to create a Sequin fi le for GenBank submission. • Conclusions: Unlike Web-based annotation packages, Plann is a locally executable script that will accurately annotate a plastome sequence to a locally specifi ed reference plastome. Because it executes from the command line, it is ready to use in other software pipelines and can be easily rerun as a draft plastome is improved.
Key words: chloroplast; GenBank; genome annotation; plastome; Sequin. many plastomes from scratch using an interactive Web application can be both time-consuming and tedious, and the annotations used may not be as detailed as the desired reference plastome annotation. A potential use case for Plann would be a phylogeographic study in which multiple (from a few to perhaps several hundred) complete plastome assemblies are obtained for a single species (or closely related species) for which a good fully annotated plastome sequence already exists. It should be emphasized that Plann is a tool for expeditiously generating annotations based on an existing reference; therefore, its annotation can only be as good as the reference annotation used. If genes are missing or poorly annotated in the reference GenBank fi le, the Plann output will also be missing genes and be poorly annotated. Genes that have many mutations in coding regions (relative to the reference plastome) may also be missed by Plann, although sequence variation in the intergenic spacers will not affect Plann at all.
Plann is unlike the widely used Web-based applications DOGMA and CpGAVAS in that Plann is not interactive and is designed to run quickly, with minimal user interaction, from the command line. Using Plann, the 10 plastome sequences in the validation test were annotated in less than fi ve minutes, compared with an hour per plastome for CpGAVAS or many hours (depending on user speed) for DOGMA. Automated commandline scripts such as Plann will make plastome annotation a quick and simple part of a workfl ow instead of a tedious and time-consuming afterthought for publication.
Unix-like platforms, including Linux and Mac OS X. To use Plann, make sure that both BLASTN and tbl2asn are directly available as executables from the command line, then execute the script plann.pl. The input fi les required are the new plastome sequence (in FASTA format), the reference plastome (in GenBank format), and the Sequin template fi le. The output is a Sequin fi le, ready for submission to NCBI, and a text report with the genes that were not aligned. These problematic genes can be manually edited in a text editor and added back to the Sequin .tbl fi le, which can then be rerun through tbl2asn. If it turns out that the sequence is incorrect, it can be edited and then rerun through Plann again.
To validate the annotations produced by Plann, we reciprocally annotated plastomes of taxa at varying phylogenetic distances. The reference GenBank records used were NC_024735.1 ( Populus balsamifera ), NC_009143.1 ( P. trichocarpa ), NC_024734.1 ( P. fremontii ), NC_024681.1 ( Salix interior ), and NC_012224.1 ( Jatropha curcas ). This analysis can be found in the repository at test/analysis.sh. The comparative results are presented in Fig. 1 . Nearly all features were successfully annotated within a genus. Even for a distantly related pair of taxa, Plann was able to identify nearly 70% of the features present in the annotation.

CONCLUSIONS
Now that whole-genome shotgun sequencing is inexpensive and widely available, many plant systematists will have access to suffi ciently deep sequencing to assemble whole plastome sequences to use in phylogenetic and phylogeographic studies. In these types of studies, it is not uncommon to generate new whole plastome sequences for many individuals. Annotating Fig. 1. For each GenBank record along the x-axis, the number of genes annotated by Plann for the raw plastome sequence is represented by a bar. Note that the self-to-self annotation is always equivalent to the number of genes present in the original GenBank record.