Envelope M protein selectively interacts with PS-containing non-MHV RNA transcript in the absence of N protein. The specific and selective interaction between M protein and PS- containing RNP complexes drives the specific packaging of PS-containing RNAs into MHV particles (28). In all known enveloped RNA viruses studied thus far, N protein or capsid protein plays an essential role in viralRNA packaging. How- ever, the role(s) of N protein in MHV RNA packaging is unknown. In coexpression experiments testing packaging of non-MHV RNA with inserted PS in the presence of combina- tions of various expressed MHV proteins, we attempted to understand how M protein selectively recognizes PS-contain- ing RNP complexes. We were particularly interested in deter- mining whether N protein is essential for this selective inter- action. DBT cells were infected with a recombinant vaccinia virus, vTF7-3, which encodes the T7 RNA polymerase (10). One hour later, the cells were independently transfected with either plasmid PS5A, which contains the entire CAT gene under the dual controls of the T7 promoter and the T7 termi- nator, or plasmid PS5B190, which carries the MHV 190-nt PS positioned downstream of the CAT gene (Fig. 1A). RNA tran- scripts are expressed from transfected PS5A and PS5B190 in vTF7-3-infected cells (28, 38). At 4 h post-vTF7-3 infection, which was 3 h post-plasmid transfection, cultures from both plasmid transfections were superinfected with one or combi- nations of three Sindbis virus expression vectors: SinM pseudovirion (expressing MHV M protein), SinN pseudovirion (expressing MHV N protein), and SinLacZ pseudovirion (en- coding the ␤-galactosidase protein) (22, 27). Cell extracts were prepared at 12 h post-Sindbis virus pseudovirion infection and used for coimmunoprecipitation analysis with anti-M MAb or control anti-H2K MAb. RNA was extracted from the immu- noprecipitated samples and treated with DNase (28, 38). Northern blot analysis with a CAT sequence
These constraints were examined in this study by evaluating in situ activities of VSV vRdRp using a minigenome assay. Our data showed that vRdRp became highly dys- regulated with large decreases in transcription and replication when the codon usage in the VSV minigenome was deoptimized (Fig. 2C). Changes in viralRNA synthesis were not related to protein translation since all viral proteins were produced by other vectors. This clearly conﬁrmed that the nucleotide sequence of the viral genome in the nucleocapsid plays an essential role in regulating viralRNA synthesis by vRdRp. In an effort to identify which steps are more affected by changes of codon usage, eight chimeric minigenomes were also created. The results showed that the regions corre- sponding to initiation and termination of gene transcription had more-dramatic detri- mental effects on vRdRp activities when CUB was deoptimized (Fig. 2C and D). In the context of the virus genome, this is consistent with the ﬁnding that vRdRp is required to accurately initiate and terminate mRNA transcription in order to balance the tran- script products from the polycistronic genome (39). For replication, elongation of cRNA and vRNA was most limited by CUB changes because the ends of the genome, which regulate the initiation and termination of genome replication by vRdRp, were not changed.
The NP protein potentiates the synthesis of cRNA, the first step in viralRNA replication, in the absence of a primer. Our main goal was to establish an in vitro system that catalyzes unprimed cRNA and vRNA synthesis using purified recombi- nant proteins. For these experiments we used purified NP that was either untagged or contained a C-terminal His tag; the same results were obtained with the two NP preparations. For cRNA synthesis, we used the U3A vRNA template, thereby eliminating termination and poly(A) addition in the absence of the NP protein (32) (Fig. 2A). Consequently, we could focus solely on functions of the NP protein in initiating cRNA syn- thesis rather than its role in antitermination at the U tract. The 50-nt U3A vRNA was first incubated with the purified viral polymerase to enable its 3 ⬘ end to bind to the PB1 subunit of the polymerase (18). This was then followed by the addition of purified NP protein, which binds to the rest of the vRNA template as well as carrying out any other function needed for unprimed cRNA synthesis. It was necessary to follow this pro- cedure to coat the vRNA template with the NP protein be- cause of its ability to nonspecifically bind any RNA at 24-nt intervals from one end to the other (1, 14, 46, 47). Conse- quently, as expected, addition of the NP protein to the U3A
Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viralRNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5 ⴕ triphosphate end groups characteristic of authentic replication products, were devel- oped. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis inter- nally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viralRNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.
RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5 ⴕ -3 ⴕ exoribonuclease, whose absence led to an ⬃ 10- to 50-fold enhancement of RNA recombination in Tomato bushy stunt virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545–10550, 2005). In this study, we found abundant 5 ⴕ -truncated viral RNAs in xrn1 ⌬ mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1 ⌬ mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5 ⴕ -truncated viral RNAs were expressed in the parental and xrn1 ⌬ mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5 ⴕ - truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viralRNA recombination by rapidly removing the 5 ⴕ -truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5 ⴕ -truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonucle- ase led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viralRNA turnover play major roles in RNA virus recombination and evolution.
The 3 ⴕ untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viralRNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555–565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that the Escherichia coli-expressed RNA-dependent RNA polymerase domain ( ⌬ 893) derived from BaMV open reading frame 1 could specifically bind to the 3 ⴕ UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3 ⴕ UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3 ⴕ UTR showed that ⌬ 893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that ⌬ 893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.
All of the previously reported recombinant RNA-dependent RNA polymerases (RdRp), the NS5B enzymes, of hepatitis C virus (HCV) could function only in a primer-dependent and template-nonspecific manner, which is different from the expected properties of the functional viral enzymes in the cells. We have now expressed a recombinant NS5B that is able to synthesize a full-length HCV genome in a template-dependent and primer- independent manner. The kinetics of RNA synthesis showed that this RdRp can initiate RNA synthesis de novo and yield a full-length RNA product of genomic size (9.5 kb), indicating that it did not use the copy-back RNA as a primer. This RdRp was also able to accept heterologous viralRNA templates, including poly(A)- and non-poly(A)-tailed RNA, in a primer-independent manner, but the products in these cases were heterogeneous. The RdRp used some homopolymeric RNA templates only in the presence of a primer. By using the 3 ⴕ -end 98 nucleotides (nt) of HCV RNA, which is conserved in all genotypes of HCV, as a template, a distinct RNA product was generated. Truncation of 21 nt from the 5 ⴕ end or 45 nt from the 3 ⴕ end of the 98-nt RNA abolished almost completely its ability to serve as a template. Inclusion of the 3 ⴕ -end variable sequence region and the U-rich tract upstream of the X region in the template significantly enhanced RNA synthesis. The 3 ⴕ end of minus-strand RNA of HCV genome also served as a template, and it required a minimum of 239 nt from the 3 ⴕ end. These data defined the cis-acting sequences for HCV RNA synthesis at the 3 ⴕ end of HCV RNA in both the plus and minus senses. This is the first recombinant HCV RdRp capable of copying the full-length HCV RNA in the primer-independent manner expected of the functional HCV RNA polymerase.
ViralRNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase- RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that sig- nificantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tis- sue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viralRNA- dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.
Given the importance of the KSHV lytic cycle, we are interested in the mechanisms underlying the regulation of viral lytic gene expression. During the lytic cycle, KSHV expresses two main regulatory proteins: ORF50 (Rta, replication and transcription activator) which initiates transcription of viral lytic genes [11,12] and ORF57 (Mta, mRNA transcript ac- cumulation), a multifunctional protein that promotes the accumulation of viral intron-less transcripts [13-17] and stimulates intron removal of viral in- tron-containing RNAs [18,19]. The intron removal function of ORF57 is especially important for the vi- rus, as pre-mRNA lacking appropriate posttranscrip- tional processing is less stable than its properly pro- cessed functional counterpart . Lytic KSHV repli- cation also promotes widespread shutoff of cellular gene expression by hyperadenylation and nuclear retention of host mRNA which leads to enhancement of cellular mRNA turnover [21-23]. Thus, it is no sur- prise that KSHV has evolved mechanisms for regu- lating the ORF57-mediated post-transcriptional viralRNA processing and accumulation. To understand how ORF57 functions in host cells is a key step to de- termine how KSHV ensures the expression of its own genes, while also shutting-off host gene expression during virus lytic infection. Previously, we have de- scribed a mechanism as to how ORF57 stimulates the expression of intron-containing viralRNA transcripts in . However, it remains unclear how ORF57 ac- cumulates intron-less viral RNAs, and whether the accumulation is mediated by interfering with nuclear export [13,16,24-26].
Using a cell-based assay for RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of noroviruses, we previously ob- served that VP1, the major structural protein of the human GII.4 norovirus, enhanced the GII.4 RdRp activity but not that of the related murine norovirus (MNV) or other unrelated RNA viruses (C. V. Subba-Reddy, I. Goodfellow, and C. C. Kao, J. Virol. 85: 13027–13037, 2011). Here, we examine the mechanism of VP1 enhancement of RdRp activity and the mechanism of mouse noro- virus replication. We determined that the GII.4 and MNV VP1 proteins can enhance cognate RdRp activities in a concentration- dependent manner. The VP1 proteins coimmunoprecipitated with their cognate RdRps. Coexpression of individual domains of VP1 with the viral RdRps showed that the VP1 shell domain (SD) was sufficient to enhance polymerase activity. Using SD chime- ras from GII.4 and MNV, three loops connecting the central ␤ -barrel structure were found to be responsible for the species-spe- cific enhancement of RdRp activity. A differential scanning fluorimetry assay showed that recombinant SDs can bind to the puri- fied RdRps in vitro. An MNV replicon with a frameshift mutation in open reading frame 2 (ORF2) that disrupts VP1 expression was defective for RNA replication, as quantified by luciferase reporter assay and real-time quantitative reverse transcription- PCR (qRT-PCR). Trans-complementation of VP1 or its SD significantly recovered the VP1 knockout MNV replicon replication, and the presence or absence of VP1 affected the kinetics of viralRNA synthesis. The results document a regulatory role for VP1 in the norovirus replication cycle, further highlighting the paradigm of viral structural proteins playing additional functional roles in the virus life cycle.
One obvious difference between the RNA molecules produced by replication and those produced by transcription is the location of their synthesis (cytoplasm versus nucleus). It is possible that the colocalization of newly replicated RNA molecules and newly translated coat protein molecules in the cytopathological structures associated with virus infection (44, 45) simply means that replicated viralRNA is the only RNA available for incorporation into the nascent capsids. However, this alone does not explain why, in the absence of replication, no nucleic acids are incorporated into capsids produced by transient expression. Other features, therefore, must also play a role. For example, RNA produced via replication has a 5=-terminal genome-linked viral protein (VPg) as opposed to a cap structure found on RNA molecules generated by transcription. Thus, the presence of VPg could, in theory, provide a means of discrim- inating between replicated RNA and that produced by transcription. VPg is believed to arise from the cleavage of an RNA-1-encoded 112K precursor (46), which also contains the sequences of the 24K protease and the 87K replicase. The 112K protein is analogous to 3BCD from poliovirus (5), which contains the sequence of VPg (3B), the viral protease (3C), and the viral polymerase (3D). In the case of poliovirus, it has been found that 3CD can stimulate the encapsidation of VPg-linked RNA, but not transcripts lacking VPg, into particles (47, 48). This has been interpreted as indicating that there is an afﬁnity between 3CD and VPg. If CPMV VPg is required for RNA encapsidation, its mere presence is clearly not sufﬁcient. Indeed, we have shown that the RNA-1 polyprotein is translated in our RNA-1 mutants, which indicates that VPg must be produced as well, but it is clearly not capable of directing encapsidation when replication is not taking place. It seems likely that any role that VPg plays in packaging is dependent on its linkage to the viral RNAs, which takes place only during replication. A further possibility is that nascent positive strands produced via replication fold into the type of hairpin structures identiﬁed as being important for packaging in human parechovirus (8), while full-length RNA molecules produced by transcription in the nucleus and then exported to the cytoplasm adopt assembly-incompetent structures. However, the fact that CPMV efﬁciently encapsidates two RNAs of different sizes that lack any sequence homology apart from at the extreme 5= and 3= ends makes the idea that the recognition of packaging signals is responsible for their speciﬁc packaging unlikely.
Many RNA viruses evolve rapidly due to a high frequency of mutations and genetic recombination as well as reassortment of genomic components (1, 52). Comparison of viralRNA genomes has revealed that recombination has shaped the evo- lution of many RNA viruses (52). RNA recombination is the process that joins two or more noncontiguous segments of the same RNA or two separate RNAs together (32). Recombina- tion events can have small or dramatic effects on viral genomes by introducing insertions or duplications, combining new se- quences, or leading to deletions or rearrangements. RNA re- combination also functions to repair truncated or damaged viralRNA molecules (16, 17, 30, 37). The repair function of RNA recombination might compensate viruses for their high mutation rate, which could introduce detrimental mutations into the viral genomes and thus reduce the fitness of viral populations (38, 39). Therefore, depending on the nature of recombining RNAs, the locations of the recombinant junction sites, and the outcome of the recombination events, RNA recombination can guard the infectivity of the viral genome or can increase genome variability. Altogether, RNA recombina- tion is a probabilistic event that can affect the population of viruses, contribute to virus variability, and function in genome repair that maintains the infectivity of RNA viruses (32, 39).
lished data). We conclude that the BrUTP-labeled sites are indeed the sites of replication. Our failure to detect P15 at these sites could have at least two possible explanations. One possibility is that P15 is a direct participant in viral replication but is only very transiently present at the viralRNA replication sites and is hence difficult to detect there. The second possi- bility, which we favor, is that P15 is never localized at the replication sites and that its effect on viralRNA replication and/or accumulation is indirect. Thus, P15 might localize to the same membranous structures as the RNA replication complex and intervenes in replication by interacting with these struc- tures to create a favorable environment for the replication process and/or to stabilize the progeny RNA prior to encapsi- dation. Another attractive hypothesis is that P15 could be a suppressor of host defense mechanisms similar or identical to posttranscriptional gene silencing (PTGS) (29). Such activity has recently been attributed to the HC-Pro of tobacco etch virus (18), the 2b protein of cucumber mosaic virus (1), and several other viral proteins (37). The preferential decrease of progeny RNA positive strands relative to negative strands for the P15 mutants is consistent with such a hypothesis, since PTGS-mediated RNA degradation would be expected to pref- erentially target the more abundant positive-sense RNA (27) in the absence of P15. Experiments are currently underway to determine if P15 plays a role in PTGS suppression during the PCV infection cycle.
was identified as the 5 ⬘ cleavage product of GFP-Cym( ⫹ ) sensor mRNA (see later). The level of GFP- PoLV( ⫹ ) mRNA remained intact and accumulated at a sig- nificantly higher level than the GFP-Cym( ⫹ ) mRNA, indicat- ing that the Cym19stop-specific siRNA-programmed RISC can cleave only the homologous sequence containing sensor mRNA (Fig. 2B, lanes 1, 3, and 5). At 3 dai, the accumulations of the two sensors were more similar to each other than at 2 dai, and the GFP expression from GFP-PoLV( ⫹ ) was slightly higher than the GFP level from GFP-Cym( ⫹ ), while the mRNA accumulation from GFP-PoLV( ⫹ ) was much higher than from GFP-Cym( ⫹ ) (Fig. 2B, lane 6 against lane 5). It is worthy of note that the accumulation of the uncleaved GFP- Cym( ⫹ ) mRNA increased at 3 dai (Fig. 2B, lanes 1, 3, and 5). This suggests that the activity of preassembled antiviral RISC lasts for approximately 2 to 3 days, since the coexpressed p19 suppressor with the sensor sequences prevents the assembly of new siRNA-containing RISC from the time point of agro- infiltration. These results also clearly suggest that the viralRNA targeting by VIGS is mediated mainly by RNA cleavage. Sequence-specific cleavage of sensor RNAs by VIGS. Al- though the obtained data suggest that the inactivation of GFP- Cym( ⫹ ) sensor and the appearance of its low-molecular-mass form is due to a sequence-specific phenomenon, we designed experiments to confirm this hypothesis and to further analyze the sequence-specific nature of VIGS. To this end, we used the previously described PoLV p14 silencing suppressor-deficient
Maximum size of reRNA which can be packaged. To define the size limits for reRNA packaging, we created constructs designed to package bacteriophage lambda RNA sequences of 0.5, 1, 1.5, 2, 3, and 4 kb. These particles were expressed and purified, and the RNA was isolated from each of these con- structs. Only the construct encoding the 0.5-kb bacteriophage lambda RNA contained a reRNA of the expected size, as determined by ethidium bromide staining. The other con- structs contained RNA which was heterogeneous in length (data not shown). Northern blotting of the purified recombi- nant RNA with probes directed to the 39 terminus of the bacteriophage lambda sequence revealed that packaging of 500 bases of RNA was very efficient but that packaging of the 1- and 1.5-kb amounts of RNA was inefficient. As the size of the reRNA was increased, greater amounts of host (E. coli) RNA was packaged in preference to the amount of reRNA that was packaged. Although the 1.0- and 1.5-kb amounts of bacterio- phage lambda RNA were detectable by Northern blotting, they were not detectable as discrete RNA species by ethidium bro- mide staining and UV fluorescence.
Identification of a candidate motif essential for type I IFN induction by positive-sense DVG RNA. We have reported that alterations in the internal (noncomplementary) region of DVG- 546 drastically affect its stimulatory capacity (26). Specifically, we found that a mutant form of DVG (DVG-324) retaining the 5= end of the internal sequence promoted high levels of expression of type I IFNs upon transfection, while a mutant DVG missing the 5= end of the internal sequence (DVG-354) showed a significant loss of stimulatory ability. These data suggest that a specific region lo- cated at the 5= end of the internal sequence plays an essential role in maximizing the stimulatory potential of DVG RNA. Additional mutant DVGs further confirmed this prediction (Fig. 2A and B). One mutant DVG that retained a shorter 5= internal sequence (DVG-268) also showed potent immunostimulatory activity, while mutant DVGs lacking either the complete internal sequence (DVG-200) or both complementary sequences (DVG-IS) demon- strated a reduced ability to stimulate antiviral genes upon trans- fection into both mouse and human cells (Fig. 2B; see Fig. S2A and B in the supplemental material). This differential activity of mu- tant DVGs was sustained over a 24 h time course, ruling out the possibility of different kinetics of IFN induction by the different mutant DVGs (see Fig. S2C). For all of these studies, in vitro- transcribed DVG (ivtDVG) RNAs were purified from gels, tested for purity and endotoxin content, and transfected into cells at equal molarity (see Fig. S3A to D).
Two additional HCV RdRps were generated to eliminate the possibility that an E. coli protein(s) responsible for TNTase activity copurified with H⌬21 but not m⌬21. First, a version lacking the C-terminal 51 amino acids, H⌬51, was produced in E. coli. Second, a full-length HCV NS5B, BacFL, was pro- duced using a recombinant baculovirus. Both proteins, H⌬51 and BacFL, were as pure as H⌬21 (Fig. 1A) and directed RNA synthesis using a number of templates, including RNA LE19 (Fig. 1D and 1E). H⌬51 and BacFL possess high levels of TNTase activity with LE19, generating labeled RNAs of 20 to 21 nt with radiolabeled rATP, rCTP, and dCTP more effi- ciently than with rUTP and dATP (Fig. 1D, lanes 2 to 7). These results are consistent with those from H⌬21, suggesting that highly purified HCV NS5B produced from either prokaryotic or eukaryotic cells possesses TNTase activity with similar sub- strate preferences.