Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV)  complexes. How different RNPs interact with each other to ensure specific genome packaging is a long-standing question and crucial to our understanding of orthomyxovirus replication and influenza virus gene reassortment. Our study of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV), shows that its nucleoprotein (NP), which forms the protein scaffold backbone of the viral RNP, has a bi-lobular structure like the influenza virus NP. Because ISAV-NP forms stable dimers in solution, we were able to determine ISAV-NP RNA binding stoichiometry by biochemical assays, electron microscopy and dynamic light scattering. Our results indicate that each ISAV-NP binds ∼12-nt RNA, shorter than the 24–28 nts originally estimated for the influenza A virus based on population average. We propose that NP-free RNA exists on orthomyxovirus RNPs, and such RNA regions likely mediate specific RNP-RNP interactions during genome packaging. Further elucidation of the RNA-mediated RNP-RNP interactions will help us determine the molecular basis of gene reassortment by orthomyxoviruses including the influenza viruses. Influenza viruses represent a serious public health concern due to their potential in causing widespread epidemics and pandemics. The genomes of the influenza A and B viruses are both comprised of eight segments of negative-sense RNA in situ hybridization (FISH) analysis of single virus particles further confirmed that the eight unique RNPs are selectively incorporated into progeny virions and most virus particles contain only one copy of each of the eight RNP complexes The segmented genome of influenza viruses offers a number of replication advantages such as gene recombination and transcriptional regulation, but at the same time it creates challenges for genome packaging. Once controversial, it is now widely accepted that the vRNPs of the influenza A viruses are specifically packaged into the virion, so that each particle contains eight unique segments of viral RNA (vRNA). Early evidence from defective-interfering RNAs (DI RNAs) and reverse genetics Early electron microscopy studies of isolated influenza A virus RNPs revealed rod-shaped structures with a double-helical arrangement Previous studies based on genome-wide stoichiometry calculations showed that there are ∼24–28 nts of RNA for each NP molecule Isavirus) is capable of causing wide-spread, severe anemia in Atlantic salmon and other salmonid species To better understand the mechanism of influenza virus RNP packaging, we performed a series of structural and functional studies of the NP from the infectious salmon anemia virus (ISAV). ISAV is the first orthomyxovirus isolated from fish, and it . These unusual features of ISAV-NP make it an excellent system for studying NP RNA binding properties for the orthomyxoviruses, as the recombinant NPs of both influenza A and B viruses are known to form multiple oligomeric species ISAV-NP was overexpressed in matogram . The ISAi.e.600EIEFDEDDEEEEDIDI616, with 12 out of the last 16 residues being either Glu or Asp); a feature that has been previously noted in many orthomyxovirus NPs. Without the last 16 residues, the ISAV-NP ΔC16 mutant also formed dimers, similar to the full-length protein . The final model contains 495 amino acid residues in total, including residues 5–25, 27–38, 41–45, 61–75, 86–112, 133–182, 201–320, 325–497, 504–530, and 542–586. Each NP molecule can be divided into three domains: an N-terminal domain, a head domain, and a body domain . While t2 in size, of which a 1518 Å2 area is mediated by the tail loop alone. Deletion of the tail loop (ΔLOOP or Δ504–530) resulted in the formation of NP monomers only  protein to 355 nM and 472 nM for the K185A/R186A and K296A/R299A mutants, respectively  . Furthermore, the N-terminal domain is structurally disordered in both the influenza A and influenza B virus NPs 230RPKR233 and 473KPKK476 have been identified in ISAV-NP A multi-sequence alignment based on secondary structure shows that a N-terminal domain is consistently found in every orthomyxovirus NP, but it is highly variable in both size and sequence . The last ordered residue in the crystal structure of ISAV-NP ΔC16 (consisting of residues 1 to 600) is residue 586, suggesting that the entire polypeptide region after residue 586 is likely to be disordered. We have shown that removing the last 16 residues of the ISAV-NP resulted in an enhanced RNA binding affinity (i.e. ∼6-fold increase for a 24-nt RNA)  was chosen for our initial assays. Interestingly, wt NP and ΔN111 exhibited similar RNA-binding affinities . When the wt NP was titrated in the solution, the fraction of bound RNA, as noted from millipolarization (mP) signal, first increased linearly and then gradually approached saturation  in the tail loop of the influenza A virus NP was found to undergo some structural rearrangements in the C-terminal part of the polypeptide, but the potential RNA binding groove remains largely intact To rule out the possibility that one NP subunit binds 24 nts of RNA and that RNA binding by one NP subunit allosterically inhibits RNA binding by the other subunit in the NP dimer, a monomeric ΔLOOP mutant  was usedwt NP for a 12-nt long RNA, which supposedly contains only one NP binding motif (dK = 488.4 nM for wt vs. 1076.1 nM for ΔLOOP NP). However, the protein binding stoichiometries of the 12-nt and 24-nt RNAs were unexpectedly different. While each 12-nt RNA bound to one ΔLOOP NP monomer, each 24-nt RNA recruited two NP ΔLOOP monomers when fully saturated , the same dimeric assemblies were observed (i.e. one RNA for four NP subunits), we observed mostly tetramers, which were presumably formed by simultaneous binding of two NP dimers to one 48-nt RNA (We then used negative-staining electron microscopy (EM) to confirm the RNA-binding stoichiometry by measuring the number of NP molecules associated with each RNA molecule. As shown in 2 oval . When thobserved . However8-nt RNA .wt NP dimer was 150.9±13.0 kD by DLS measurement. The calculated molecular weight of a dimer is 136.5 kD, and the slight difference could be explained by the non-globular shape of the dimer. When the 48-nt RNA was added to the NP sample, the molecular weight of the complex was increased to 299.7±16.2 kD, which matches closely to the calculated molecular weight of two ISAV-NP dimers binding to a 48-nt poly(C) RNA (2×136.5+14.8 = 287.8 kD). Our results from EM and DLS once again confirm that one ISAV-NP molecule binds to a 12-nt RNA.Similar results were also obtained from size measurement using dynamic light scattering (DLS) . AssuminEM images of ISAV RNPs released from virions showed double-helical structure with similar dimensions compared to those from the influenza A viruses. For instance, it was reported that the longest RNPs of ISAV were ∼120 nm long i.e. based on single-stranded RNAs bounds to nucleocapsids from the tobacco mosaic virus and also rhabdoviruses), each NP molecule should bind no more than ∼12 nts of RNA, consistent with our stoichiometric measurements. It has also been noted by Jennings et al. that the length of 22–26 nts exceeds the dimension of NP, so RNA coiling or looping may be necessary for RNP assembly Since only the head and body domain of ISAV-NP affect equilibrium-state RNA binding, ISAV-NP should serve as a valid model for studying the NP-RNA binding for other orthomyxoviruses including the influenza viruses. We conclude that each ISAV-NP monomer, and possibly each influenza A virus NP, bind ∼12 nts of RNA. This stoichiometry of binding is also supported by the observation that the potential RNA-binding groove of the ISAV and the influenza A virus NP is ∼60 Å long. Assuming that bound nucleotides are ∼5 Å apart (i.e. ∼24 nts vs. ∼12 nts for each NP).The cryo-EM reconstruction of influenza A virus RNPs shows that NP molecules follow a regular double-helical arrangement, with the potential RNA binding groove facing outward e.g. ISAV, influenza A viruses, etc) genomic RNA likely exists in a NP-free state in the context of RNP. Such NP-free RNA regions are probably rich in secondary or tertiary structures, as it has been shown that double-stranded RNAs bind less favorably to NP compared to single-stranded RNAs. We envision that there is an uneven distribution of NP along the viral RNA that varies depending on the secondary/tertiary structure of the RNA. Because nascent v/cRNA molecules are encapsidated by NP co-transcriptionally, we assume that only local RNA structures occur in RNPs. Although little is known about the details of such RNA structures, presumably they need to be stable enough to resist denaturation by NP binding, as it has been reported that the influenza A virus NP was able to melt the viral RNA panhandle structure Based on our stoichiometry measurements and the rationale as stated above, we speculate that a fraction of the orthomyxovirus . PCR products were digested with NcoI and XhoI (New England Biolabs) for 2 hours, and ligated into the pET28b+ vector for expression in Rosetta 2 E. coli cells (DE3) (Novagen). For the ISAV ΔLOOP mutant, a short linker sequence “Gly-Gly-Gly-Gly-Ser” was used to replace the “tail-loop” (aa 504–530).The full-length ISAV-NP (aa 1–616), the ΔC16 NP (aa 1–600), and the ΔN111 NP (aa 112–616) were cloned with an N-terminal 6×His tag. The polymerase chain reactions were performed using the 600 nm of 0.8. SeMet-substituted NP proteins were expressed in M9 minimal medium supplemented with seleno-methionine (ACROS organics). Cells were collected and sonicated in lysis buffer containing 300 mM NaCl, 5 mM Imidazole, 10% glycerol (v/v), and 50 mM Tris·HCl, pH 7.5. The lysate was centrifuged at 40,000× g for 30 min and the supernatant was used for further purification. Recombinant NP proteins were purified using a HisTrap™ HP Column, a HiTrap™ Heparin HP Column, and finally a Superdex™ 200 Column . Purified proteins were at least 95% pure according to Coomassie-stained SDS–polyacrylamide gels. All proteins were concentrated to 10 mg/ml and stored at 4°C.The expression of NP proteins was induced with 0.1 mM IPTG at 15°C for 24 hours after the cell density had reached an OD18. Although the DNA oligo could not be seen in the electron density, its presence was required for crystal formation. The best ΔC16 crystals were grown at 10°C for two weeks using the sitting-drop vapor diffusion method with the drop made of equal volume of protein solution  and well solution . To improve crystal diffraction, an additional dehydration step was performed by transferring crystals to a mother liquor containing 0.8 M lithium chloride, 0.1 M sodium citrate, pH 4.0, 9% PEG 6000 and 20% PEG 400. After 24 hours, the crystals were directly frozen in liquid nitrogen.Crystals of ISAV-NP ΔC16 were obtained from a protein-ssDNA complex containing the oligo dCDiffraction data were collected at the Beamline 4.2.2 at the Advanced Light Source, Berkeley. All diffraction data were processed using the program HKL2000 e.g., a 12mer RNA has the sequence of 5′–(AC)6–3′) to avoid potential secondary structures that may interfere with protein binding.5′-fluorescein-labeled RNA oligos were purchased from Thermo Fisher Scientific and Sigma-Aldrich. The sequences of the RNA oligos used are simple “AC” repeats (dK). NPs were titrated into the binding solution until the millipolarization (mP) stabilized. All of the experiments were carried out at 25°C. The data were plotted and analyzed by using the equation P = {(Pbound−Pfree)[protein]/(dK+[protein])}+Pfree, where P is the polarization measured at a given total protein concentration, Pfree is the initial polarization of fluorescein-labeled RNA without protein bound, Pbound is the maximum polarization of RNA when all are bound by protein, and [protein] is the protein concentration. The free and total protein concentrations were assumed to be equal since the dK was at least 10-fold higher than the concentration of fluorescein-labeled RNA. The hyperbolic curves were fitted by nonlinear least-squares regression analysis assuming a bimolecular model of pseudo first order such that the dK values represent the protein concentrations at which half of the ligands are in the bound-state.For the affinity measurements shown in dK. The titration curves showed a linear increase (first phase) in millipolarization until RNA-binding sites were saturated (second phase). Data points belonging to each phase were separately fitted by linear least-squares regression analysis to produce the inflection point, at which point the RNAs were stoichiometrically bound with protein.For the FA experiments used to determine the binding stoichiometry, the assay conditions were identical to those used for the binding affinity determination, except that the RNA concentration was increased to 60 nM and the NaCl concentration was reduced to 50 mM. These adjustments were made to ensure that the RNA concentration was at least 20-fold higher than the The ISAV-NP ΔC16:48 nt-RNA sample was prepared by mixing the protein with the 48 nt-ssRNA at a 4∶1 molar ratio in 100 mM NaCl and 25 mM HEPES, pH 7.5. The ΔC16:24 nt-RNA complex was prepared by mixing protein with RNA at a molar ratio of 2∶1. Both samples were incubated at 25°C for 30 min and purified by gel filtration before use.Carbon-coated copper grids (Electron Microscopy Sciences) were glow discharged at 5 mA for 1 min. 3 uL of protein samples at 0.02 mg/mL was added onto glow-discharged copper grids, washed twice with distilled water, and stained with freshly prepared 1.5% uranyl acetate solution for 1 min. Images were taken with a JEOL 2010 electron microscope  operated at 100 KeV using a magnification of 40,000×.g for 30 min to remove large aggregates prior to the experiments. The DLS experiments were carried out at 25°C in 100 mM NaCl and 25 mM HEPES, pH 7.5. For each sample, 20 molecular weight readouts were calculated with each being the average of five individual measurements. The 20 readouts were further averaged to obtain the mean and standard deviation of the sample molecular weights.The ISAV-NP ΔC16:48 nt-RNA sample was prepared in the same manner as for EM. DLS experiments were performed using a DynaPro99 Dynamic Light Scattering system from Wyatt. All of the samples were centrifuged at 22,000×Figure S1Calculated electronic potential for an ISAV-NP monomer. Positively charged residues are highlighted. The diagram on the left is the same as the one shown (DOCX)Click here for additional data file.Figure S2wt and mutant ISAV-NP. RNA binding affinity measurements for the wt NP and its two double mutants, K185A/R186A and K296A/R299A, were performed by FA using a 24-nt RNA.RNA binding by (DOCX)Click here for additional data file.Figure S3wt NP, ΔN111 and ΔC16 were performed by FA using a 20-nt RNA oligo.ISAV-NP RNA binding. RNA binding affinity measurements for the (DOCX)Click here for additional data file.Figure S4wt NP were performed by FA using four poly(C) oligos ranging from 15 to 30 nucleotides long. (A) and (B) are plotted against a narrower and broader X-axes, respectively.ISAV-NP DNA binding. DNA binding affinity measurements for the (DOCX)Click here for additional data file.Figure S5wt NP (blue), ΔLOOP (red) and ΔC16 (green) were kept at 5 µM concentration. Protein buffer was 200 mM potassium phosphate pH 7.5. Measurements were done at room temperature.Far-UV CD Spectra of ISAV NP. All three NP proteins, including the (DOCX)Click here for additional data file.Table S1Crystallographic data statistics.(DOCX)Click here for additional data file.


ORF73 latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) is targeted to the nucleus of infected cells where it binds to chromatin and mediates viral episome persistence, interacts with cellular proteins and plays a role in latency and tumorigenesis. A structurally related LANA homolog has been identified in the retroperitoneal fibromatosis herpesvirus (RFHV), the macaque homolog of KSHV. Here, we report the evolutionary and functional conservation of a novel bi-functional nuclear localization signal (NLS) in KSHV and RFHV LANA. N-terminal peptides from both proteins were fused to EGFP or double EGFP fusions to examine their ability to induce nuclear transport of a heterologous protein. In addition, GST-pull down experiments were used to analyze the ability of LANA peptides to interact with members of the karyopherin family of nuclear transport receptors. Our studies revealed that both LANA proteins contain an N-terminal arginine/glycine (RG)-rich domain spanning a conserved chromatin-binding motif, which binds directly to importin β1 in a RanGTP-sensitive manner and serves as an NLS in the importin β1-mediated non-classical nuclear import pathway. Embedded within this domain is a conserved lysine/arginine-(KR)-rich bipartite motif that binds directly to multiple members of the importin α family of nuclear import adaptors in a RanGTP-insensitive manner and serves as an NLS in the classical importin α/β-mediated nuclear import pathway. The positioning of a classical bipartite kr-NLS embedded within a non-classical rg-NLS is a unique arrangement in these viral proteins, whose nuclear localization is critical to their functionality and to the virus life cycle. The ability to interact with multiple import receptors provides alternate pathways for nuclear localization of LANA. Since different import receptors can import cargo to distinct subnuclear compartments, a multifunctional NLS may provide LANA with an increased ability to interact with different nuclear components in its multifunctional role to maintain viral latency. Kaposi's sarcoma (KS) is a multifocal vascular neoplasm that develops in conjunction with HIV infection and AIDS. Epidemiological data strongly supports the role of the human rhadinovirus, Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV), as the causative agent of KS M. nemestrina, (RFHVMn) M. nemestrina), and is associated with retroperitoneal fibromatosis (RF), a KS-like tumor of macaques in vitro and to the nucleus of RFHVMn-infected RF tumor cells in vivo where it is believed to perform functions similar to KSHV LANA in the maintenance of viral latency We have previously sequenced the ORF73 LANA homolog of the retroperitoneal fibromatosis-associated herpesvirus Nuclear localization of large proteins, such as LANA, requires a nuclear localization signal (NLS) that mediates binding to members of the karyopherin family of nuclear transport proteins. The karyopherins transport NLS-containing cargo proteins through the nuclear pore into the nucleus where the cargo protein is released to function The importin β superfamily of karyopherins consists of more than 20 distinct receptors related to importin β/importin β1, the first identified receptor (see review 10–12(K/R)3/5 where at least 3 of 5 consecutive residues are arginine or lysine after a variable linker region 10–12KRRK for binding to importin α Classical NLSs that bind to importin α consist of short stretches of basic amino acids, typically lysine-rich, which may occur as single or bipartite motifs. Monopartite motifs contain a common structure of a hexapeptide or heptapeptide with three to five basic amino acids either flanked by an N-terminal proline or glycine or containing an internal proline Non-classical NLSs that bind directly to importin β family members are less well defined and show specificity for individual importins. Arginine is the predominant basic amino acid in these motifs, which are often much longer than the classical lysine-rich motifs. The arginine-rich NLSs of HTLV-1 Rex protein RKRNRSP” motif located at aa 24–30 EKRPRSP” matching the consensus importin-α binding motif and has been shown to bind importin α1 and importin α5 for nuclear import Previous studies have suggested that the N- and C-terminal regions of KSHV LANA each contain a putative NLS and independently localize to the nucleus RKRNRSP” motif of KSHV LANA is not sufficient to induce nuclear localization of a fused EGFP dimer nor can it bind to members of the importin family for nuclear import. Instead, our results indicate that the “RKRNRSP” motif is part of a classical Lys/Arg-rich bipartite NLS (kr-NLS), which is conserved in the LANA homolog of macaque RFHVMn and interacts with multiple members of the importin α adapter family for nuclear import by the classical importin α/β-mediated pathway. Furthermore, this bipartite kr-NLS is embedded in a larger domain rich in arginines and glycines (rg-NLS), which interacts with importin β1 directly to utilize a non-classical nuclear import pathway. Thus, these LANA homologs have evolved a conserved mechanism for interacting with multiple importin-mediated transport pathways that allow them to traffic to the nucleus, a critical step in establishing and maintaining viral latency in an infected cell.Here, we report that the previously characterized “in vitro and in vivoRKRNRSP”  24TKRCLPP30” that is positionally aligned with the “24RKRNRSP30” of KSHV LANA contains only two of the four basic residues predicted to be functional components of the NLS  and the “PSORT” (http://psort.nibb.ac.jp/form.html) programs. “PSORT” predicted the nuclear sorting of both KSHV and RFHVMn LANA. While the “predictNLS” software was unable to identify the experimentally determined “24RKRNRSP30” NLS motif of KSHV LANA, it identified a Lys/Arg-rich sequence “40ARKRRKG46” in RFHVMn LANA, Region II , the Region II sequence 41LQPRRKH47 (ksII41–47), or the entire peptide sequence from Region I to Region II (ksI+II20–49). An alanine and glycine substituted peptide “AGGAGAG” derived from Region II of RFHVMn LANA was used as a negative control EGFP fusion peptide. The EGFP fusion constructs were transfected into Vero cells and allowed to express EGFP for 12–18 hours, as described in To identify functional NLS motifs, we used an expression system that produces an N-terminal test peptide fused to the enhanced green fluorescence protein (EGFP) to determine nuclear targeting. EGFP fusions with KSHV LANA peptides were prepared containing either the Region I 20–49(EGFP), containing both Regions I and II was targeted to the nucleus, as shown by the co-localization of EGFP and Topro-3 fluorescence (24–30(EGFP), containing only the 24RKRNRSP30 NLS motif of Region I, was strongly targeted to the nucleus (41–47(EGFP), containing only Region II, showed a strong cytoplasmic expression of EGFP  positionally corresponding to the 24RKRNRSP30 NLS of KSHV, the putative Region II NLS peptide 40ARKRRKG46 (rfII40–46), or the entire peptide from Region I to Region II (rfI+II20–48) (see 20–48(EGFP) into Vero cells gave a strong nuclear EGFP fluorescence (24–30), unlike the Region I peptide of KSHV, showed minimal ability to localize EGFP to the nucleus, as significant cytoplasmic fluorescence was detected in every cell. However, some nucleolar accumulation was observed (40ARKRRKG46 NLS in Region II (rfII40–46), demonstrated a strong nuclear and nucleolar-like targeting of EGFP fluorescence  see . Transferescence , very sirescence . In contobserved . The putrescence , similar peptide . These r peptide . Thus, R24–30(EGFP) and rfII40–46(EGFP) were transfected into Cos7 cells and the lysates were analyzed by Western blot for expressed protein using the anti-EGFP antibody. Both lysates showed abundant amounts of expressed EGFP-fusion protein ((1–462), was also purified. This deletion mutant contains aa 1–462 of importin β1, corresponding to HEAT repeats 1–11, and lacks the C-terminal domain, which interacts with importin α 24RKRNRSP30 peptide of KSHV and either importin α1, α3, or α5 ((1–462) (40ARKRRKG46 peptide of RFHVMn and importins α1 or α5 ((1–462) (To delineate the mechanism of nuclear import induced by the monopartite Lys/Arg-rich NLS motifs of KSHV (Region I) and RFHVMn LANA (Region II), the ability of these motifs to interact with members of the importin family of nuclear transporters was assessed. The constructs ksI((1–462) , lane 6,α1 or α5 , lane 2,20–49 and rfI+II20–48, respectively, were assayed for the ability to bind equivalent amounts of GST-importins α3 and β1Δ(1–462). Cos7 lysates of both constructs showed abundant amounts of the EGFP fusion proteins (20–49(EGFP) and rfI+II20–48(EGFP) was detected to both importin α3 and importin β1Δ  and RFHV1–50(EGFP)) were transfected into Cos7 cells and cell lysates were prepared. Western blot analysis revealed substantial amounts of the EGFP-fusion proteins (1–51(EGFP) and RFHV1–50(EGFP) was detected to importins α1, α3, α5, α7 ((1–462) ((1–462) deletion mutant is unable to interact with importin α, the binding of the KSHV and RFHVMn LANA constructs to importin β1 was direct.To examine the ability of the Region I/II bipartite NLS motif of KSHV and RFHV to bind to members of the importin family, plasmids expressing EGFP fused to the N-terminal 51 and 50 amino acids of KSHV and RFHV, respectively,  , lane 7.(1–462), we investigated whether the interaction was sensitive to competition by the small GTPase Ran. Ran loaded with GTP dissociates NLS cargoes from importin β-related import receptors (1–462) immobilized on glutathione beads was incubated with lysates of Cos7 cells expressing KSHV1–51(EGFP), washed and further incubated with increasing amounts of RanQ69L-GTP. Bound EGFP was detected by Western analysis. Strong binding of the KSHV1–51(EGFP) to importin β1Δ(1–462) was detected in the absence of RanQ69L-GTP (1–51(EGFP) to importin β1 was not only direct but specific.To determine the specificity of the interactions between the KSHV LANA N-terminal domain and importin β1ΔQ69L-GTP , lane 1,Q69L-GTP , lane 7.Q69L-GTP , lane 4,1–51(EGFP) and RFHV1–50(EGFP) to bind to importin α, using importin α3, which showed the most robust binding in (1–462) immobilized on glutathione beads, and then incubated with 80 µM RanQ69L-GTP. KSHV1–51(EGFP) interacted strongly with importin β1Δ(1–462) ((1–462) was completely inhibited by RanQ69L-GTP (1–50(EGFP) construct  , lane 1 Δ(1–462) , lane 3.Q69L-GTP , lane 2,Q69L-GTP , lane 4.onstruct .1–329; FLAG-LANAN(1–462) immobilized on glutathione beads. Bound protein was detected by Western blot analysis using an anti-FLAG antibody. The full-length N-terminal domain of KSHV LANA interacted strongly with importin α3  or alanine-substituted peptide fusion constructs were transfected into Cos7 cells and examined by confocal microscopy. Surprisingly, none of the single or triple alanine substitutions in either Region I, Region II or both Region I and II eliminated the ability of the 51 amino acid peptide to target 2×EGFP to the nucleus   , lane 1.(1–462) , lane 2 (1–462) . Arginin1–24(EGFP) construct used in this study and expressed it in Cos7 cells. The cell lysate was incubated with GST-tagged importin β1Δ(1–462) immobilized on glutathione beads. Western blot analysis of the bound proteins revealed substantial amounts of bound KSHV1–24(EGFP) ((1–462) and the ksI+II20–49 peptide ((1–462) and the ksI24–30 peptide, itself  , lane 1. peptide , lane 10, itself , lane 6.f  and accumulation in nucleoli, confirmed by co-localization of B23.1-RFP and nucleolin-RFP, we believe this resulted from diffusion into the nucleus and retention through the peptide interactions with other cellular nuclear and/or nucleolar constituents. We did not see any significant differences in the accumulation of 1×EGFP and 2×EGFP fusion protein controls throughout the cell nucleus and cytoplasm after 24 hours. However, we did detect differences in the ability of the 1×EGFP and 2×EGFP to localize to the nucleus and nucleolus when fused to different targeting peptides. Our data suggests that care should be taken when interpreting nuclear localization studies with a single EGFP.We further examined the ability of the 40ARKRRKG46” alone, which was predicted to function as a monopartite NLS in our bioinformatic analysis. This peptide has an additional lysine residue compared to the Region I 24RKRNRSP30 peptide of KSHV, which may have slightly increased its affinity to importins.A previous study noted the presence of Lys/Arg-rich motifs in Regions I and II of KSHV LANA, suggestive of a bipartite NLS 10–12KRRK) consensus sequence for classical bipartite NLSs that interact with importin α. Studies have shown that the N-terminal “KR” motif of bipartite NLSs binds the P1′ and P2′ positions of the minor binding site in importin α, while the larger “KRRK” motif binds the P2–P5 positions of the major binding site in importin α 378EKRPRSP384” motif, which is very similar to the 24RKRNRSP30 of KSHV LANA. Analysis of the flanking sequences in EBNA-1 revealed a downstream “PRR” motif that is also found in the Region II basic cluster of KSHV LANA, indicating that the EBNA-1 NLS also matches the bipartite consensus , as did the N-terminal 24 aa (KS1–24), the 30 aa across Region I and II (KSI+II20–49) and the 51 N-terminal aa (KS1–51). In all cases, the binding was inhibited by RanQ69L-GTP, demonstrating its specificity. The intact 329 aa N-terminal domain of KSHV LANA was also able to bind importin β1Δ(1–462), albeit more weakly. The β1Δ(1–462) mutant is a truncation that contains only the N-terminal domain and HEAT repeats 1–11 of the full-length importin β1 Our studies showed that the KSHV mut7 was able to strongly bind importin β1Δ1–51 N-terminal domain revealed the presence of an extended stretch of arginines and glycines, even after the alanine substitutions of the Lys/Arg motifs in Regions I and II in KSHV mut7. Similar Arg-rich and Arg/Gly-rich sequences are characteristic of non-classical NLS motifs in proteins that bind importin β1 directly, including the tumor suppressor RASSF5 (1–6)Q(θ/R/K)(θ/R/K)(θ/R/K), where θ represents a hydrophobic amino acid, is present in KSHV and RFHVMn LANA and other importin β1-binding proteins  and RFHVMn (40ARKRRKG46) suggesting an underlying ability to interact with nucleolar components. However, the full-length LANA is actively excluded from the nucleolus  and “PSORT” (http://psort.nibb.ac.jp/form.html) programs.The sequences of the ORF73 LANA homologs from KSHV and RFHVMn were aligned using ClustalW. In order to identify possible nuclear localization sequences, the LANA sequences were analyzed using the web-based “predictNLS” . The plasmids were constructed by annealing pairs of oligonucleotides (labeled a and b) for each test construct see . The ann0–48 see . The EGFapaIa (GGGCCCGCCACCATGGTGAGCAAG-3′5′- ATC) and pEGFPBamHIb (GGATCCCCTTGTACAGCTCGTCCATGC-3′5′-TCAC) that contained ApaI and BamHI restriction sites. The resulting DNA product was digested with ApaI and BamHI and inserted in-frame and upstream of the existing EGFP gene within the pEGFP-N2 vector. To create 2×EGFP fused to KSHV LANA 24–30 or 1–51, the oligos ksI 24–30 a and b or KS 1–51 a1–4, b1–3  , 100 U/ml penicillin, 100 µg/ml streptomycin, 1.0 mM HEPES, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate).4) were plated onto 17 mm diameter circular spots drawn with the ImmEdge pen (Vector Labs) on 60 mm dishes. In some cases, the cell populations were synchronized by serum starvation for 22–24 hours. The different pEGFP expression constructs (0.5 µg) were transfected using either Lipofectamine 2000 (Invitrogen) or TransIt LT1 (Mirus) per manufacturer's instructions. Cells were incubated at 37°C for 12–24 hrs and treated with 100 µg/ml of cycloheximide (Sigma) for 2–4 hrs prior to processing for microscopy to allow post-translational nuclear accumulation of the EGFP fusion proteins. Cells were fixed with 8% para-formaldehyde, permeabilized and analyzed using confocal fluorescence microscopy (Zeiss), as described previously Vero cells  and FlagM2 (Sigma) and the DyLight 680 anti-mouse secondary antibody (Rockland) were utilized at a 1∶10000 dilution in Western blot analysis.Plasmids expressing the GST-tagged importins α1, α3, α5, and α7 were a gift from R. Fagerlund and were described previously pET28a-6×His-RanQ69L was a gift from D. Forbes . 6×His tagged RanQ69L was purified on Talon resin, eluted with imidazole elution buffer (Qiagen), desalted using a Centricon 10 concentrator column and loaded with GTP as previously described Figure S1Preparation of GST-importin-sepharose beads. GST and GST-importin fusion constructs were expressed in E. coli HB101 under IPTG induction, as described in (1–462) importin. Bead volumes containing equivalent amounts of GST or GST-importin fusions, estimated from the Western blot, were used in the GST-pull down experiments.(TIF)Click here for additional data file.


The website contains a help page with explanations regarding the exact usage.The mechanism of RNA thermometers is a subject of growing interest. Also known as RNA thermosensors, these temperature-sensitive segments of the mRNA regulate gene expression by changing their secondary structure in response to temperature fluctuations. The detection of RNA thermometers in various genes of interest is valuable as it could lead to the discovery of new thermometers participating in fundamental processes such as preferential translation during heat-shock. RNAthermsw is a user-friendly webserver for predicting the location of RNA thermometers using direct temperature simulations. It operates by analyzing dotted figures generated as a result of a moving window that performs successive energy minimization folding predictions. Inputs include the RNA sequence, window size, and desired temperature change. RNAthermsw can be freely accessed at  Innovative experimental protocols e.g.,  are becoIt is well known that a structure prediction program based solely on conformational rules and thermodynamics will become increasingly inaccurate for long RNA sequences that are several hundred nucleotides long RNAthermsw uses a sliding window approach to traverse a region of interest in the genome that may contain an RNA thermometer. Each time, it performs two folding predictions by energy minimization inside the given window. The first is with a default temperature and the second is with a specified change in temperature that is intended to approximately simulate a heat-shock or a cold-shock effect. The change in temperature is determined by the user according to the biological context and the biochemical experiment that is performed, noting that it is easier to perform computational simulations to explore a variety of temperature ranges than to conduct wet lab experiments. The difference between the two secondary structures predicted is then calculated by the base pair distance, after which a dot is displayed with intensity that is proportional to the distance. If there is no change in secondary structure then the dot is white (invisible), while for the largest distance the dot is black. If the dots are plotted on a graph in which the x-axis corresponds to the left-most position of the window inside the region of interest and the y-axis corresponds to the right-most position, as in http://www.cs.bgu.ac.il/~rnathemsw/RNAthemsw/help.html. The server itself is accessible at http://www.cs.bgu.ac.il/~rnathemsw/RNAthemsw/ (note the slash sign at the end). The output of each run is a dotted figure in which the formation of noticeable clusters, if available, are looked for. The most interesting locations of the clusters can be used for experimental validation.Additional information and a detailed example of software usage are available at drosophila melanogaster HSP70 gene that is equal in its sequence length to that of the homo sapiens HSP70 gene and raising the temperature difference in humans to be the same as in Drosophila. A signal was still found for a potential RNA thermometer appearing in the HSP70 of Drosophila that does not appear in the HSP70 of humans.For method illustration, Heat-Shock Protein) genes in various eukaryotic organisms has been outlined in the previous section in relation to contemporary high-throughput sequencing attempts to analyze RNA secondary structures genome-wide The reason for examining HSP  that sacrifices efficiency with the purpose of gaining better accuracy.E.coli ibpA from E.coli ibpA from Finally, although no running example on a previously known RNA thermometer using RNAtips is available in E.coli ibpA in SalmonellaBradyrhizobium japonicumE.coli ibpA known RNA thermometer. Without this check, RNAthermsw can be used to search for completely new RNA thermometers that have not been discovered before, but the identification of previously known RNA thermometers in new locations should proceed with caution.The basic implementation of RNAthermsw does not rely on known RNA thermometers as potential guides to search for new ones. In fact, aside of the As more RNA thermometers are discovered, it could be valuable to insert an option into RNAthermsw to make use of known structural information, similar to the approach taken in File S1Supplementary information. The file contains the relevant chapter (Chapter 4) from reference (PDF)Click here for additional data file.


Nicotiana benthamiana, NbHsp90, was identified in association with partially purified replicase complexes from BaMV-infected tissue, and shown to specifically interact with the 3′ untranslated region (3′ UTR) of BaMV genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA (satBaMV RNA) or that of genomic RNA of other viruses, such as Potato virus X (PVX) or Cucumber mosaic virus (CMV). Mutational analyses revealed that the interaction occurs between the middle domain of NbHsp90 and domain E of the BaMV 3′ UTR. The knockdown or inhibition of NbHsp90 suppressed BaMV infectivity, but not that of satBaMV RNA, PVX, or CMV in N. benthamiana. Time-course analysis further revealed that the inhibitory effect of 17-AAG is significant only during the immediate early stages of BaMV replication. Moreover, yeast two-hybrid and GST pull-down assays demonstrated the existence of an interaction between NbHsp90 and the BaMV RNA-dependent RNA polymerase. These results reveal a novel role for NbHsp90 in the selective enhancement of BaMV replication, most likely through direct interaction with the 3′ UTR of BaMV RNA during the initiation of BaMV RNA replication.Host factors play crucial roles in the replication of plus-strand RNA viruses. In this report, a heat shock protein 90 homologue of  Nicotiana benthamiana, NbHsp90, specifically interacts with the 3′ UTR of Bamboo mosaic virus (BaMV) genomic RNA, but not with the 3′ UTR of BaMV-associated satellite RNA or that of other viruses. We further demonstrate that NbHsp90 specifically involves in the immediately early stage of BaMV RNA replication. NbHsp90 directly interacts with the BaMV 3′ UTR through the domain E, the key structural differences that distinguishes the BaMV 3′ UTR from the satBaMV 3′ UTR, which might contribute to NbHsp90's differential requirement for BaMV and satBaMV replication. Our work revealed a new role of Hsp90 in the interaction with RNA molecules, and demonstrated the differential requirement of Hsp90 in the replication of BaMV and satBaMV RNAs, which provide additional leverage for understanding the complex interactions between host, virus and its associated satellite RNA.Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone in prokaryotes and eukaryotes, and regulates diverse cellular processes through ensuring the correct folding of numerous client proteins. However, there are no reports of direct interactions between Hsp90 with viral RNA. Here, we report that a new member of the Hsp90 proteins of  The five open reading frames (ORFs 1 to 5) of the BaMV genome encode ORF1 protein (155 kDa), TGBp1 (28 kDa), TGBp2 (13 kDa), TGBp3 (6 kDa), and the capsid protein (25 kDa), respectively cis-acting elements responsible for efficient replication Flock house virus RNA polymerase synthesis and for efficient infection in Drosophila cells Influenza A virus polymerase complex by binding to PB1 and PB2 polymerase subunits Heat shock protein 90 (Hsp90) is a highly conserved molecular chaperone in prokaryotes and eukaryotes, and regulates diverse cellular processes through ensuring the correct folding of numerous client proteins In this article, we report a direct and specific interaction between Hsp90 and the 3′ UTR of BaMV RNA, and illustrate a novel and required function for Hsp90 during the early stages of BaMV replication by using virus-induced gene silencing and Hsp90-specific inhibitors. Our findings reveal a unique and specific role for Hsp90 in viral RNA recognition and replication, and suggest that replicase complexes recruit different host factors for replication of BaMV and satBaMV. We propose a model to illustrate the stages at which Hsp90 is most likely involved in BaMV RNA replication, and to further describe the differential requirement of Hsp90 in the replication of BaMV and satBaMV RNAs.Results from previous studies suggested that satBaMV evolved a distinct 3′ UTR RNA structure compared to that of BaMV N. benthamiana and thus designated as NbHsp90. There are three publicly available full-length coding sequences of known N. benthamiana Hsp90 family proteins, namely NbHsp90-1, -2, and -3, which are reported as AY368904, AY368905, and GQ845021, respectively N. benthamiana Hsp90 proteins, the NbHsp90 identified in this study shared the highest amino acid sequence similarity with NbHsp90-2, with an E-value of 1.28×10−6 and a total of 23 NbHsp90 derived peptides as identified by MALDI-TOF MS, and covering 28% of the full-length NbHsp90-2 , middle (M), and C-terminal (C) domains based on alignment results with yeast Hsp90, whose crystal structure was determined from a complex of an ATP-analogue with co-chaperone p23/Sba1 in planta, the TRV-based VIGS system was used to generate NbHsp90-knockdown plants. Phytoene desaturase (PDS)-knockdown plants, which exhibit the photo-bleaching phenotype 7 days post agro-infiltration (dpai), were used as indicators for the VIGS process. To examine the RNA silencing of NbHsp90, the relative level of Hsp90 mRNAs was examined at 7 dpai by real-time PCR and semi qRT-PCR  alone served as a negative control and was used for normalization. The results revealed that BaMV RNA accumulation was significantly reduced for both GA and 17-AAG treatments in a dose-dependent manner, which suggests that accumulation of BaMV RNA depends on the Hsp90 function  and a His(6) fusion protein (Hsp90-His(6)), respectively. GST-RdRp was bound to resin and mixed with Hsp90-His(6). After incubation, GST-RdRp was eluted from the resin and Hsp90-His(6) in the eluted sample was analyzed by immunoblotting. Specific signal of Hsp90-His(6) was detected only when both GST-RdRp and Hsp90-His(6) were present  . This doHsp90 has been proposed to assist in the assembly of viral replication complexes Based on results presented in this study, we propose a model  to illusin cis and in trans replication processes have been previously reported. For example, the genome of Red clover necrotic mosaic virus (RCNMV) comprises bipartite RNAs, RNA1 and RNA2. RCNMV RNA2 does not encode replicase and thus must exploit viral replicase proteins encoded by RNA1 to replicate in trans by their cognate helper viruses and host plants for replication in trans and directly interacts with the RNA2 3′ UTR but not with RNA1 cis-preferential function of the virus-encoded proteins has been reported for several positive-strand RNA viruses cis-preferential function of BaMV replicase by specifically binding to the BaMV pseudoknot domain, which is absent in the satBaMV 3′ UTR. Thus, BaMV may retain the replication competency in the presence of satBaMV RNA. It has been demonstrated that ribosomal RNA (rRNAs) serves as a scaffold in ribosomes for the proper positioning and folding of ribosomal proteins SatBaMV RNA may employ replicase complexes consisting of host factor(s) distinct from those used by BaMV. The involvement of different replicases or host factors for et al, GenBank acc. AF272736), and CMV strain NT9 BaMV strain S and the associated satBaMV RNA isolate F4 in vitro RdRp assays were described previously Methods for preparing BaMV RdRp and 32P-labeled RNA probes (25 fmol) in binding buffer  were added to the BaMV RdRp preparation and incubated at room temperature for 10 min. The mixture was placed in an ice bath and illuminated under a UV lamp at 254 nm wavelength  for 20 minutes. The samples were then treated with RNase A (10 µg) and RNaseT1 (0.5 unit) for 30 min at 37°C, boiled in protein sample buffer for 5 min and analyzed by electrophoresis with a 10% polyacrylamide gel containing 1% SDS. Radioactive images were scanned and quantified using the BAS-1500 bioimaging analyzer . BamHI-linearized pT7r138/Bam, XbaI-linearized psatBaMV/3′ UTR, and BstNI-linearized pT7CMV/tRNA were transcribed with T7 RNA polymerase in the presence of [α-32P]UTP 32P-labeled RNA probes corresponding to BaMV 3′ UTR, satBaMV 3′ UTR, and CMV 3′ UTR, respectively. In the competition reactions, various amounts of unlabeled competitor RNA were pre-incubated with the proteins for 10 min prior to the addition of 32P-labeled RNA probe. Description of competitor RNA preparations can be found in the Supporting Information  for 2 h and washed with renaturation buffer three times (10 min per wash). The membrane was then air dried and analyzed by PhosphorImager .Details for protein expression and purification are described in the Supporting Information . PurifieTobacco rattle virus (TRV)-based virus induced gene silencing (VIGS) system 5′- GCTCTAGAAGCAAGAAGACCATG-3′) and reverse primer NbHsp90S-R (5′- TCCCCCGGGTTAGTCAACTTCC-3′), followed by cloning of the amplified fragment into the pTRV2 vector. The pTRV1, pTRV2, and pTRV2/NbHsp90 plasmids were individually introduced into Agrobacterium tumefaciens strain C58C1 by electroporation. For VIGS assays, A. tumefaciens cultures (OD600 = 1) containing pTRV1, pTRV2, or pTRV2/NbHsp90 were mixed in a 1∶1 ratio as indicated, and co-infiltrated by syringe onto three leaves of each test plant (N. benthamiana). At 7 days post agro-infiltration (dpai) the third and fourth leaves above the infiltrated sites were mechanically inoculated with 0.5 µg virions of BaMV, PVX, or CMV. Two and six days later, total RNA was extracted from the virus-inoculated leaves of three independent plants. BaMV, PVX, and CMV RNA accumulations were determined by Northern blot assay.The 39 primer. For real-time PCR, the Hsp90 mRNA levels were monitored by forward primer Hsp90-908F (5′-AGGGTCAGCTGGAGTTCAA-3′), and reverse primer Hsp90-1098R (5′-GGGAAGATCCTCGGAATCCAC-3′). For a negative control, we used PCR without prior reverse transcription. The actin mRNA level was determined by real-time PCR with forward primer Actin-F (5′-GATGAAGATACTCACAGAAAGA-3′) and reverse primer Actin-R (5′-GTGGTTTCATGAATGCCAGCA-3′) for normalization of the specifically silenced gene.At seven dpai, total RNA of the third and fourth leaves above the infiltrated leaves were extracted for detection of Hsp90 mRNA expression by real-time PCR. First-strand cDNA was synthesized by reverse transcription using 1 µg of total RNA as the template and oligo d(T)32P-labeled probes, specific for the detection of (+)-strand BaMV and satBaMV, were transcribed from HindIII-linearized pBaHB and EcoRI-linearized pBSHE using SP6 and T7 RNA polymerase, respectively, as described previously et al, unpublished), with HindIII, followed by transcription with T7 RNA polymerase in the presence of [α-32P] UTP. Hybridization signals were detected and quantified by using a PhosphorImager .Total RNA was extracted from inoculated leaves at 2 and 6 dpi and from protoplasts at 24 hpi N. benthamiana as previously described 5 protoplasts. BaMV, PVX, or CMV RNA was purified as described previously DraIII and SacI.Protoplasts were isolated from HIS3 and lacZ reporter gene expression in Saccharomyces cerevisiae. Preparation of yeast competent cell, yeast transformation, selection of interaction, and β-galactosidase filter assay were carried out according to the user manual provided by Invitrogen. The bait and prey designate plasmids combined as indicated were transformed simultaneously into an L40 cell and plated on -Trp/Zeo300 for selection of successfully co-transformed cells. The colonies grown on the selected plate were transferred to the filter to analyze β-galactosidase activity. Additionally, three colonies were selected at random, dissolved in water and plated on -Trp-His/Zeo300 for interaction selection. Descriptions of plasmids and their construction are detailed in the Supporting Information  and an activation domain (AD). These were fused as two separated hybrid proteins referred to as bait and prey, respectively. A positive interaction causes ormation .(6) fusion proteins (1 µg each) and further incubation with gentle shaking for 2 h. The proteins bound to beads were washed with 1× PBS and eluted with elution buffer . The eluted proteins were resolved by 10% SDS-PAGE and analyzed by immunoblotting using an anti-His antibody (LTK BioLaboratories).Details for protein expression and purification are described in the Supporting Information . GST or Figure S1Schematic representation of the secondary structures of the 3′ UTRs of BaMV, satBaMV, and PVX. (A) The secondary structure of the BaMV 3′ UTR, established by enzymatic and chemical probing, comprises four stem-loops  and a pseudoknot (Domain E) B) The secondary structure of the satBaMV 3′ UTR, confirmed by enzymatic probing, comprises stem-loops A, B and C C) The secondary structure of the PVX 3′ UTR, established by enzymatic and chemical probing, consists of stem-loops 1, 2 and 3 (TIF)Click here for additional data file.Figure S2Comparison of the NbHsp90 amino acid sequences with those of three known isoforms. The full-length amino acid sequences of all published Hsp90 isoforms, NbHsp90-1, NbHsp90-2, and NbHsp90-3, and that of the clone from our lab, NbHsp90, were aligned using CLUSTAL W. The alignment output is ordered by the similarity between NbHsp90 and NbHsp90-1, -2, and -3. NbHsp90 is most similar to NbHsp90-2. Residues are numbered below the alignment. Completely conserved residues from the 4 sequences are lettered in gray. Residues that are identical to NbHsp90 are lettered in black and similar residues are lettered in white over a gray background. Different residues are lettered in white over a black background. The black lines above the sequence show the peptides identified by MALDI-TOF MS.(TIF)Click here for additional data file.Figure S3Knocking down Hsp90 expression in N. benthamiana reduces BaMV RNA accumulation. (A) Semi-quantitative RT-PCR analysis of Hsp90 mRNA levels in silenced (TRV1/TRV2-Hsp90) and non-silenced  plants. Actin mRNA was used as an internal control. Lane NC represents the negative controls in which the reverse transcriptase-free RT reaction mix was used as a template in the PCR reaction (36 cycles). (B) Quantification of the relative accumulation of BaMV, PVX, and CMV viral RNA in the control and NbHsp90-silenced plants at 2 or 6 dpi. All data are mean averages of three experiments and normalized against those of TRV1/TRV2 control plants.(TIF)Click here for additional data file.Figure S4Inhibition of NbHsp90 with GA and 17-AAG treatments reduces BaMV RNA accumulation in N. benthamiana protoplasts. Quantification of relative accumulation of BaMV, PVX, and CMV viral RNAs in control and Hsp90 inhibitor-treated protoplasts was shown. The data are mean averages of three independent experiments and normalized against those of DMSO-treated control protoplasts.(TIF)Click here for additional data file.Figure S5Effect of 17-AAG on satBaMV RNA accumulation in protoplasts. The relative accumulation of satBaMV RNA in protoplasts treated with DMSO or 17-AAG at various concentrations as indicated at the bottom. The satBaMV RNA accumulation concentration in DMSO treated protoplasts is designated as 100%. The data represent the mean averages of three independent experiments and standard deviations are indicated.(TIF)Click here for additional data file.Text S1Details for plasmid construction and recombinant protein purification.(DOC)Click here for additional data file.


Viral protein synthesis is completely dependent upon the translational machinery of the host cell. However, many RNA virus transcripts have marked structural differences from cellular mRNAs that preclude canonical translation initiation, such as the absence of a 5′ cap structure or the presence of highly structured 5′UTRs containing replication and/or packaging signals. Furthermore, whilst the great majority of cellular mRNAs are apparently monocistronic, RNA viruses must often express multiple proteins from their mRNAs. In addition, RNA viruses have very compact genomes and are under intense selective pressure to optimize usage of the available sequence space. Together, these features have driven the evolution of a plethora of non-canonical translational mechanisms in RNA viruses that help them to meet these challenges. Here, we review the mechanisms utilized by RNA viruses of eukaryotes, focusing on internal ribosome entry, leaky scanning, non-AUG initiation, ribosome shunting, reinitiation, ribosomal frameshifting and stop-codon readthrough. The review will highlight recently discovered examples of unusual translational strategies, besides revisiting some classical cases. No virus encodes its own ribosome. Indeed, it has been proposed that the distinction between cellular life and the virus world could be based simply on whether an entity encodes ribosomes or a capsid . Neverth7G) and a 3′ poly(A) tail. Translation can be divided into four stages: initiation, elongation, termination and ribosome recycling. The initiation step  and the A-site peptidyl–tRNA to the P-site, thus also moving the mRNA through the ribosome and opening the A-site for the next round of elongation. Elongation continues until a termination codon  enters the A-site. These codons are recognized by eukaryotic release factor eRF1, which, together with eRF3 and GTP, mediates termination of translation and release of the newly synthesized protein via hydrolysis of the P-site peptidyl–tRNA  or the use of segmented genomes where most segments are monocistronic (e.g. reoviruses and orthomyxoviruses). Another common strategy is to encode long polyproteins that are subsequently processed by virus-encoded or host proteases to generate the viral proteome (e.g. picornaviruses and flaviviruses). However, the use of these mechanisms has consequences. Viruses with segmented genomes have to ensure the correct packaging of the different segments, or must be able to tolerate the reduction in specific infectivity if segments are packaged randomly or individually into separate virions. Polyprotein expression can be considered to be an inefficient way of exploiting the available resources of the host cell as the mature virus proteins are produced in equal amounts, even though the enzymic proteins are often required in much smaller quantities than the structural proteins. Moreover, while some viral proteins may be expressed from sgRNAs, in probably all positive-strand RNA viruses the components of the replication complex must still be translated from the genomic RNA. Non-canonical translational mechanisms provide a number of alternative ways to express multiple proteins from a single mRNA.Coronaviridae and Reoviridae). Thus there is strong selective pressure to optimize their coding capacity, for example via the utilization of overlapping ORFs. Non-canonical translational strategies such as leaky scanning, ribosomal frameshifting and alternative initiation are essential in facilitating access to such ORFs. Non-canonical translational mechanisms may also help to overcome the challenges imposed by the marked structural differences present in many viral transcripts in comparison with typical cellular mRNAs. For example, packaging and/or replication signals within the 5′UTR of the genomic RNA or RNA segments can inhibit scanning-dependent translation initiation. Ribosome shunting or internal ribosome entry sites (IRESes) can be employed to circumvent such impediments. Non-canonical translational mechanisms may also play roles in regulating the expression level and/or timing of expression of various proteins.RNA viruses also have very compact genomes, with the largest around 30 kb (e.g. some members of the families It should be noted that many RNA viruses lack the machinery to add a 5′ cap and/or poly(A) tail to their transcripts and have evolved alternative mechanisms for ribosome recruitment and/or mRNA circularization. Similarly, several viruses have evolved proteins and or RNA structural elements that further enhance translation of the viral mRNAs. Due to space limitations, such mechanisms will mostly not be discussed here. Many excellent reviews covering these topics, besides some cautionary critiques, are available .i–GTP ternary complex (reviewed by Flaviviridae) requires fewer initiation factors , recruits 40S subunits directly, and places the initiator AUG into the ribosomal P-site without any requirement for scanning . On the other hand, in foot-and-mouth disease virus, two in-frame AUG codons are used as alternative initiation sites to produce different isoforms (Lab and Lb) of the leader protease  that directs translation of the 3′ ORF encoding structural proteins is very unusual. It is short  in comparison to picornavirus IRESes  and compact, but is folded elegantly by virtue of RNA pseudoknotting into a structure that can partly mimic E- and P-site tRNAs, including the P-site codon : anticodon duplex  that overlaps the 5′-proximal region of the polyprotein ORF . There iIn the scanning model of initiation, 40S ribosomal subunits bind close to the 5′ cap and scan linearly until they encounter the first AUG codon . HoweverOne of the earliest examples described is found in segment S1 of mammalian orthoreovirus . Here, tCaliciviridae), translation of an ORF overlapping the capsid coding sequence appears to initiate at the third AUG codon on the sgRNA  can also increase the efficiency of leaky scanning. This has been demonstrated in both tymoviruses and in segment 6 of influenzavirus B . Indeed, there are a number of cases of leaky scanning – including the PB1-F2 protein of influenza virus A – where there are one or more intervening AUG codons between the initiation codons of the ORFs that encode functional products . Such AUi, a tRNA that differs from the standard (elongation) Met–tRNA. However, initiation does not necessarily have to occur at an AUG codon. Near-cognate codons, such as CUG and ACG, can under certain circumstances also be recognized by Met–tRNAi. Initiation at a non-AUG codon normally requires a strong context (e.g. an A or G at −3 and a G at +4) and is enhanced when an RNA structure (e.g. a stem–loop) is able to form at a distance of approximately 14 nt 3′ of the initiation codon, so that it is positioned at the entrance of the mRNA channel when the potential initiation codon is in the P-site of the ribosome . An upstream in-frame ACG codon is used to initiate translation of C′, an N-terminally extended version of the C protein , involves three consecutive ORFs where translation of the first ORF initiates at an AUU codon , the second ORF initiates at an AUG codon with a poor context, and the first two ORFs contain no other AUG codons, despite spanning around 895 nt, thus allowing leaky scanning also to the third ORF  also lack a separate sgRNA for coat protein expression and, like the panicoviruses, these and several related viruses also appear to use non-AUG initiation to express the second movement protein . Nearly all plant viruses encode one or more movement proteins that facilitate cell-to-cell movement through plasmodesmata. For many plant viruses, this takes the form of the ‘triple gene block’ – an evolutionarily conserved module that comprises three proteins, TGB1, TGB2 and TGB3  or via reinitiation . The pgRNA is capped and has a long 5′UTR, much of which is predicted to fold into a large stem–loop structure. A short ORF terminates just upstream of the stem–loop  in Sendai respirovirus , the σC i–GTP ternary complex. Such short ORFs are thought to be widely used in cellular genes to regulate expression of downstream protein-encoding ORFs in response to the cellular environment , the 40S subunit may remain associated with the message, resume scanning, and reinitiate translation at a downstream AUG codon (reviewed by Lagovirus and Sapovirus), ORF2 is contiguous with ORF1 so that some capsid protein is also translated from the genomic RNA as a fusion with the non-structural polyprotein, even though the major source of capsid protein translation is still the sgRNA  coding sequence is used to translate the polymerase (Pol) – which is required in much lower quantities – from the single genomic RNA . Here, reinitiation is mediated by a viral protein ‘transactivator viroplasmin’ or TAV  and re-pairing in the −1 frame (XXX_YYY), thus allowing for perfect re-pairing except at the wobble positions . How theTotiviridae of dsRNA viruses make use of −1 frameshifting to express their RdRp or reverse transcriptase  is encoded as a separate ORF, two frameshifts are required to express the Gag–Pro–Pol polyprotein. There are numerous potential advantages in using frameshifting as an expression strategy. In retroviruses and totiviruses, for example, it allows the virus to generate a defined ratio of Gag : Gag–Pol that is likely to be optimized for virion assembly and allows facile targeting of the replicative enzymes to the virion core. It also obviates the need to produce a separate mRNA for expression of the viral polymerase. Similarly, in many positive-strand RNA viruses, frameshifting may serve to produce the polymerase at a fixed ratio relative to other components of the replication complex  frameshifting appears to be utilized to express the viral polymerase. However, in viruses these mechanisms have been far less well-studied than −1 tandem slippage and remain poorly understood, in part because the frameshift efficiencies, where investigated, often seem to be very low. In members of the  of ORF1 .Totiviridae utilize −1 tandem slippage to express a Gag–Pol fusion, or reinitiation to express Gag and Pol separately, a few members apparently utilize +1 or −2 frameshifting. Trichomonas vaginalis virus 1 (genus Trichomonasvirus) is particularly interesting. Here, ORF2 is in the +1 frame relative to ORF1 and nucleotide sequence analysis indicates that frameshifting is most likely to occur on a conserved CC_CUU_UUU sequence  was shown to be translated and essential for virus infectivity (pipo) is expressed as part of an approximately 25 kDa product that is believed to correspond to a fusion of PIPO with the N-terminal region of P3 (i.e. P3N–PIPO). The frameshifting mechanism has not yet been determined, but current evidence suggests that it occurs at the level of translation rather than transcription, and most likely involves a highly conserved GAA_AAA_A motif at the 5′ end of the pipo ORF  and its flanking nucleotides, especially the immediately adjacent 3′ base  and are a feature common to many viral readthrough cases – notably in the tobamoviruses, poleroviruses and luteoviruses  with replication  members of the taxa Iflavirus, Dicistroviridae, Tetraviridae, Rotavirus, Cypovirus and Totiviridae, with some viruses having multiple stop–carry on cassettes (Stop–carry on is mediated by the amino acid motif D(V/I)ExNPGP, which, together with less-conserved but nonetheless functionally important upstream amino acids also within the ribosome exit tunnel , prevents formation of a peptide bond between glycine and the final proline . NonetheAlthough this review has focused on the unusual translational mechanisms that viruses employ to cope with the unique constraints imposed by their compact genomes and atypical mRNAs, a number of RNA viruses have evolved various non-translational mechanisms that in some ways achieve similar results. As discussed previously, many viruses make use of sgRNAs, segmented genomes and post-translationally cleaved polyproteins in order to express the multiple proteins necessary for their replicative cycle. In addition, retro-transcribing viruses, besides a small proportion of RNA viruses, including orthomyxoviruses (e.g. influenzavirus) and bornaviruses, enter the host-cell nucleus where they make use of the host-cell splicing machinery for producing alternative transcripts.Mononegavirales), notably viruses in the subfamily Paramyxovirinae, which includes measles virus (genus Morbillivirus), mumps virus (genus Rubulavirus) and parainfluenzaviruses (genus Respirovirus). In these viruses, programmed polymerase slippage or stuttering occurs at a specific site  during transcription of the phosphoprotein mRNAs, leading to the efficient insertion of one or more extra Gs in a proportion of transcripts  and programmed RNA editing. The most common form of RNA editing in vertebrates is editing of adenosine to inosine by ADARs . Some such viruses are highly divergent from known viruses and are likely to provide examples of new translational mechanisms, besides many new variations of previously identified mechanisms. This is particularly so for viruses of organisms (or organelles) with unusual components of the translational apparatus, such as unusual ribosomes or unusual tRNA types and abundances. Other newly discovered viruses are related more closely to known viruses but can provide a broader phylogenetic baseline for computational comparative analyses that can be used to detect undiscovered elements in economically and medically important species.Together, it is clear that RNA viruses provide a fascinating plethora of examples of non-standard mechanisms for gene expression. Traditionally, virus research has focused on those species that are most relevant to humankind – that is, human viruses and the viruses of commercially important plants and animals, besides viruses of human parasites such as The discovery of novel translational elements is increasingly driven by bioinformatic analysis of sequence databases, but new data resources  involve a significant proportion of translating ribosomes and tend to be subject to strong purifying selection and phylogenetic conservation. Not surprisingly, there are continua in the dimensions of efficiency, functionality and evolutionary conservation, and it is not always obvious whether a given case of non-canonical translation is programmed or incidental.The study of non-canonical translation can lead to the development of extremely valuable tools for molecular biological research and biotechnology. A case in point is the use of stop–carry on cassettes for equimolar co-expression of multiple proteins from a single transcript. Non-canonical translational mechanisms (in particular frameshifting in HIV) have also been proposed as potential targets for antiviral drugs. This is particularly attractive if it can be demonstrated that a given mechanism is not utilized for host gene expression. Finally, by acquiring a greater understanding of the extent to which the translational machinery can be subverted from canonical cap-dependent scanning initiation and triplet decoding, and the mechanisms for achieving such subversions, one will also develop a greater understanding of the canonical mechanisms of eukaryotic translation – arguably (together with counterparts in bacteria and archaea) the most important process in the modern biosphere.


Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco ( Nicotiana benthamiana plants accumulated increased amount of PSTVd genomic RNA comparing to the wild-type control, supporting the contention that viroid hairpin-derived siRNAs can reduce the accumulation of viroid by RNA silencing system.Viroids are the smallest known infectious agents of plants, and induce disease in a wide range of hosts including many crop species. They have been identified as non-coding, circular, single stranded RNAs ranging in size from 246 to 401 nt Nicotiana plants. Furthermore, a specific siRNAs produced in squash stock has been detected in phloem sap from a grafted cucumber scion RNA silencing by siRNAs in plants has the ability to spread systemically. This does not involve gradual cell-to-cell spread. The silencing signal travels along the vascular system and induces silencing in sink tissues Therefore, virus and/or viroid-derived siRNAs transported from stock harboring the siRNAs producing system would be expected to counteract the accumulation of invader RNA in the scion. If this is indeed the case, resistance of non-transgenic scion to virus and/or viroid infection might be improved by grafting on genetically-modified rootstock. Here we present for the first time the evidence that viroid-specific small RNA signals supplied from genetically-modified rootstock expressing non-infectious form of hairpin viroid RNA can attenuate the accumulation of viroid RNA in the scion.N. benthamiana companion cells.The expression of a truncated, near full-length, hpPSTVd-ΔTLE was controlled by a companion cell-specific promoter, pCoYMV N. benthamiana were obtained. Although a certain (ca. 5.3∼12.2%) proportion of the four T1 seedlings exhibited abnormal phenotypes such as dwarfism, delayed rooting, an aberrant leaf shape and so on, none of the T2 seedlings obtained exhibited any obvious phenotypical abnormalities. Lines 24 and 32 accumulated detectable levels of PSTVd-specific siRNAs by RNA-gel blot analysis, while the remaining two lines (11 and 141) did not show detectable levels of PSTVd-siRNAs , which are genes homologous to the tomato RNA-binding protein SlVirp1 gene SlVirp1 mRNA is decreased in PSTVd-infected tomato leaves NbBRP1 and 2 was inversely up-regulated in CoYMV:hpPSTVd-ΔTLE lines , whose mRNA level was reported to have been increased by virus infection NbAGO1 mRNA in hpPSTVd-ΔTLE lines was almost equivalent to that in control plants , in our previous study The results presented here provided a potentially useful technique for preventing plant from virus and/or viroid infections, in which we have constructed a hair-pin structure of near full-length (non-infectious form) of PSTVd and expressed under the control of companion cell–specific CoYMV promoter in EcoRV site of the pBluescript II SK+ plasmid . Then the CAT1 intron AatII/HindIII sites of the plasmid. Finally, an additional PSTVd-ΔTLE sequence, amplified by PCR using primers tPSTVdaFw and tPSTVdaRv, was integrated into the HindIII/KpnI sites, to make a hairpin (hp) structure of PSTVd-ΔTLE, in which a pair of PSTVd-ΔTLE is located in both boundaries of CAT1 intron by head to head orientation. The hpPSTVd-ΔTLE fragment from the plasmid was inserted into the BamH/KpnI sites of CoYMV-nos ter A truncated, near full-length, PSTVd-Intermediate strain sequence  was performed as described previously −2s−1.Transformation of tobacco  infected with PSTVd-Intermediate strain (accession no. AY937179) in 150 µl of 50 mM phosphate buffer (pH 7.0) containing 1 mg/ml bentonite. The 7-th true leaf of one-month-old plants were dusted with 600-mesh carborundum powder, and then gently rubbed with the inocula. Diethylpyrocarbonate-treated water was used for mock inoculation.Extraction of small RNAs and detection of siRNAs were performed essentially as described by Kasai et al. N. benthamiana BRP, AGO1, ZIGA3, and NAPDH genes was obtained from the EMBL data base. The primers used for qPCR are described in Total RNA was extracted using TRizol reagent , and treated with TURBO DNA-free . In accordance with the manufacturer’s instructions, cDNA was synthesized with 1 µg RNA as a template using Superscript VILO (Invitrogen). The qPCR analysis was performed essentially as described in Kasai et al. N. benthamiana plants was assessed by blot analyses. Two discs (1.0 cm in diameter) were sampled from the upper most expanding leaves and subjected to extraction of the RNA fraction, as referenced The amount of PSTVd in Micrografting was performed essentially as described by Kasai et al. Figure S1Genomic PCR of the CoYMV:hpPSTVd-ΔTLE lines. (A) Schematic representation of each PCR fragment. (B) Genomic PCR analysis of four independent transgenic lines using three primer pairs. Quantity of genomic DNA was confirmed by NbUbi. Only IR3’ fragment was performed by 40 cycles and others were by 35 cycles.(TIF)Click here for additional data file.Figure S2The expression of possible target genes of PSTVd-mediated RNA silencing in CoYMV:hpPSTVd-ΔTLE plants. (A) Locations of potential mRNAs in tomato targeted by srPSTVd. (B) Alignment of possible srPSTVd target sites in tomato and N. benthamiana (left). Expression levels of three genes in transgenic tobacco whole plants harvested at one month after sowing. The levels of these mRNAs were analyzed by qRT-PCR. Error bars, s.d. (n = 3). **; significant difference from Empty (P<0.05). The amounts were normalized on the basis of NbUbi.(TIF)Click here for additional data file.Figure S3Dot-blot analyses of PSTVd RNA in CoYMV:hpPSTVd-ΔTLE lines. Transgenic lines were inoculated with PSTVd. At 7 and 14 dpi, leaf disks were sampled and the extracted RNA was dot-blotted. The membrane was hybridized with the PSTVd negative strand RNA probe. 5.8S rRNA was used as an internal control for the amounts of total RNA.(TIF)Click here for additional data file.Figure S4Dot-blot analyses of PSTVd RNA in scions grafter onto respective stocks. (A) Scion WT plants were inoculated with PSTVd (ca. 6.7 µg/20 µl) and the dot-blotted membrane was hybridized with the PSTVd negative strand RNA probe. (B) 5.8S rRNA was used as an internal control for the amounts of total RNA.(TIF)Click here for additional data file.Table S1Sequences of primers used in gPCR and qRT-PCR.(TIF)Click here for additional data file.


We thus probed the sequence space that is compatible with HIV-1 replication as an initial test of the function of these three regions. We also analyzed the impact of selected sequences on the secondary structure of the leader RNA, that is the individual hairpin motifs and the proposed LDI:BMH riboswitch that controls multiple leader RNA functions.The 5’ untranslated leader of the HIV-1 RNA genome contains several highly conserved structures that control late virus replication steps. The DIS, SD and Ψ hairpins control the processes of RNA dimerization, splicing and packaging, respectively. Relatively few studies have focused on the short single-stranded domains that connect these hairpin structures. Using the in silico using RNA secondary structure software.The domains upstream of the DIS , between DIS and SD  and between SD and Ψ  were randomized in pLAI. These virus DNA libraries were transfected into the SupT1 T cell line and the supernatant was passaged for multiple weeks to select the most replication-competent variants. Interesting variants were recloned into pLAI to confirm the phenotype. The impact on RNA secondary structure was probed Although the sequence of all 3 motifs is highly conserved among natural HIV-1 isolates, quite different results were obtained for these regions. A strict requirement for the wild-type sequence (GCAGGA) was observed for region 1, although two point mutants (GCAGUA and GCAAGA) were also selected. A more relaxed sequence requirement was apparent for region 2, as a larger variety in sequences was selected compared to region 1. In sharp contrast, the highly conserved A5-stretch of region 3 did not reappear in SELEX. None of the selected sequences affected the folding of the individual hairpin motifs or the global LDI:BMH equilibrium of the leader RNA.In silico analysis indicates that sequences are avoided in all 3 single-stranded domains that affect the local RNA folding of the hairpins or the LDI: BMH riboswitch. Surprisingly, a large variety of region 3 sequences support HIV-1 replication, despite the strong conservation of the A5 stretch in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure of HIV-1 to acquire an A-rich RNA genome.Our results show a strong sequence requirement for the DIS hairpin flanking regions 1 and 2, whereas the sequence of region 3 (between the SD and Ψ hairpins) seems to have a less prominent role. We postulate that the sequences of region 1 and to a lesser extent 2 are important for the binding of specific protein factors that support leader RNA mediated functions.


Profiling gene expression in human ocular tissues provides invaluable information for understanding ocular biology and investigating numerous ocular diseases. Accurate measurement of gene expression requires high-quality RNA, which often is a challenge with postmortem ocular tissues.later or preserved after initial storage at 4 °C to create a range of DP times from 2 to 48 h. Ten ocular tissues were dissected from each eye. After total RNA was extracted from each dissected ocular tissue, the RNA integrity number (RIN) was determined using an Agilent Bioanalyzer.We examined the effect of various death to preservation (DP) times on the RNA quality of ten different ocular tissues. We used 16 eyes from eight different human donors. The eyes were preserved immediately in RNAThe RIN values from corneal and trabecular meshwork tissues were significantly (p<0.05) higher than those from the ciliary body at an earlier DP time (<6 h), but were not different among all tissues after 8 h. Interestingly, the RIN values from non-vascularized tissues were significantly (p=0.0002) higher than those from vascularized ocular tissues at early DP times (<6 h). The RIN value from the cornea was significantly (p<0.05) higher at short DP times compared to longer DP times. The RIN values from corneal tissues were significantly correlated to DP time according to regression analysis (p<0.05).In this study, we determined RNA quality from postmortem ocular tissues with various DP times. Our results emphasize the need for rapid preservation and processing of postmortem human donor eye tissues, especially for vascularized ocular tissues. Messenger ribonucleic acid (mRNA) mediates informational transfer from genomic DNA to biologic effector protein molecules. Gene expression profiling using RNA isolated from cells and tissues has been extensively used to study the development, differentiation, and effects of exogenous factors such as growth factors and drugs, as well as to better understand disease pathogenesis. Although isolating RNA from fresh cell cultures is relatively straightforward, harvesting RNA from tissues, particularly human tissues, is more challenging. RNA integrity in tissues depends on the postmortem time to preservation, the metabolic profiles of the tissues, endogenous RNase activity, and natural RNA degradation.Numerous studies have been performed to study mRNA expression profiles using various techniques, including reverse transcription–polymerase chain reaction (RT–PCR), RNA differential display, DNA microarrays, and RNAseq. In particular, microarray analysis of mRNA expression has been used to identify genes associated with aging , retinalGAPDH. RNA integrity is also tested by visualizing 28S and 18S rRNA after agarose gel electrophoresis. This method is now automated using a microcapillary electrophoresis system to provide an RNA integrity number (RIN) [RNA is progressively degraded in postmortem tissues and during RNA extraction. In many cases, RNA extracted from donor eye tissues is not of optimal quality, which complicates gene expression analysis. Several methods are used to determine RNA quality. A commonly used technique is reverse transcriptase PCR amplification of housekeeping genes such as β-actin or er (RIN) .later, and the RNA extraction technique all affected overall RNA quality. Malik and colleagues examined the effects of DP time on RNA quality in the retina and retinal pigment epithelium of pig eyes [Several studies have been performed to establish optimal conditions for RNA extraction from human ocular tissues. Wang and colleagues tested several different RNA preservation methods for human donor ocular tissues to extract high-quality RNA . They fopig eyes . ImmediaAlthough many of the eye tissues are vascularized, the eye also contains avascular tissues . DP time may differentially affect RNA quality in vascularized versus non-vascularized tissues. In the current study, we examined RNA quality in ten different vascularized and avascular human ocular tissues at various postmortem times using RIN analysis.later  as described earlier [later and stored at 4 °C. The contralateral eye was placed in a moist chamber at 4 °C for 8–48 h before being placed in 30 ml of RNAlater. The paired eyes were then shipped on ice to UNTHSC for dissection.Donor eyes were provided by the Lion’s Eye Institute for Transplant and Research . As shown in  earlier . Paired later until >12 h DP time. Each dissected tissue was then placed in a 1.5 ml tube filled with RNAlater and stored in −80 °C until RNA extraction was performed.The following ocular tissues were carefully dissected from each donor eye: iris (I), sclera (S), trabecular meshwork (TM), cornea (C), retina (R), optic nerve head (ONH), optic nerve (ON), lens (L), choroid/retinal pigment epithelium (C/R), and ciliary body (CB). The majority of the eyes were pseudophakic, and the crystalline lens was present in only four donor eyes. In some eyes, clean tissue dissection was difficult, especially in eyes that had not been placed in RNAEach eye tissue was homogenized in 1 ml of Iso-RNA Lysis Reagent  using a TissueLyser II . Total RNA was further extracted using RNeasy Micro Kits (Qiagen) as described in the manufacturer’s guide. Briefly, 50 μl of 4-Bromoanisole  was added to tissue homogenates and mixed gently. After centrifugation at 12,000 ×g for 15 min at 4 °C, the aqueous phase was separated and mixed with the same volume of 100% ethanol. The aqueous phase and ethanol mixture was passed through an affinity column for RNA and washed. The column was washed again after DNase treatment and dried to remove the remaining ethanol. Total RNA was eluted in DNase/RNase free water.The RNA integrity number (RIN) was determined in ten ocular tissues from 16 donor eyes using an Agilent 2100 Bioanalyzer  with RNA nano- or picochip kit systems depending on the RNA concentration of each ocular tissue . First, 2 value and the significance between the DP time and the RIN. Data were also subjected to the paired Student t test or one-way analysis of variance (ANOVA) followed by a post hoc test (Bonferroni) for differences in the RINs between tissues. All data are expressed as means ± standard error of the mean (SEM). A p value of 0.05 or less was considered statistically significant.Statistical analysis was performed using GraphPad Prism Version 5.0 . For determining the time-dependent changes in the RIN, linear regression was performed to determine the Rlater within 6 h. of DP. RNA isolated from the cornea and the TM had significantly higher RIN values than RNA isolated from the ciliary body . In contrast, the other ocular tissues did not show significant correlations between the RIN and the DP time.Shorter DP times enhanced RIN values in RNA isolated from the cornea (p<0.05), TM (p=0.0607), and ON, but had only modest to no effects on RNA quality from the iris and choroid/RPE . Represelater preservation. Ocular tissue RNA quality was determined as RIN values using a commercial bioanalyzer. We found that RNA quality deteriorated rapidly and was associated with DP time. There was a statistically significant correlation in RIN values with DP times in corneal tissues, but surprisingly not in the other ocular tissues. RNA quality was significantly higher at shorter DP times for the non-vascularized ocular tissues compared to the vascularized ocular tissues.We examined the effect of postmortem time on the quality of RNA isolated from human ocular tissues subsequent to RNARho was detected at all postmortem time ranges out to 48 h. However, in isolated RPE cells, the RPE-specific RPE65 mRNA showed relatively rapid decay after death. Their results demonstrated that RNA degradation varied by tissue and by the specific RNA analyzed. We also examined the effects of postmortem time on RNA integrity using ten different ocular tissues. One of the paired eyes was placed in RNAlater within 2–6 h, while the contralateral eye was placed in a moist chamber at 4 °C, simulating normal storage conditions for enucleated human eyes. These eyes were then placed in RNAlater 8–48 h postmortem (The quality of the postmortem human DNA was previously determined from the retina and iris. Wang et al. showed that total DNA from the postmortem human retina was significantly superior in quality and quantity compared to that from the postmortem human iris . More restmortem . We werestmortem . Howeverstmortem . RNA frostmortem . We specstmortem .later as soon as possible after enucleation to prevent potential loss of RNA quality. Importantly, an incision should be made through the sclera into the vitreous to facilitate penetration of this reagent into the eyes, and eyes should be kept at 4 °C until dissected, preferably no longer than 1 month. RNA quality diminished quickly, particularly in highly vascularized ocular tissues. Our findings provide guidance for analyzing gene expression from human donor ocular tissues.In this study, we determined the RNA integrity of ten human ocular tissues isolated from human donor eyes preserved at various postmortem times. From our data, we conclude that donor eyes should be preserved in RNA