We highlight how these elements may interact with host translation factors and speculate on their contribution for achieving translational control. We also describe other translation strategies used by plant viruses to optimize the usage of the coding capacity of their very compact genomes, including leaky scanning initiation, ribosomal frameshifting and stop-codon readthrough. Finally, future research perspectives on the unusual translational strategies of +ssRNA viruses are discussed, including parallelisms between viral and host mRNAs mechanisms of translation, particularly for host mRNAs which are translated under stress conditions.Viral protein synthesis is completely dependent upon the host cell's translational machinery. Canonical translation of host mRNAs depends on structural elements such as the 5′ cap structure and/or the 3′ poly(A) tail of the mRNAs. Although many viral mRNAs are devoid of one or both of these structures, they can still translate efficiently using non-canonical mechanisms. Here, we review the tools utilized by positive-sense single-stranded (+ss) RNA plant viruses to initiate non-canonical translation, focusing on  We also describe other protein translation strategies used by plant viruses to optimize the usage of the coding capacity of their very compact genomes, including leaky scanning initiation, ribosomal frameshifting and stop-codon readthrough.Viruses usurp the metabolism of the host cell in their own benefit. Viral mRNA translation is a paradigmatic illustration of this, as the hallmark of viruses is that their genomes do not code for a protein synthesis apparatus. Thus, viruses have evolved many subtle ways to use and control the translational machinery of their hosts ppp(5′)N cap structure, and a poly(A) tail at the 3′ end, which are critical cis-acting elements during canonical translation. Traditionally, translation is divided into four distinct steps: initiation, elongation, termination and ribosomal recycling. Translation initiation is the rate limiting and most highly regulated step -binding protein (PABP) to the 3′ poly(A) tail  bind to the A-site through the second codon of the mRNA Lewin, . After tcis in the 5′-UTR, for example internal ribosome entry sites (IRESes) or genome-linked viral proteins (VPgs), in the 3′-UTR, for example cap-independent translation elements (3′-CITE) or tRNA-like structures (TLS), and also in intergenic regions, for example intergenic IRESes  tail. These can be mediated by stimulators present in es Table .Potyviridae is the largest among plant viruses with RNA genomes. The potyviral genome acts as mRNA and codes for a single polyprotein which is cleaved by viral proteases rendering 10 final functional proteins  is only 22 amino acids (aa) long, while that of potyviruses consists of around 192 aa.The family Tobacco etch virus  showed that its 5′-UTR contains a sequence that was able to enhance translation 8- to 21-fold in tobacco protoplasts  (see Section on CITEs) and the polypyrimidine-rich tracts located in both IRES elements found in Blackcurrant reversion virus  , Turnip mosaic virus , and Triticum mosaic virus    replication  Table  have beee) Table , and IREe) Table . The 131e) Table ; this RNPicornaviridae, and lack strong structure or conserved sequence, and AUG triplets  . The exceptionally long (739 nt) 5′-UTR is much longer than that of other potyvirids and translation initiates at the 13th AUG triplet  VPg binds eIF4E and eIFiso4E and enhances viral translation in plants   RNA, so the VPg acts as a functional analog of the cap  VPgs adopt a compact three-helical bundle structure, Murine norovirus  VPg has only two helices  harbors two IRESes that stimulate the synthesis of the CP and movement protein (MP), 75 and 148-nucletotides long, respectively    . This IRES, which is in a highly unexpected location, 22 nt downstream of the start codon and within a region of the PLRV RNA genome that is characterized by non-canonical translation mechanisms such as −1 ribosomal frameshifting, leads to translation of replication-associated protein (Rap1)  ORF from undetectable amounts of degraded RNA. This alternative mechanism of expression may still be biologically relevant, or simply an artifact of the assays, but would not result from an IRES.Bromoviridae and the genera Tobamovirus and Tymovirus possess a 5′ cap structure but lack a 3′ poly(A) tail. In contrast, they contain tRNA-like structures (TLSs) at their 3′ termini that perform many viral processes, such as (i) serving as a telomere by interacting with CTP:ATP nucleotidyl transferase which adds CCA in a non-templated fashion to the 3′ end , TMV and Brome mosaic virus . Because of their multiple functions, it has been difficult to tease out the mechanisms of each role, but the translational enhancement structures and mechanisms have been well characterized for TMV and TYMV  IRES of dicistroviruses  RNA requires the viral CP for efficient translation and infection. The 3′-UTR of AMV also plays a role in translation due to its ability to bind the CP, adopting the CP-binding (CPB) conformation. This binding avoids the minus-strand promoter activity and enhances translation, possibly acting as a mimic of the poly(A) tail  elements, but contain in their 3′ ends structured RNA elements capable of enhancing translation in the absence of cap . Most 3′-CITEs have in common their ability to bind translation initiation factors of the eIF4E or eIF4G families, as well as the presence of small sequence stretches within or near the 3′-CITE capable of base-pairing to sequences in the 5′-UTR of the mRNA to establish long-distance RNA:RNA interactions  and is located in a 120-nt sequence termed translation enhancer domain (TED)  was recently shown to harbor a 3′-CITE in the TED class  and Melon necrotic spot virus   present in the 3′-UTRs of In vitro binding assays revealed that Ma5TE forms a complex with eIF4F and this interaction was mapped to a conserved guanosine residue located in a Ma5TE internal loop  Xinjiang by interfamilial recombination, conferring to the recipient MNSV isolate the advantage to translate efficiently and infect resistant melon varieties  is one of the best-characterized 3′-CITEs and is found in all members of the Luteovirus, Dianthovirus, Alphanecrovirus, Betanecrovirus, and Umbravirus genera . The BTE binds preferentially to the eIF4G subunit of the eIF4F heterodimer , in which mutations in potential complementary loops had no effect on translation and possess the longest BTE and 3′-CITE  and other viruses belonging to the genus Tombusvirus, contain 3′-CITEs resembling Y-shaped structure (YSS), formed by three helical regions. The efficiency of translation controlled by the YSS of TBSV depends on a long-distance interaction with the 5′-UTR of the genome. Mutational analysis of TBSV YSS showed that alterations in junction residues between helices and in a large asymmetric bulge in the major supporting stem disrupted translation  requires addition of the eIF4F or eIFiso4F complex to a factor-depleted wheat germ extract to promote efficient translation  was first identified in Panicum mosaic virus   , harbors a PTE which participates in a long-distance RNA:RNA interaction with a hairpin located in the p26 ORF , contains an internal T-shaped structure (TSS) that consists of three hairpins, two pseudoknots and multiple unpaired single stranded linker regions  genomes contain a VPg linked to their 5′ end, thus are uncapped but polyadenylated requiring also cap-independent translation mechanisms. The two genomic RNAs (gRNA) of Blackcurrant reversion virus , have translation enhancing sequences in their 5′- and 3′-UTRs. The 5′ leader sequences of the two gRNAs of BRV contain IRES elements that facilitate translation when placed either at the 5′-end of a non-capped reporter RNA or internally between two reporter genes  initiation of translation at multiple start codons in different reading frames, by leaky scanning of ribosomes, (ii) frameshifting by a portion of the ribosomes during the elongation phase of translation, or (iii) generating subgenomic mRNAs that allow translation of each ORF from a separate mRNA. The latter will not be discussed, as it is not a translational control mechanism.Potato virus X  and Barley stripe mosaic virus  and the TGB2 of Peanut clump virus  are expressed by leaky scanning  contains a non-canonical ORF for its TGB3 protein  and Maize chlorotic mottle virus  were suggested to be initiated in a GUG or CUG start codons, accomplished by leaky scanning aAUGGCg initiation context Figure . If the Scheets, .Turnip rosette virus  to establish systemic infection  are predicted to use a +1 frameshift to synthesize their viral replicases  to translate overlapping ORFs. This recoding event can occur in the + or − direction relative to the normal 0 frame of mRNA translation by shifting the ribosome in one or two nucleotides forward or backward. Productive frameshifting normally competes poorly with standard decoding, so the efficiency of frameshifting in viruses varies from 1% in BYDV to 82% in cardioviruses ; and a downstream secondary structure element separated from the slippery sequence by a spacer region of 5-9 nt Dinman, . In planThe -1 PRS stimulatory elements of BYDV, PEMV-RNA2 and RCNMV fold into a stem-loop with an internal bulge in a similar manner  and PEMV1  form h-type pseudoknots  employs a complex series of downstream interactions. A stable bulged readthrough stem-loop (RTSL) immediately downstream of the leaky stop codon contains a G-rich bulge which must base pair to a distant readthrough element (DRTE) located 3 kb downstream in the structure required for replication initiation , IRES, and CITE (Karetnikov et al., From the point of view of the cellular translational machinery and how viruses use it, the described diversity of translation mechanisms points toward the different ways that viruses use and control the basic translation machinery of the cell, but it also seems to point toward the existence of a diversity of associations of RNA and protein translation factors used for the uninfected cell to synthesize proteins from different mRNA populations under different micro-environmental conditions and/or subcellular locations. It is tempting to speculate that during evolution plant viruses may have adopted cellular preexisting mechanisms to translate their proteins; quite likely, there is a significant overlap between translation mechanisms of viral RNAs and translation of cellular mRNAs in uninfected cells under abiotic stress conditions (Spriggs et al., cis-acting translation elements identified in plant viruses may contribute to the design of tools for synthetic biology (Ogawa et al., Last but not least, the diversity of MM wrote Sections Intergenic Region Enhancers to Optimization of Viral mRNA Coding Capacity, WM, VT, and MA edited and added specific information to all sections, MA supervised MM writing and wrote Sections Introduction and Perspectives.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Similar to eukaryotic mRNA, the positive-strand coronavirus genome of ~30 kilobases is 5’-capped and 3’-polyadenylated. It has been demonstrated that the length of the coronaviral poly(A) tail is not static but regulated during infection; however, little is known regarding the factors involved in coronaviral polyadenylation and its regulation. Here, we show that during infection, the level of coronavirus poly(A) tail lengthening depends on the initial length upon infection and that the minimum length to initiate lengthening may lie between 5 and 9 nucleotides. By mutagenesis analysis, it was found that (i) the hexamer AGUAAA and poly(A) tail are two important elements responsible for synthesis of the coronavirus poly(A) tail and may function in concert to accomplish polyadenylation and (ii) the function of the hexamer AGUAAA in coronaviral polyadenylation is position dependent. Based on these findings, we propose a process for how the coronaviral poly(A) tail is synthesized and undergoes variation. Our results provide the first genetic evidence to gain insight into coronaviral polyadenylation. Posttranscriptional modifications occurring in the nucleus of eukaryotic cells include cleavage of the 3′ end of nascent mRNAs and the addition of a poly(A) tail –5. The pcis-acting element required for replication may also be used for polyadenylation in RNA viruses. For example, the hexamer AAUAAA in bamboo mosaic virus and a domain immediately upstream of the poly(A) tail in coxsackievirus B3 have been shown to function as cis-acting elements involved in both negative-strand RNA synthesis and polyadenylation [As opposed to mRNAs used only for translation, polyadenylation of viral RNA in RNA viruses may be involved in both translation and replication , 18. RNAnylation , 25.betacoronavirus, subfamily Coronavirinae, family Coronaviridae and order Nidovirales, is a 5’-capped and 3’-polyadenylated positive-strand RNA virus. Although the mechanism for coronaviral polyadenylation remains unknown, a stuttering mechanism based on the short poly(U) stretch found in the negative-strand genome has been postulated [Bovine coronavirus (BCoV), a stulated . Moreovestulated , 27. Sucstulated . HoweverIn the current study, we determined that both the poly(A) tail and hexamer AGUAAA are important elements responsible for the polyadenylation of coronavirus. The efficiency of poly(A) tail elongation during infection depends on the initial poly(A) tail length at the time of infection. Based on these findings, we propose a process for how the coronaviral poly(A) tail is synthesized and undergoes variation. The results presented here provide the first genetic evidence that will help in elucidating coronaviral polyadenylation.7 PFU/ml was used as a helper virus throughout the study. Human rectal tumor (HRT)-18 cells  were maintained in DMEM supplemented with 10% fetal bovine serum and used for BCoV infection as described [A plaque-purified Mebus strain of bovine coronavirus (BCoV)  (GenBank accession no. U00735) at 3 × 10escribed –30.SpeI and MluI. The digested fragment was then cloned into SpeI- and MluI-linearized pW-25A to generate the mutant pR-25A. Mutants of pM1-25A, pM2-25A, pM3-25A, pM4-25A, pM5-25A, pPAS-R-25A and pPAS-PAS-25A were similarly constructed, except for the corresponding oligonucleotides used in the first and second reactions, as described in Construction of defective interfering (DI) RNA pW-25A  -25A, pR-25U, pR-25C, and pR-25G, PCR was also performed using a similar method but with pR-25A DNA as the template. This strategy was also applied to generate mutants pM1-15A, pM2-15A, pM3-15A, pM4-15A, pM5-15A, pPAS-R-15A and pPAS-PAS-15A, but the DNA templates for these mutants were pM1-25A, pM2-25A, pM3-25A, pM4-25A, pM5-25A, pPAS-R-25A and pPAS-PAS-25A, respectively, and the oligonucleotides were TGEV 7(-) and BM15A(+).To generate the constructs pW-0A, pW-5A, pW-8A, pW-12A, pW-15A, pW-18, pW-20A, pW(C)-25A, pW-25U, pW-25C, pW-25G, pW-random and pW-polyCC, PCR was performed using pW-25A DNA as the template with the oligonucleotide TGEV 7(-) and the appropriate oligonucleotide that binds to the terminal sequence of the 3’ UTR, as described in in vitro, all DNA constructs  tail, or with only poly (U), poly(C) or poly(G) tail) were linearized with MluI. The linearized DNA was transcribed in vitro with the mMessage mMachine T7 transcription kit (Ambion) according to the manufacturer's instructions and passed through a Biospin 6 column (Bio-Rad), followed by transfection [5 cells/dish) were infected with BCoV at a multiplicity of infection of 5 PFU per cell. After 2 hours of infection, 3 μg of transcript was transfected into mock-infected or BCoV-infected HRT-18 cells using Lipofectine (Invitrogen) [To synthesize transcripts sfection . For traitrogen) , 36.To prepare RNA for the identification of DI RNA poly(A) tail length, RNA was extracted with TRIzol (Invitrogen) at the indicated times after transfection of DI RNA constructs into BCoV-infected HRT-18 cells; the virus within the transfected cells is referred to as virus passage 0 (VP0) . SupernaAmong the PCR-based methods for the determination of poly(A) tail length –41, a he5 cells/dish). For RNA stability assay, 3 μg of DI RNA transcript was transfected into the HRT-18 cells. HRT-cells were incubated with RNase A  for 15 min prior to extraction of cellular RNA. For a replication assay, after 2 h of infection with BCoV at a multiplicity of infection of 5 PFU per cell, 3 μg of DI RNA transcript was transfected into the BCoV-infected HRT-18 cells. Tthe supernatant was harvested at 48 hpt (VP0), and 500 μl was used to infect freshly confluent HRT-18 cells in a 35-mm dish (VP1). Total cellular RNA was extracted with TRIzol (Invitrogen) at 48 hpi of VP1, and 10 μg was electrophoresed through a formaldehyde-agarose gel. The RNA was transferred from the gel to a Nytran membrane by vacuum blotting, and the blot was probed with 5’-end 32P-labeled oligonucleotide TGEV8(+) for 16 h. The probed blot was washed and autoradiographed at -80°C for 24 h.Detection of the reporter-containing DI RNA was performed essentially as described previously , 36. In ®Universal PCR Master Mix (Applied Biosystems) using primers MHV3’UTR3(-) and BCV23-40(+). The real-time PCR amplification was performed according to the manufacturer’s recommendations in a LightCycler® 480 instrument (Roche Applied Science).To determine the replication efficiency of DI RNA, the cDNA prepared as described above from head-to-tail ligation RNA collected at 1 and 24 hpi of VP1 was used for real-time PCR amplification with TagMancis-acting elements required for replication in coronaviruses [betacoronavirus and that the replication efficiency of this MHV 3’ UTR-containing BCoV DI RNA is similar to that of wild-type BCoV DI RNA [In a previous study, we demonstrated that the length of the coronaviral poly(A) tail on both viral RNAs and DI RNA is regulated during infection; that is, the poly(A) tail length is increased in the early stage of infection and then decreased after the peak tail length in the later stage of infection , 27. To aviruses , 47–49. aviruses , 31, 32 aviruses , 27. It V DI RNA , were deV DI RNA , the polcis-element involved in coronaviral polyadenylation, the hexamer in BCoV DI RNA W-25A was mutated from AGUAAA to UCAUUU; the resulting DI RNA was designated R-25A  hexamer AAUAAA or its variant  located10–30 nts upstream of the poly(A) cleavage site , 10 and ed R-25A . RNA wased R-25A , the reped R-25A , suggested R-25A , replicaed R-25A  and recoed R-25A  may not Since the hexamer AGUAAA may not be required for coronaviral polyadenylation when the poly(A) tail on input DI RNA is 25 nts long, we hypothesize that the AUGAAA hexamer may be critical when the tail length is short. That is, it may be possible that both the hexamer AGUAAA and poly(A) tail may contribute to coronaviral polyadenylation through concerted action when the tail is at a certain length shorter than 25 nts on input DI RNA. This hypothesis is based on the results that (i) a poly(A) tail was not synthesized for W-0A , even thDiscussion for more details). In addition, the unidentified poly(A) tail from R-5A may be attributed to the undetected RT-PCR product due to the poor replication efficiency. For the factor of recombination, no RT-PCR product was observed  tail length, the DI RNA stability, replication efficiency and recombination are also factors affecting the synthesis of poly(A) tail on DI RNA, DI RNAs W-25A, R-5A, R-15A and R-20A with various length of synthesized poly(A) tail  at 24 hpi of VP1  were selobserved , suggestobserved . TherefoTo test further the role of hexamer AGUAAA and poly(A) tail in coronaviral polyadenylation, 25 nts of the W-25A poly(A) tail was first replaced with the same length of a poly(U), poly(C) or poly(G) tail to create mutant W-25U, W-25C or W-25G, respectively . Moreovecis-acting element in coronaviral polyadenylation. Additionally, different levels of substitutions within the hexamer AGUAAA were also performed in W-25A  tail site, where the original hexamer AGUAAA is located, was replaced with UCAUUU, and the sequence at 49 to 54 nts upstream of the poly(A) tail site was substituted with hexamer AGUAAA to create mutant PAS-R-15A  and the cis-acting elements responsible for eukaryotic mRNA polyadenylation [betacoronavirus C, betacoronavirus A and deltacoronavirus, respectively. Other hexamer variants, which differ from hexamer AAUAAA, AGUAAA or AUUAAA by one nucleotide, have also been identified in alphacoronavirus, betacoronavirus B, betacoronavirus D, and gammacoronavirus. Interestingly, although the positions of these hexamers in coronaviruses can be from 24 to 57 nts upstream of the poly(A) tail, the motifs are at similar positions within the same genus or lineage. However, it remains to be determined whether the hexamer has similar functions in coronaviruses other than BCoV.The consensus PAS hexamer AAUAAA, located 10–30 nts upstream of the poly(A) cleavage site, is one of the nylation , 8. Varinylation –11. In tIn this article, we provide genetic evidence of how coronavirus lengthens its poly(A) tail during the infection cycle and how the hexamer AGUAAA and poly(A) tail act in concert to accomplish coronaviral polyadenylation. The characterization of two polyadenylation-related elements, AGUAAA and the poly(A) tail, will help in a better understanding of the mechanism of coronaviral polyadenylation.In the previous study , it was In the present study, we demonstrated that both the poly(A) tail and hexamer AGUAAA are involved in coronaviral polyadenylation and the two elements appear to be able to functionally compensate for each other when one is missing or modified, as exemplified by DI RNA mutants R-25A and W-25U. Based on present results, we propose that both the hexamer AGUAAA and poly(A) tail contribute to virus survival under diverse environments. For example, a poly(A) tail length shorter than 20 nts was consistently found for coronaviral RNA collected from (i) BCoV-infected HRT-18 cells at 2 hpi, (ii) W-25A-transfected BCoV-infected HRT-18 cells at 72 hpi of VP1 and (iii) mouse brain at 5 days postinfection with MHV-A59 . MoreoveUnlike most mammalian mRNAs with ~250 nts of poly(A) tail, positive-strand RNA viruses such as piconaviruses, have heterogeneous lengths of natural poly(A) tail ranging from about 10 to 120 nts long –57. FuncIn the present study, we found that the replication efficiency of DI RNA mutants R-25A (36%)  and W-25It has been shown that with reverse genetic approaches the deletion of nts 30–170 upstream of poly(A) tail do not affect the growth function in tissue culture for MHV , 61. It Based on the evidence shown in this study and in others, we propose a model for coronaviral polyadenylation . First, Accordingly, under the argument of constitution of a stable complex for efficient coronavirus polyadenylation, this model may explain (i) why a poly(A) tail was synthesized from poly(A)-deficient DI RNA constructs W-25U, W-25C, and W-25G, rather than poly(A)-deficient DI RNA W-0A, even though they all contain a hexamer AGUAAA; and (ii) why a poly(A) tail was found for R-15A and R-18A, but not R-25U, R25C and R25G, despite the fact that they all lack the hexamer AGUAAA. In the cases of W-25U, W-25C and W-25G, which contain the hexamer AGUAAA but lack a poly(A) tail, it is speculated that in spite of different affinity the binding of proteins  to In conclusion, we for the first time determined the viral RNA elements involved in coronaviral polyadenylation. Future works may elaborate on the identification of the cellular and viral proteins participating in the synthesis of the coronaviral poly(A) tail as well as the detailed mechanism of coronaviral polyadenylation and its regulation.S1 Fig(A) The structure of BCoV DI RNA and its composition relative to the BCoV genome are illustrated in the upper panel. The engineered BCoV DI RNA with the MHV 3’ UTR (W-25A) used in this study is shown in the lower panel. To differentiate the origin of the poly(A) tail between the helper virus BCoV genome and BCoV DI RNA, the latter was engineered to carry the mouse hepatitis virus (MHV) 3’ UTR with which an MHV-specific primer can be used for RT-PCR to determine the length of the DI RNA poly(A) tail. (B) Schematic illustration of the experiment showing the transfection of BCoV-infected HRT-18 cells with DI RNA and the passage of the resulting BCoV progeny. (C) Method for determining the coronaviral poly(A) tail length. The decapped and head-to-tail ligated BCoV DI RNA was used as the template for RT-PCR with primer 1 (for RT) and primer 2 followed by sequencing.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A) and (B) Upper panel: detection of DI RNAs W-0A and W-5A at the different time points of VP1 by RT-PCR. (A) and (B) Lower panel: quantitation of the synthesis of DI RNAs W-0A and W-5A at the different time points of VP1 by qRT-PCR.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(A) Detection of DI RNA synthesis at 1 and 24 hpi of VP1 by RT-PCR. (B) The relative levels of DI RNA synthesis at 1 and 24 hpi of VP1 as quantitated by qRT-PCR. (C) Sequence analysis of DI RNA at 24 hpi of VP1. The sequence within the hexamer AGUAAA of individual DI RNA different from that of DI RNA W-15A is underlined.(TIF)Click here for additional data file.S6 Fig(A) Detection of DI RNA synthesis at 1 and 24 hpi of VP1 by RT-PCR. (B) The relative levels of DI RNA synthesis at 1 and 24 hpi of VP1 as quantitated by qRT-PCR. (C) Sequence analysis of DI RNA at 24 hpi of VP1. The sequence of individual DI RNA different from that of DI RNA W-15A is underlined.(TIF)Click here for additional data file.S7 Fig(A) BCoV-infected HRT-18 cells at 2 hpi (13 nts). (B) W-25A-transfected BCoV-infected HRT-18 cells at 72 hpi of VP1 (13 nts). (C) Mouse brain at 5 days postinfection with MHV-A59 (17 nts) (shown in negative strand). (D) Delayed brain tumor (DBT) cells with MHV persistent infection of 97 days (15 nts) (shown in negative strand).(TIF)Click here for additional data file.S8 Fig(A) Upper panel: synthesis of W-25A, W-25U, W-25C and W-25G at 48 hpi of VP1 as detected by Northern blot analysis. Lower panel: the relative levels of RNA synthesis. (B) Quantitation of the synthesis of DI RNAs W-25U, W-25C, R-25U and R-25C at 24 hpi of VP1 by qRT-PCR.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens.Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays  were obtained from both MB  and PB  patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite’s stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite’s stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing.The specificity and sensitivity for determining the presence and susceptibility of Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy. Mycobacterium leprae could undermine existing leprosy control measures. PCR/DNA sequencing is currently the method of choice for drug-resistance surveillance in leprosy; however, this technique is not available to most endemic communities and is not cost-effective for large sampling surveys. Therefore, there is a need for drug-susceptibility tests for M. leprae that could be applicable to large sampling studies, particularly in low-resource areas, where leprosy is endemic, resistance appears to be low, but drug resistance prevalence is likely to be underestimated. Our study demonstrated the utility of qPCR-HRM DST of qPCR-RLEP positive specimens as a reliable screening tool that improves the applicability in endemic regions and reduces cost and time for drug susceptibility screening from a variety of specimen types. To improve the overall reliability we recommend that all mutants be subjected to DNA sequencing, thereby providing valuable information to improve patient treatment outcome and to the global context of leprosy drug resistance monitoring.Despite three decades of effective treatment with multidrug therapy (MDT), leprosy persists as a public health problem in many regions of the world. The recent increase in relapse cases, MDT treatment failures, and the emergence of drug-resistant strains of  Mycobacterium leprae strains in relapse cases  may serve as the source of M. leprae DNA.A recent report has described a novel, ‘single-tube’, Real-Time qPCR high resolution melt (HRM) analysis method for anti-leprosy drug susceptibility testing DST . qPCR-HRM. leprae from skin biopsy tissues using either a conventional phenol/chloroform DNA extraction or a column-based DNA purification protocols from fresh-frozen tissues, or FFPE tissue sections, or archival Fite’s-stained FFPE sections on glass slides.Therefore, the purpose of the current study was to explore the usefulness of qPCR-HRM DST to predict DDS, RMP and OFX susceptibility in M. leprae drug susceptibility directly from clinical specimens. Results also defined the specificity of the assay for M. leprae as well as demonstrated that conventional phenol based extraction and ethanol precipitation of DNA is a suitable method for this analysis. In addition to ethanol-fixed and fresh specimens, frozen, FFPE sections and Fite’s-stained FFPE sections from glass slides appear to be suitable specimens for this assay.Results of this study support previous reported data that the qPCR-HRM DST assay strongly correlates with PCR/direct DNA sequencing for detection of The Guide to Care and Use of Laboratory Animals, Eighth Edition.All procedures involving human subjects including biological sample collections and testing were performed following approval from the governing human subjects’ research ethical committees: in Brazil by the Institutional Review Board at the Federal University of Uberlandia; in the United States by the Institutional Review Board of the Tulane School of Medicine, New Orleans, LA. Written informed consent procedures were carried out in Brazil. In the United States the study was determined to be exempt from consent because archival diagnostic specimens were used, accessed through a de-identified database. Animal procedures were performed under a scientific protocol reviewed and approved by the NHDP Institutional Animal Care and Use Committee (Assurance #A3032-01), and were conducted in accordance with all state and federal laws in adherence with PHS policy and as outlined in M. leprae Thai-53 drug-susceptible reference strain was propagated through serial passage in nu/nu mice  and freshly harvested bacilli purified from hind footpads were stored at 4°C and used within 48 hr of harvest, as previously described [2 asphyxiation and the hind feet were removed and soaked in 70% ethanol and Betadine to kill surface contaminants. The skin was removed aseptically and the tissue was excised, minced and homogenized in 10 ml of RPMI-10% FBS medium. Tissue debris was removed by slow speed centrifugation (50 x g for 10 sec) and the bacilli-rich supernatant was enumerated by direct counting following staining using the Fite's variation of Ziehl-Neelsen method. The bacteria were resuspended for 8 min in 0.1N NaOH, and washed 3 times in Tris EDTA (TE) buffer to remove extraneous mouse tissue and DNA adsorbed to the bacilli.The escribed . BrieflyM. leprae DNAs from 19 M. leprae reference strains , including those containing the most common DRDR mutations leading to DDS, RMP and OFX resistance, and susceptible strains, were obtained from the Leprosy Research Centre (LRC), National Institute of Infection Diseases, Tokyo, Japan, the Laboratory of Molecular Biology Applied to Mycobacteria (LABMAM), FIOCRUZ, Rio de Janeiro, and the Institute Lauro de Souza Lima (ILSL), Bauru, Brazil. These DNA samples were used to validate the qPCR-HRM assay for drug susceptibility in M. leprae.A panel of purified M. leprae-specific RLEP quantitative RT-PCR assay [M. lepromatosis (patient specimen), M. avium, M. intracellulare, M. kansasii, M. lepraemurium, M. lufu, M. marinum, M. simiae, M. smegmatis, BCG-Pasteur, M. ulcerans, M. bovis, M. gordonae, M. fortuitum, M. haemophilum, M. tuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Clostridium perfringens.A panel of DNAs purified  from other mycobacterial strains and bacteria often found in the skin, was obtained from the NHDP-LRB Biobank and used to test the specificity of the qPCR-HRM DST assay. All DNAs were tested for 16S rDNA PCR/direct DNA sequencing to confirm species and as a control for DNA amplification, and for the M. leprae. The code was broken when all data was compiled for final analysis. These specimens were derived from multibacillary  and paucibacillary  leprosy patients. The procedures for collection and processing of the biopsy specimens are described below.In this study, 211 skin biopsy specimens obtained from untreated leprosy cases for routine diagnosis of leprosy from Brazil and the U.S. Remaining specimens were “blinded” using a coding system and sent to the NHDP for qPCR-HRM DST for M. leprae strain Thai-53 using the DNeasy Blood and Tissue Kit®  and a modified protocol. Briefly, serial dilutions of M. leprae (starting with 2 x 107) were pelleted at 10,000 x g for 10 min and resuspended in 180 μl Buffer ATL and 20 μl proteinase K, and processed using the DNeasy Blood and Tissue Kit. Samples were incubated for 16 hr at 56°C. Proteinase K was inactivated by incubation at 95°C, 10 min and DNA was purified using the DNA spin column and eluted in 100 μl of AE as per manufacturer’s recommendations.DNA was purified from mouse footpad-derived Fresh skin biopsies were obtained from 172 patients and immediately after excision skin biopsies were frozen by immersion into liquid nitrogen and stored at -20°C for up to one week prior to processing (fresh-frozen). Biopsies were finely minced on a sterile petri dish (or glass side) with a single use disposable sterile scalpel or blade and transferred to a vial containing 500 μl of lysis buffer , 30 μl of proteinase K (10 mg/ml) and 40 μl SDS (10%). Tissues were lysed for 12 hr at 56°C and stored until use at -20°C. Proteinase K was inactivated by incubation at 95°C for 10 min and 250 μl Tris-HCl (1M pH 8), 250 μl chloroform/isoamyl alcohol (24:1), and 500 μl Tris buffered phenol (pH 8) were added to each specimen. The solution was homogenized and centrifuged at 10,000 x g for 10 min. The aqueous phase containing DNA was removed and precipitated with an equal volume of ethanol. The pellet was washed with 75% ethanol, followed by a second spin at 10,000 x g for 10 min, air dried, and resuspended in 50 μl of ultrapure water then stored at -20°C.DNA was prepared from FFPE skin biopsy tissues from 36 leprosy patients. Briefly, 10 μm thick sections were obtained from paraffin blocks using a microtome. Sections were trimmed of excess paraffin and treated with 1 ml xylene for 30 min. Xylene was removed after centrifugation at 10,000 x g, 10 min, room temperature. Xylene extraction was repeated, xylene was removed and tissues were rinsed in 100%, then 70% ethanol and washed in 1 ml 1x TE Buffer . Tissue was finely minced and added to a 1.5 ml conical microfuge tube containing 180 μl Buffer ATL and 20 μl proteinase K and processed using a modification of the DNeasy Blood and Tissue Kit protocol as described above.DNA was prepared from three archival Fite’s acid-fast stained glass slides containing FFPE sections of skin biopsies from MB leprosy patients. These slides had been stored at room temperature for 1wk, 5 wk and 20 yr, respectively. To remove sealant, slide cover and paraffin from slides, the slides were soaked for 24–48 hr in xylene in separate containers. Coverslips were removed and slides were incubated for an additional 30 min in fresh xylene and rinsed in 100% ethanol and then 70% ethanol. A drop of Buffer ATL was added to the tissue and the tissue was scraped off of the slide with a sterile scalpel and transferred to a 1.5 ml conical microfuge tube containing 180 μl Buffer ATL and 20 μl proteinase K. The DNA was purified using a modification of the DNeasy Blood and Tissue Kit protocol as described above.M. leprae DNA using the quantitative real-time PCR-RLEP assay (qPCR-RLEP) as previously described [M. leprae after three cycles of freeze-thaw . Then 1 μl was added to PCR reagents to a 96-well plate and amplified. These dilutions represented 2.0 x 107–4 x 103M. leprae/ml equivalents. All samples were run on an ABI 7500 Fast PCR Real-Time System  in duplicate.Purified DNA from all types and formats of processed clinical specimens was initially evaluated for the presence of escribed . BrieflyM. leprae reference panel of purified DNAs using the q-PCR-HRM DST assay was performed as previously described by Li et al. (2012) with the following modifications. The qPCR reaction included: 10 μl of MeltDoctor HRM Master Mix , forward and reverse primers (0.5 μl each of 10 μM stocks) targeting DRDR fragments in the folP1, rpoB or gyrA associated with mutations resulting in DDS, RMP, or OFX resistance, respectively . The software assembles curves with similar profiles into distinct groups. The curves of the control WT and mutant strains were designated as reference profiles. Data that were similar to the WT reference control were assigned to the drug-susceptible group and data that resembled the mutant reference control were assigned to the “variant” (V) drug-resistant group. For better visualization the melting curves matching each group were color coded.folP1 DRDR pre-melt (80.8°C to 81.3°C) and post-melt (83.4°C to 83.9°C); rpoB DRDR pre-melt (85.4°C to 85.9°C) and post-melt (88.1°C to 88.6°C); gyrA DRDR pre-melt (81.2°C to 81.6°C) and post-melt (83.4°C to 83.8°C).After standardization, qPCR-HRM DST was performed on DNA from clinical specimens. The software default setting of automatic selection of the melting region (between the pre- and post-melt temperatures), produced acceptable results for reference samples with purified DNA applications. Initially the start and end temperatures of the melting regions were established using these settings. These were then adjusted manually to increase the stringency of the software group assignment, particularly for clinical samples. The baseline settings of the pre-melt and post-melt temperature gates for the qPCR-HRM assay were as follows: folP1, rpoB and gyrA DRDRs was performed on all samples. PCR amplicons corresponding to the DRDRs in rpoB, folP1 and gyrA genes were investigated by direct sequencing of PCR amplicons obtained using a modification of the WHO guidelines for Global Surveillance of Drug Resistance in Leprosy [gyrA DRDR sequence was obtained from a subset (80%) of the samples. Briefly, the PCR reaction mix included 25 μl of AmpliTaq Gold 360 PCR Master Mix , forward and reverse primers (2.5 μl each of 10 μM stocks), nuclease-free water (15 μl), and DNA from samples (5 μl). PCR cycling parameters were: 95°C for 2 min followed by 45 cycles of 95°C for 10 sec, 58°C for 30 sec, and 72°C for 30 sec and then a final extension step at 72°C for 7 min. PCR products were loaded in 2% agarose gel for confirmation of the fragment amplification. Amplified products were purified through QIAquick PCR Purification Kit (QIAGEN). DNA concentrations were determined using NanoDrop 8000 spectrophotometer  and sequenced by capillary electrophoresis using BigDye Terminator v3.1 cycle sequencing kit in the ABI Prism 3130 Genetic Analyzer (Applied Biosystems). Sequence data was analyzed using the nucleotide database in the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov) to identify mutations associated with drug resistance.PCR for  Leprosy . Since OM. leprae Thai-53 type strain  and drug-resistant (n = 14) reference strains demonstrated that all characterized drug-resistant strains had distinct variant (“V”) HRM assignment group based on melt curve profiles from that of the drug-susceptible, WT e strain . In addipoB DRDR . The speM. leprae was defined using purified DNA from a variety of other mycobacterial and bacterial species. Results demonstrated that only M. lepromatosis generated a PCR amplicon in the qPCR-HRM in folP1 and rpoB DRDRs, with no amplification in gyrA. In each of these two assays M. lepromatosis DRDR was assigned to a distinct “V” HRM profile from that of M. leprae (folP1 and rpoB from M. leprae and M. lepromatosis demonstrated multiple base-pair mismatches between these two strains (data not shown). It is of note that RLEP results demonstrated that M. lepromatosis presented no DNA amplification for the M. leprae-specific gene fragment, as observed for all mycobacterial and bacterial species tested by qPCR-RLEP.The specificity of the qPCR-HRM DST for detection of . leprae . AlignmeM. leprae qPCR-HRM DST for DDS, RMP and OFX was conducted on 211 RLEP positive clinical specimens preserved using a variety of procedures including: freezing, FFPE, and Fite’s-stained FFPE sections on glass slides and processed using either conventional or column-based DNA purification protocols. Results demonstrated that 6/14 (42.9%) TT and 29/41 (70.7%) BT patient specimens and 154/156 (98.7%) MB specimens gave HRM results for all three drugs (M. leprae/ml (enumerated by qPCR RLEP assay) did yield HRM results, the majority of those containing ≤ 350 M. leprae/ml did not yield reliable results in the all three qPCR-HRM DST assays.The ee drugs . The twoee drugs  and contWhen HRM profiles were initially compared to DNA sequencing results there was a 97% correlation between the two assays. Five specimens giving a “V” HRM profile in the qPCR-HRM assay gave the susceptible genotype in DNA sequencing  0.2°C, which generated a HRM difference in fluorescence plot as a melting curve shape above the WT profile  generated distinct “V” HRM profiles in lP1 DRDR . These m profile . This ishenotype . All spegenotype .M. leprae, RMP-R and RMP-S DNAs were combined using 10-fold dilutions of templates resulting in ratios 9:1 (WT:RMP-R) to 9:1 (RMP-R:WT) ratios. The DNA mixtures containing equimolar DNA concentrations were analyzed by both qPCR-HRM DST and PCR/direct DNA sequencing. Results demonstrated that the qPCR-HRM DST for RMP could detect as little as 10% of the RMP-R genotype in a background of 90% RMP-S genotype  was received from the LRC laboratory and used to establish the qPCR-HRM DST assay using the ABI 7500 Fast Real-Time PCR Instrument and ABI HRM software. In addition, four additional multi-drug-resistant isolates from the LABMAM laboratory were added to this library. Even though these mutants had not been previously evaluated in qPCR-HRM DST, our results demonstrated that all mutant strains within this combined collection were correctly identified by this technique. The mutations associated with drug resistance in the rpoB, folP1 and gyrA DRDRs of M. leprae have been associated with moderate to high levels of drug resistance in M. leprae using mouse footpad DST. No mutations have been identified in low level dapsone-resistant M. leprae.To standardize the qPCR-HRM DST of folP1 missense mutations within codons 53 and 55 have been associated with the development of resistance [folP1 mutation types (ACC→ATC) in codon 53 and (CCC→CTC and CCC→CGC) in codon 55. In addition, one of four clinical specimens generating a “V” folP1 profile in the current study contained a folP1 mutation type (ACC→GCC) in codon 53. Its HRM curve was located above and right of that of the WT strains. This was most likely due to the substitution of a guanine base for an adenine base in codon 53 of the folP1 of this M. leprae strain, resulting in an increased melting temperature of its DNA heteroduplex. Together, these four folP1 mutation types cover 91% of the DDS-R mutants described worldwide [folP1 mutation type (CCC→CGC) in codon 55 was found in three characterized Brazilian DDS-R isolates. This mutant was difficult to discern by HRM, as it was initially assigned to the WT group. This was most likely due to the minimal change in the melting temperature of its duplex containing a substitution of a guanine base for a cytosine base. It was necessary to adjust software parameters for the gate region between the pre- and post-melt temperatures in order for this mutant to be grouped as a “V” profile. Thereafter, these adjustments defined the baseline settings as described in the methods section. The presence of double folP1 mutation types (ACC→CTC) in codon 53 and (CCC→CTC) in codon 55, as well as, a mixed infection with WT type allele in codon 53 was also confirmed in one isolate. Together these data confirmed earlier observations that the q-PCR-HRM DST was highly specific for detection of DDS susceptibility in M. leprae [For DDS resistance sistance , 21. We orldwide , 22. The. leprae .rpoB mutation types (ACC→ATC) in codon 433, (GGC→CAC) in codon 448, (CAC→TAC) in codon 451, and (TCG→TTG) in codon 456 have been previously associated with the RMP-R phenotype of M. leprae and have previously shown to generate distinct “V” HRM profiles [rpoB profile contained multiple rpoB mutation types including: (ACC→ATC) in codon 433, (GGC→CAC) in codon 448, and (CAC→TAC) in codon 451. In addition, this isolate also had resistance to DDS (folP1 mutation type CCC→CTC in codon 55) and OFX (gyrA mutation type GCA→GTA in codon 91). qPCR-HRM DST gyrA confirmed initial results that qPCR-HRM DST detects a mutation in codon 91 (GCA→GTA) in clinical isolates; which occurs in more than 90% of the reported mutations associated with the development of OFX resistance in M. leprae [folP1, rpoB and gyrA DRDRs in mixed infections of WT and resistant M. leprae. For example, in strain Ze-4 the melting curve for gyrA DRDR differed from that of the WT and the expected mutant. This was assigned to a “V” profile. DNA sequencing analysis of this mutant confirmed mixed alleles in codon 91 of the gyrA. In addition, the mixed infection of DDS-R and DDS-S M. leprae was also confirmed in Ze-5. Thus, these results reinforce that HRM clustering can be sensitive to the presence of multiple alleles, as reported by Li et al. (2012).For RMP resistance, profiles . Resultsprofiles . In addi. leprae , 23. TheM. leprae genotype in 90% susceptible genotype was sufficient to convert the WT profile to “V” profile in the HRM assay for DST. In contrast, DNA sequencing DST required as much as 30% of the resistant allele for detection of the mixed genotypes. This confirms that qPCR-HRM DST analysis may enable detection of minor populations of mutant alleles in a WT background and thus the emergence of drug resistance.Further analysis of mixed alleles demonstrated that as little as 10% resistant M. leprae infection, possibly in animal models experiments. This qPCR-HRM strategy for DST directly from clinical samples, could be potentially optimized to analyze RMP-R in M. tuberculosis, once it has been demonstrated that the conventional in vitro culture methods may allow one strain to predominate, hindering the detection of resistant strains [Taken together, these results confirmed the initial observations of Li et al. (2012), demonstrating a strong correlation between qPCR-HRM DST results and that of PCR/DNA sequencing from characterized isolates. Future studies to explore the HRM capability in genotyping lower proportions of drug-resistant strains in mixed infections within the same patient are needed to understand the clonal complexity in the course of  strains .M. leprae DNA was detected using qPCR RLEP. The two MB specimens that did not produce qPCR-HRM DST results were from mid-borderline (BB) leprosy patients with low bacterial numbers. Four of the clinical specimens from MB patients generated “V” HRM profiles consistent with DDS-R leprosy. One of these specimens also contained an rpoB “V” HRM profile. qPCR-HRM DST results were obtained for all three drugs from 63% of all PB leprosy patient biopsies in which M. leprae DNA was detected using qPCR RLEP. Although results were obtained from PB specimens with as little as 30 M. leprae (enumerated by qPCR-RLEP), more consistent results were obtained from samples with ≥ 350 bacteria. No drug resistance was detected in the PB group. DNA sequencing confirmed the genotypes of these clinical specimens and our data confirm the initial report of Li et al. [qPCR-HRM DST results were obtained for all three drugs from 99% of all MB leprosy patient biopsies in which i et al. .M. leprae directly from clinical specimens. The first of these was the purity of the DNA in the samples to be analyzed for HRM analysis. A column-based DNA purification protocol has been recommended for sample processing for qPCR-HRM DST analysis [Several other key factors were critical to the success of qPCR-HRM DST of analysis . HoweverM. leprae and thereby is used extensively to stain for detection of M. leprae. Results demonstrated that the method of fixation does not appear to have an effect on the ability to generate qPCR-HRM DST results. The number of bacilli (~ 350) appears to be the limiting factor. Therefore, combining these data with that of previous published data suggest that tissues preserved for histopathology , ethanol-fixed and fresh-frozen tissues are all suitable specimens for qPCR-HRM DST of M. leprae.The initial study also evaluated ethanol-fixed skin biopsy tissues as a source of DNA for the qPCR-HRM DST . The curM. leprae directly in clinical specimens is the specificity of the qPCR amplification for M. leprae DRDRs. Since the dye used in this assay binds nonspecifically to dsDNA and therefore, any dsDNA PCR product can emit fluorescence, the current study characterized the specificity of the qPCR-HRM DST assays for M. leprae. After testing 20 other bacterial and mycobacterial DNAs and biopsy specimens containing some of these mycobacterial species , M. lepromatosis was the only other mycobacterial species that generated amplicons in qPCR HRM DST. This was not surprising because M. leprae and M. lepromatosis are highly related mycobacterial species which both cause leprosy and are now referred to as the Leprosy Complex [M. lepromatosis DRDRs 53I(ATC); and Ry-6 containing the high frequency gyrA mutant allele, A(GCA)91V(GTA). Control samples were critical to establish reproducible derivative melting curve plots, which could then be used as reference peaks at the expected melting temperature for each specific DRDR fragment of M. leprae. The appropriate gate for each allele could be set and therefore further define the parameters for assigning WT and “V” profiles of the unknowns. In addition, to prevent the interference with the analysis of other samples on the plate, any sample that did not amplify before the Ct value = 36 in the respective qPCR assay for each of the DRDRs or that did not give the expected peak in the derivative melt curve plot were excluded from analysis.Proper gating of the HRM data for the difference plots was another key factor affecting the performance of the qPCR-HRM DST using clinical specimens. This was vastly improved by the addition of two dilutions of an appropriate known drug-susceptible and -resistant control DNAs on each qPCR-HRM plate. These controls consisted of the DRDRs of: Ze-4 containing the high frequency rpoB mutant allele S(CTG)456L(TTG); Ai-3 containing the high frequency M. leprae has broad applicability and can dramatically reduce the cost and time involved with DNA sequencing by only sequencing those specimens that generate the “V” HRM profile, especially in resource poor endemic regions thereby providing valuable information to improve patient treatment outcome and to aid in the global context of leprosy drug resistance.In summary the qPCR-HRM DST is a ‘single-tube’ assay that can identify genetic variants in drug “target” genes by post-PCR analysis of the shapes and melting temperatures of amplicon melting curves. Since PCR amplicons and melting curves are generated in the same instrument, there are no operator-dependent sample manipulations after the qPCR reaction is assembled and no need for additional reagents or supplies to determine drug susceptibility. This reduces the risk of amplicon cross contamination and the cost of DNA sequencing. In addition, 42 samples (tested in duplicate) can be investigated at the same time in a single 96-well plate within 3 hr. Therefore, qPCR-HRM DST lends itself to high throughput screening of leprosy drug resistance. The estimated cost of a single reaction using qPCR-HRM DST is ~ $3 (U.S. dollars). In contrast, PCR/DNA sequencing DST includes post PCR amplicon purification and quantification prior to DNA sequencing. The estimated cost for analysis of a single specimen is $22 (U.S. dollars). These data strongly suggest that qPCR-HRM DST for S1 Table(XLSX)Click here for additional data file.S1 FigrpoB DRDR primer sequences in M. leprae. Green colored bases denote rpoB codons that are associated with rifampin resistance in M. leprae.Yellow colored bases denote (TIF)Click here for additional data file.


Hfq is a critical component of post‐transcriptional regulatory networks in most bacteria. It usually functions as a chaperone for base‐pairing small RNAs, although non‐canonical regulatory roles are continually emerging. We have previously shown that Hfq represses IS10/Tn10 transposase expression through both antisense RNA‐dependent and independent mechanisms. In the current work, we set out to define the regulatory role of Hfq in the absence of the IS10 antisense RNA. We show here that an interaction between the distal surface of Hfq and the ribosome‐binding site of transposase mRNA (RNA‐IN) is required for repressing translation initiation. Additionally, this interaction was critical for the in vivo association of Hfq and RNA‐IN. Finally, we present evidence that the small RNA ChiX activates transposase expression by titrating Hfq away from RNA‐IN. The current results are considered in the broader context of Hfq biology and implications for Hfq titration by ChiX are discussed. Hfq binds sRNAs and their partner mRNAs and facilitates intermolecular base‐pairing. This typically affects translation and/or transcript stability. Hfq contains three RNA‐binding surfaces, all of which play a role in promoting base‐pairing between RNAs. The ‘top’ and ‘bottom’ of the toroidal‐shaped Hfq homohexamer are termed the proximal and distal RNA‐binding surfaces respectively. The proximal surface binds short U‐rich sequences typically found in sRNAs while the distal surface binds longer ARN repeats  typically found in mRNAs  to inhibit translation. As the Spot42 pairing region in sdhC is too far upstream of the TIR to influence translation, it was inferred that stable association of Hfq with sdhC was sufficient to compete with 30S ribosomal subunit binding  that caused restructuring of the mRNA within the 5′ untranslated region (5′UTR), which ultimately prevented Hfq binding  that is transcribed from the opposite strand of DNA relative to the transposase . MCI can be explained by the fact that transposase is a cis‐acting protein whereas the asRNA is trans acting , but not completely abrogated, when transposition was measured for IS10 in single copy. These observations were consistent with Hfq contributing to MCI, but also playing a role in down‐regulating IS10 transposition independent of the MCI pathway  in the absence of RNA‐OUT transposase, expression increased sixfold in −hfq; (ii) there is an Hfq‐binding site in RNA‐IN that overlaps the TIR; and (iii) Hfq status has only a subtle effect on steady‐state RNA‐IN transcript level , we asked if regulation was maintained when RNA‐binding surfaces of Hfq were mutated. Our expectation was that if a trans‐encoded sRNA is involved in this pathway, all three surfaces would be critical for regulation. In particular, we expected the proximal surface to be important for stabilizing any involved sRNAs and the lateral surface for catalyzing pairing with RNA‐IN.To gain further insight into how Hfq regulates IS−hfq phenotype. We constructed a chromosomal IS101–339‐lacZ translational fusion with a single bp change (HH104) in the promoter for RNA‐IN, which increases transcription ∼ 100‐fold , proximal (K56A) or lateral (R17A) surface  and mutant forms of Hfq to complement an lacZ expression increased almost 13‐fold in the absence of Hfq, and that regulation was fully restored when HfqWT was expressed from a plasmid. In contrast, none of the Hfq variants were able to fully complement −hfq, with HfqY25A being the most impaired and HfqR17A functioning the most like WT. Importantly, the reduced function of the mutant proteins cannot be attributed to protein expression as the Hfq levels were not significantly different for WT versus the mutant forms of Hfq . As the results showed that the integrity of the lateral site is not important for regulation and the integrity of the proximal site is less important than that of the distal site, our data suggested that Hfq repression was sRNA independent.We show in Fig. 10 transposition assay. As IS10 transposition frequency is directly proportional to transposase expression , RNase T1 or RNase V1.We have previously shown that Hfq binds a 14‐nt A‐rich region of RNA‐IN, which overlaps with the ribosome‐binding site (RBS), as well as an 8‐nt U‐rich region overlapping with codons 5–8 of the transposase coding region .Our first objective was to define the relative binding affinity of sites 1 and 2 for Hfq. We show by electrophoretic mobility shift assay (EMSA) in Fig. Fig. S2), we conclude that IN:Hfq‐1 is formed through Hfq binding to site 1 and IN:Hfq‐2 is formed through Hfq binding to both site 1 and site 2; note we have not looked at the importance of site 3 in IN:Hfq complex formation. Moreover, Hfq binds site 1 with a much higher affinity than it binds site 2, and Hfq binding to site 2 appears to be dependent on Hfq first binding site 1. As formation of IN:Hfq‐2 increased in a concentration‐dependent manner, it is likely that occupancy of site 2 depends on recruitment of a second Hfq hexamer to IN:Hfq‐1, as opposed to the unoccupied proximal surface of an Hfq hexamer bound at site 1 and engaging site 2.Based on these results, as well as footprinting experiments  into the promoter region of RNA‐IN, which destabilizes RNA‐OUT while having only a subtle effect on RNA‐IN transcription  to the steady‐state level of fusion transcript provides a measure of the translation efficiency. For example, if expression was low and transcript levels were high, this would be indicative of low translation efficiency. The presence of Hfq in cells expressing the WT reporter decreased translation efficiency approximately 2.5‐fold, consistent with Hfq interfering with transposase translation. For the M5 reporter, translation efficiency was greatly reduced compared with the WT reporter, which is not unexpected given that 5 of the 8 nt changes in this construct are in the RBS. Importantly, the translation efficiency did not further decrease in the presence of Hfq. Thus, Hfq is unable to down‐regulate translation when site 1 is mutated. Translation efficiency for the M2 reporter was intermediate to that of the WT and M5 reporters, indicative of site 2 playing a more minor role compared with site 1 in Hfq‐directed repression of translation.In the above experiment, the ratio of transposase‐10 transposase expression in vivo, and that the most important Hfq‐RNA interaction for this response is between the distal surface of Hfq and site 1, which includes the RBS of RNA‐IN. In addition, results from Fig. 10 transposase expression by interfering with IS10 transposase translation. To further test the hypothesis that Hfq binding to site 1 inhibits RNA‐IN translation, we performed in vitro toeprinting assays. We show in Fig. lpp or usg mRNA, inhibition of the toeprint signal was significantly weaker . For example, at the same ratio of Hfq:mRNA that gave greater than 90% inhibition for IS10, only 50% inhibition was observed for lpp and usg . Taken together, the results in this section show that Hfq can inhibit 30S ribosome binding to RNA‐IN in vitro and that this inhibition requires an available distal surface on Hfq. Furthermore, as we have shown that there is a high‐affinity Hfq‐binding site (site 1) that spans the RBS of RNA‐IN and engages the distal binding surface of Hfq, we conclude that Hfq binding to this site is responsible for blocking translation initiation.We reasoned that because the distal surface of Hfq interacts with site 1 of RNA‐IN, then the distal surface of Hfq would be critical for blocking 30S ribosome binding to RNA‐IN. Accordingly, a competitor RNA that is specific for the distal RNA‐binding face of Hfq should relieve the ‘toeprint repression’ afforded by Hfq. We show in Fig. E. coli, there is a concern that any phenotype observed in an −hfq strain might be the result of dysregulation of a factor under Hfq control. As we are proposing that Hfq binds directly to the 5′UTR of RNA‐IN to block translation, we thought it important to look for Hfq‐RNA‐IN binding in vivo where RNA‐IN would have to compete with other cellular RNAs for Hfq binding. We performed an Hfq‐RNA immunoprecipitation experiment (RIP) with −hfq cells containing a chromosomal copy of IS10HH104‐kan and a plasmid encoding FLAG‐tagged Hfq . PCR reactions were then analyzed on an agarose gel. We show that RNA‐IN was strongly enriched in the Hfq IP compared with control reactions  were subject to RIP, no Hfq was detected in the IP fraction  and accordingly no RNA‐IN was detected by RT‐PCR  where RNA‐IN was amplified from total cDNA with gene‐specific primers , providing important determinants for Hfq binding in vivo  or a vector control  ChiX may base‐pair with RNA‐IN to increase ribosome accessibility, as seen in the In the absence of Hfq, ChiX exhibited high reactivity to lead with the exception of nucleotides 17–22, 56–65 and 70–78, which is consistent with a mostly unstructured RNA containing a 5′stem‐loop and a Rho‐independent terminator . We show that the distal surface of Hfq binds Hfq‐I, while binding of Hfq‐II requires an intact proximal surface. In addition, a third Hfq binding site (Hfq‐III) was identified that includes the poly(U) tract following the Rho‐independent terminator, which accordingly interacts with the proximal surface of Hfq.We also performed footprinting experiments with Hfq‐binding face mutants and ChiX RNA . This indicates that ChiX does not base‐pair with the first 160‐nt of RNA‐IN, a conclusion that is also supported by the absence of an additional complex (i.e. ChiX:RNA‐IN binary complex or ChiX:Hfq:RNA‐IN ternary complex) in the EMSA (bottom panel). Addition of RNA‐IN did, however, reduce the lead footprint in the A‐rich Hfq‐binding site of ChiX (compare lane 6 with lanes 7–8) and the amount of ChiX:Hfq complex formed (compare lanes 3 and 4 in the EMSA), consistent with ChiX and RNA‐IN competing for Hfq binding.in vitro data that RNA‐IN can compete with ChiX for Hfq binding, we performed an RIP in −hfq cells containing the IS101–339‐lacZ translational fusion, FLAG‐tagged Hfq and the ChiX overexpression plasmid or a vector control. RNA recovered from the Hfq IP was analyzed directly by Northern blot or primer extension to detect ChiX and IN‐lacZ respectively; the 5S rRNA and lpp were also analyzed as negative controls , we calculated that ChiX was enriched 365‐fold in the Hfq IP, while RNA‐IN was enriched 12‐fold. We presume that the relatively low amount of 5S rRNA and lpp mRNA that were detected in the IP represents non‐specific interactions with Hfq in vivo or during the IP procedure.Based on our sodB, ptsG and maeA) interact solely with the distal surface of Hfq  .As ChiX was the only sRNA in our screen that titrated Hfq away from RNA‐IN and ChiX is unique among sRNAs in that it contains a distal Hfq‐binding site, we wondered if other RNAs that interact with the distal surface of Hfq would increase transposase expression through an Hfq‐titration mechanism. Most Hfq‐binding mRNAs , and as pairing proceeds, Hfq is released from the sRNA–mRNA duplex, whereupon it can catalyze additional pairing reactions  that were frequently associated with the Shine–Dalgarno sequence. Notably, 18% of all Hfq‐associated mRNAs contained Hfq‐binding sites located within the TIR   was not produced. This was meant to mimic a situation where IS10 is in single copy and the antisense RNA has little to no effect on transposase expression  in part by decreasing the number of mRNAs bound to Hfq as well as specific competition with the sRNAs CyaR and InvR  it possesses both distal and proximal Hfq‐binding sites. Moreover, in a recent study that compared the ability of a set of sRNAs to compete for Hfq binding, it was established that ChiX was at the top of the hierarchy, while SgrS was at the bottom. This same work also showed that the A‐rich region of ChiX that we designated Hfq‐I interacts with the distal surface of Hfq and provides important determinants for Hfq competition  is a negative regulator of genes involved in chitobiose utilization. ChiX is constitutively expressed but its levels increase substantially in stationary phase , and ChiX levels are regulated by an ‘anti‐sRNA’, chbBC . P1 transduction was then performed to convert DBH331 to DBH337 (−hfq). λDBH504 was created by crossing IS10HH104‐kan from pNK1223 onto λNK1039 in DBH60; kanR lysogens were selected by replica plating and then phage stocks (λDBH504) were prepared from these lysogens. For β‐galactosidase assays with chromosomal IS10HH104‐lacZ, DBH107 was lysogenized with λRS271 (obtained from DBH90 via spontaneous phage release) to create DBH287. DBH287 was subjected to recombineering (details available upon request) to remove the G8 mutation creating DBH298. P1 transduction was then performed to convert DBH298 (+hfq) to DBH299 (−hfq).For mating out experiments, DBH33 was lysogenized with λDBH504 to create DBH331 (hfq gene (including the P3 promoter) from pDH700, pDH701 and pDH713 , M2  and M5 . PCR products were digested with EcoRI and HindIII and cloned into the same sites in pDH858. M5 and M2 were originally identified as increased expression mutants in a transposase expression screen. The transposase gene used was derived from a library of sequences generated by mutagenic PCR.The multi‐copy IS74 Jain, . First, sodB, ptsG and maeA were constructed as previously described , pDH868 (INM2‐160) and pDH875 (INM5‐160) and primers oDH515 (IN‐160 and INM2‐160) or oDH510 (INM5‐160) with oDH199. RNA‐IN was generated by in vitro transcription and internally labeled with [α32P]‐UTP (for EMSA) or 5′labeled with [γ32P]‐ATP (for footprinting) as previously described Acetate (Sigma‐Aldrich) for 3 min at ambient temperature, which was stopped by addition of ethylenediaminetetraacetic acid (EDTA) to a final concentration of 50 mM. Following ethanol precipitation, RNA footprinting samples were resuspended in denaturing load dye  and resolved on a 10% polyacrylamide gel containing 7 M urea. EMSA was performed essentially as described . Apparent dissociation constants were determined as previously described  was removed from binding reactions, mixed with native load dye  and resolved on a 6% polyacrylamide TBE gel.The ChiX −1) and tetracycline (10 μg ml−1). Saturated overnight cultures were used to seed subcultures (1:40 dilution), which were grown to mid‐log phase (OD600 = 0.4–0.6). IPTG (1 mM) was added to the subculture to induce sRNA expression. The Miller assay was performed as previously described  supplemented (where necessary for plasmid selection) with ampicillin  and plated on M9‐glucose supplemented with thiamine (1 μg ml−1), arginine (40 μg ml−1), kanamycin (50 μg ml−1) and tetracycline (15 μg ml−1). Donor colonies were grown overnight to saturation in LB supplemented with kanamycin (50 μg ml−1) and tetracycline (15 μg ml−1) and the recipient strain (HB101) was grown in LB supplemented with streptomycin (150 μg ml−1). Donor and recipient strains were subcultured in LB without antibiotics, and then grown and mixed for mating as previously described  and streptomycin (150 μg ml−1) or streptomycin plus kanamycin (50 μg ml−1) for ‘total exconjugates’ and ‘hops’ respectively. The transposition frequency was calculated by dividing the number of SmRKanR colonies by SmR colonies (‘hops’ per ‘exconjugate’).The mating out assay was performed essentially as previously described (Ross −1) to OD600 = 0.6 at which time 600 μl of cells was added to 300 μl of RNA lysis buffer  and boiled for 1 min. Samples were chilled on ice for 30 s and then sequentially extracted twice with acid phenol (pH 4.3), once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with 2‐butanol followed by ethanol precipitation. Residual genomic DNA was removed with TURBO DNase  prior to primer extension analysis. Five or 10 μg of total RNA  and oDH482 (lpp) and SuperScript III (Invitrogen) according to the manufacturer's instructions.Cells were grown in LB supplemented with ampicillin  was annealed to 5′32P‐labeled oDH511 (RNA‐IN), oDH482 (lpp) and oDH555 (usg) in toeprint buffer  by heating to 95oC for 1 min followed by snap‐cooling on ice for 2 min. While on ice, magnesium acetate was added to a final concentration of 10 mM and dNTPs to 0.5 mM. Reactions were then incubated at 37oC for 5 min. Hfq (0.5–4 pmol of hexamers) or buffer was added to reactions, which were incubated for another 15 min at 37oC, followed by addition of 30S ribosome  and incubation at 37oC for 5 min. Initiator fMet‐tRNA  was added and reactions were incubated for a further 15 min before addition of 200 U of SuperScript II (Invitrogen) and a final incubation of 10 min at 37oC. Reactions were stopped by addition of 100 μl of stop solution  followed by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Samples were resuspended in denaturing load dye  and resolved on a 10% polyacrylamide gel containing 7 M urea. Dried gels were exposed to a phosphorimager storage screen, imaged with a Storm imager and quantitated with ImageQuant .The 30S ribosomal subunit was prepared as previously described  was transformed with plasmids expressing untagged Hfq (pDH904) or Hfq with a C‐terminal 3xFLAG tag (pDH909). Cells were grown to mid‐exponential phase (OD600 = 0.5) in LB supplemented with tetracycline (15 μg ml−1) at which point 50 OD600 of cells was collected by centrifugation and washed once in Tris‐buffered saline  and resuspended in 400 μl of lysis buffer  with 40 U of RNasin (Promega) and 2 U of TURBO DNase . Cells were mixed with 400 μl of zirconia/silica beads  and lysed by vortexing , after which 800 μl of lysis buffer was added followed by centrifugation . An aliquot of the cleared lysate (100 μl) was phenol extracted and ethanol precipitated (‘input RNA’). ANTI‐FLAG® M2 magnetic beads  were added to 800 μl of cleared lysate and samples were incubated at 4oC with rotation for 4 h. Beads were washed five times with 1 ml of lysis buffer, and resuspended in 400 μl of lysis buffer. Hfq‐bound RNA (‘IP RNA’) was recovered by phenol extraction and ethanol precipitation. Following precipitation, residual DNA was removed with TURBO DNase  and samples were ethanol precipitated and finally resuspended in diethylpyrocarbonate (DEPC)‐treated ddH2O. RNA concentration was determined with a NanoPhotometer (Implen).DBH337  or pDH875 (INM5‐lacZ). Cells were grown in LB supplemented with tetracycline and ampicillin (100 μg ml−1) and IP was performed as above.For the RIP with WT and mutant ISacZ Fig. B, DBH12 −hfq; contains IS10HH104‐kan lysogen) transformed with pDH909 (Hfq‐3xFLAG) and pDH765 (ChiX) or pDH763 (vector). IP was performed as above. Total input RNA (10 μg) or RNA recovered from the IP (0.3 μg) was analyzed directly by Northern blot (ChiX and 5S rRNA) or primer extension (RNA‐IN and lpp).The RIP with ChiX overexpression used DBH337  and SuperScript III (Invitrogen) according to the manufacturer's instructions. Enzyme was omitted for the no reverse transcription controls (−RT). Reverse transcription reactions were purified with a PCR purification kit (Qiagen) and eluted in 50 μl of ddH2O. The first 160‐nt of RNA‐IN was detected by 28 cycles of PCR using 4 μl of cDNA as a template and primers oDH199 and oDH483 (WT) or oDH517 (M5). A portion of the 16S rRNA (nt 1071–1425) was detected by 18 cycles of PCR using primers oDH204 and oDH205. PCR reactions were analyzed on a 2% agarose TBE gel.An RNA adapter  was ligated to input (10 μg) or IP (1 μg) RNA using T4 RNA ligase. Adapter‐ligated RNA was purified with an RNeasy Mini Kit (Qiagen), eluted in 30 μl of DEPC ddH32P‐labeled oDH298 in ULTRAhyb‐oligo buffer  at 42oC. ChiX and the 5S rRNA were detected by probing the membrane with an internally 32P‐labeled antisense RNA [templates were generated by PCR with genomic DNA template and primers oDH234 and oDH235 (5S rRNA) or oDH308 and oDH309 (ChiX)] in ULTRAhyb buffer  at 68oC. Membranes were washed according to the manufacturer's instructions. Northern blots were exposed to a phosphorimager storage screen and imaged with a Storm imager .A 3.5 μg of total RNA Fig. B was denSupporting InformationClick here for additional data file.


Picornavirales genome encapsidation and will assist further work in the development of CPMV as a biotechnological tool.Cowpea mosaic virus (CPMV) is a picorna-like plant virus. As well as an intrinsic interest in CPMV as a plant pathogen, CPMV is of major interest in biotechnology applications such as nanotechnology. Here, we report high resolution cryo electron microscopy (cryo-EM) maps of wild type CPMV containing RNA-2, and of naturally-formed empty CPMV capsids. The resolution of these structures is sufficient to visualise large amino acids. We have refined an atomic model for each map and identified an essential amino acid involved in genome encapsidation. This work has furthered our knowledge of  Picornavirales, with a relatively simple, non-enveloped capsid that has been extensively studied. The Picornavirales, which contains >8 families and hundreds of distinct viruses, includes members that infect vertebrates (e.g. foot and mouth disease virus and poliovirus), insects  and plants (e.g. bean pod mottle virus (BPMV), rice tungro spherical virus).Cowpea mosaic virus (CPMV) is a plant-infecting member of the order Comoviridae subfamily and, in common with all members of the Picornavirales, has a positive-sense, single-stranded RNA genome. For CPMV, the genome is bipartite, with RNA-1 (6 kb) and RNA-2 (3.5 kb) being separately encapsidated . The trefoil-shaped RNA density was not seen, but rather a dodecahedral cage of RNA density was observed, with discrete bridges of density to the protein capsid that implicated several amino acid residues in genome binding.In addition to this wealth of X-ray evidence, recently we have determined two high resolution cryo electron microscopy (cryo-EM) structures for CPMV, with a 3.4 Å structure of the RNA-1 containing bottom fraction (CPMV-B) and a 3.0 Å structure of a recombinant empty virus-like particle (eVLP)9. This results in eVLPs composed of L and S subunits which have an identical sequence to the three forms of WT CPMV (T/M/B). However, the eVLP retains the C terminal segment better than WT CPMV particles that contain RNA (CPMV-B/M), suggesting an allosteric effect of genomic RNA in accelerating polypeptide backbone cleavage. Indeed in the recent high resolution EM structure of the CPMV eVLP, we were able to resolve the structure of some of this segment for the first time , alternative proteolysis, or the different timescales for specimen preparation . However, given that a 4.4 Å structure of the cleaved particle, that is essentially identical to the X-ray structure, was refined from a subset of the cryo-EM dataset which generated our uncleaved eVLP map, we suggest that timescale is the most important factor 11. CPMV-M is also of particular interest in biotechnology as it contains a smaller genome segment (RNA-2) than CPMV-B (by 2.5 kb) allowing additional RNA sequences to be inserted into RNA-2 without affecting its ability to be incorporated into particles13. Therefore, CPMV-M is an ideal candidate for recombinant virus production for nucleic acid delivery. In addition, as CPMV replication proteins are encoded by RNA-1, preparations of CPMV containing only recombinant RNA-2 are incapable of causing productive infection14.As well as its inherent interest as a plant pathogen and model system for animal picornaviruses, in recent years CPMV has been extensively used as a tool for biotechnology. A recombinant eVLP has been designed and can easily be produced 16 . A total of 24,976 particles were automatically picked16 from the CPMV-M dataset. Classification (both 2D and 3D) was used to select a homogenous subset of particles. The homogenous subset  was used for 3D refinement. The resulting density map was sharpened using an empirically-derived B-factor of −144.1 Å2, to 3.94 Å  and aspartic acid (D) in an infectious clone of RNA-2  it is possible the introduction of N174D may interfere with R17 by formation of a salt bridge which could explain the more severe effect of N174D compared with N174A. Reduced RNA encapsidation has previously been shown to adversely affect CPMV yield and systemic spread6, thus the phenotypes of these mutants are consistent with reduced RNA encapsidation. In addition, mutations of N174 in eVLP (to A or D) do not affect capsid assembly (not shown), showing these mutations do not substantially change the protein:protein interactions which hold the capsid together. Together these data suggest Asn174 is important for genome encapsidation. The cryo-EM reconstruction of CPMV-B shows a large bridge of density between encapsidated RNA-1 and a tryptophan residue (W190) in the L subunit of the capsid8; by contrast, no interaction between RNA and W190 can be visualised in the CPMV-M structure  to select a homogenous subset of particles . The homogenous subset  was used for 3D refinement. The refined CPMV-T EM density map was sharpened using empirically-derived B-factor of −185.8 Å2 to 4.25 Å resolution . A total of 1,324 micrographs of CPMV-T Fig. , bottom 17. This atomic model was used because a simple examination of the EM density map revealed that extra C-terminal density, similar to that in the CPMV eVLP EM derived model was present. Coot18 was used to remove residues 1 to 7 and 189 to 202 from the S subunit where no density was visible in the CPMV-T map. New regions of density in the C terminus of the S subunit were visible and the position of three amino acids  were built into the model using Coot18. The resulting model was fitted into the cryo-EM density using the Rosetta ‘relax’ protocol19, generating the model presented in Fig. 21 confirms this, and shows that much of the map is at ~3.5 Å  identified a number of different C termini in the S subunit fast electrophoretic band; however, cleavage between Ser183 and Thr184 was not identified10. We therefore suggest that residues 184–189 are present in the particle, but disordered and missing from the density map. The absence (or lack of defined structure) of these amino acids potentially leaves a gap in the capsid shell and so the inside of the capsid is accessible to the exterior environment. Analysis of Brome Mosaic Virus (BMV), a virus with three genomic RNA segments, which are packaged separately, has shown that the different genomic RNAs are released at different times, helping to regulate the timing of gene expression25. The different structures of CPMV-B and CPMV-M could provide a structural basis for such effects, with the CPMV-M capsid appearing to be less stable than the CPMV-B capsid. However, further studies will be required to validate this idea, and identify the routes for allosteric communication that allow an effect on capsid structure to be driven by different genomic RNA binding on the inside surface of the capsid.The structure of the majority of the CPMV-M and CPMV-T capsid is essentially identical, but the C terminal segment of the S subunit shows a number of differences Fig. . The C t183 Fig. , which i10 is consistent with the conformation seen in the CPMV-B8 and the CPMV-M map presented here, confirming previous proteomic studies that show the S-subunit has been cleaved10. In the cryo-EM structure of eVLP a number of important amino acids were identified in the C terminus extension. Mutational analysis of these amino acids  demonstrated they were involved in genome encapsidation and/or capsid assembly and the role of the C terminal extension is to stabilise the interactions between the S subunits at the pentamer8. The same interactions are also visible in the CPMV-T map presented in this study8.In the CPMV-T map, EM density up to residue Thr184 can be visualised, after which the density becomes disordered and the next residue that can be convincingly built into the CPMV-T map is Pro188. The final visible residue is Ile197 Fig. . Thus, a26. By contrast, both the naturally occurring CPMV-T and the engineered eVLP8 which lack any RNA are cleaved more slowly, and thus retain the C-terminus for longer. Cleavage of the C terminal peptide reveals a large hydrophobic patch on the surface of CPMV. The presence of an intact C-terminal sequence appears to be important for efficient intercellular movement of the virus, since genetic removal of this region results in impaired movement of the virus within the plant6, a phenomenon associated with virus aggregation within cells . Thus, the C-terminal sequence may have a role in occluding this hydrophobic patch thereby preventing aggregation and enhancing viral movement, rather than facilitating direct interactions between the C-terminus and the viral movement protein.Previous work shows that the C-terminus of the S subunit is proteolytically sensitive in all CPMV particles, but more so in those that contain genomic RNA8 structure (which encapsidates RNA-1) the most prominent RNA:protein interaction is between W190 of the L subunit. Intriguingly, density linking W190 to the RNA density is not visible in the structure of CPMV-M (containing RNA-2) reported here. CPMV-M cannot replicate alone in host cells as it does not contain sequences encoding the replication proteins. CPMV-M is therefore an ideal candidate for drug or nucleic acid delivery27. RNA-2 has been modified for use in diagnostic RT-PCR14 and more recently, it has been modified to remove the region encoding the viral movement protein, thereby abolishing the ability of the virus to move from cell to cell28. This study provides the first structural analysis of purified CPMV-M particles. This information should aid the further development of CPMV-M as a particle for the encapsidation of specific RNA molecules for diagnostic or therapeutic purposes.In this study, we have produced the first near atomic resolution structures of CPMV-M and CPMV-T. These structures have permitted us to identify key differences between the forms of CPMV. The CPMV structures are similar, particularly in the L subunit. The main differences are between the empty CPMV particles (CPMV-T and eVLP) and CPMV particles containing RNA (CPMV-B and CPMV-M). We have demonstrated the importance of specific amino acids in genome encapsidation. Of particular interest is N174 in the L subunit, which was identified by its interaction with density attributed to RNA. Mutational analysis demonstrated N174 is involved in RNA encapsidation but not particle assembly. In the CPMV-BN. benthamiana with CPMV was initiated by agroinfiltration of plants with pBinP-S1NT and pBinP-S2NT29 and the resulting virus particles were purified30. The individual components were separated by centrifugation on 42%, 49%, 57% and 65% (w/v) CsCl gradients31.Infection of HT using the GENEART® Site-Directed Mutagenesis System (Invitrogen™) according to manufacturer's protocol. Primers for site-directed mutagenesis were designed using QuikChange Primer Design Program. To examine the effect of the mutations on virus infectivity and RNA encapsidation, the mutant forms of pEAQ-RNA-2 were co-infiltrated into N. benthamiana with pBinP-S1NT and any particles produced were purified as described for WT CPMV (above).Point mutations were introduced into the coat protein-coding region of either pEAQ-RNA-2, a plasmid containing a full-length copy of CPMV RNA-2 in pEAQ-−/Å2 and a magnification of 125,085x. The final object sampling was therefore of 1.10 Å/pixel. A total of 1,323 (CPMV-T) and 1,759 (CPMV-M) exposures were recorded using the EPU automated acquisition software on a 17 Hz FEI Falcon II direct electron detector. Each exposure movie had a total exposure of one seconds and contained seven images.Cryo-EM grids were prepared by placing 3 μl of ~7.9 mg/ml CPMV-M or ~1.92 mg/ml CPMV-T onto 200 mesh grids with 2 μm holes . Grids were glow discharged for ~20 seconds prior to plunge freezing in liquid ethane cooled by liquid nitrogen, using a FEI Vitrobot IV at 100% relative humidity, chamber temperature of 4 °C. Data was collected on an FEI Titan Krios  transmission electron microscope at 300 kV, using an electron dose of 42 e15 and the contrast transfer function of each determined using CTFFIND332 any images showing signs of significant astigmatism were discarded. All subsequent image processing steps were performed using RELION (v1.3)33 unless otherwise stated. Approximately 1,000 particles were manually picked and classified using reference-free 2D classification. The resulting 2D class average views were used as templates for automated particle picking16  symmetry imposed. For both CPMV-M and CPMV-T, the initial starting model was the CPMV eVLP structure (EMD-30148) filtered to ~60 Å resolution, a resolution at which almost all information other than size, shape and symmetry of the structure are removed. After each round, the best classes/class was taken to the next step of classification. To correct for mechanical drift, beam-induced movement and radiation damage, statistical movie processing and particle polishing procedures were implemented34. As CPMV particles are readily visible even in individual movie frames, a running average of three frames was used in the calculations. Post-processing was employed to appropriately mask the model, estimate and correct for the B-factor of the maps35. The final resolution was determined using the ‘gold standard’ Fourier shell correlation (FSC = 0.143) criterion33 4.25 Å for CPMV-T and 3.94 Å for CPMV-M. Local resolution was estimated using the ResMap wrapper in RELION21.Drift-corrected averages of each movie were created using MOTIONCORR8) was fitted into the CPMV-M EM map using Chimera as a simple examination revealed that no density for the C-terminus was present. Residues present in the CPMV-B model but not in the CPMV-M EM map (residues 184 to 189 of the S subunit) were deleted using Coot18 and the resulting model was refined using the ‘relax’ protocol in Rosetta19. The eVLP CPMV EM derived atomic model (PDB: 5a338) was fitted in to CPMV-T EM map because a simple examination of the EM density map revealed that extra C-terminal density, similar to that in the CPMV eVLP EM derived model was present. Residues in the CPMV eVLP model not present in CPMV-T map were deleted (residues 1 to 5 and 198 to 202 of the S subunit) using Coot18. Amino acids that were previously not visualised (residues 184 and 188–189) were added and modeled in Coot18. The model was subsequently refined using the ‘relax’ protocol in Rosetta19 and assessed for quality using MolProbity (Table 17.During model refinement, the EM density map was fixed and the atomic model was refined against that fixed map. The EM derived atomic model of CPMV-B (PDB: 5a32Supplementary Information