Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.     

Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the top of the terminal 3' stem-loop structure

Dengue virus (DEN) is a major public health problem worldwide and causes a spectrum of diseases, for which no antiviral treatments exist. Peptide-conjugated phosphorodiamidate morpholino oligomers (P-PMOs) complementary to the DEN 5' stem-loop (5'SL) and to the DEN 3' cyclization sequence (3'CS) inhibit DEN replication, presumably by blocking critical RNA-RNA or RNA-protein interactions involved in viral translation and/or RNA synthesis. Here, a third P-PMO, complementary to the top of the 3' stem-loop (3'SLT), inhibited DEN replication in BHK cells. Using a novel DEN2 reporter replicon and a DEN2 reporter mRNA, we determined that the 5'SL P-PMO inhibited viral translation, the 3'CS P-PMO blocked viral RNA synthesis but not viral translation, and the 3'SLT P-PMO inhibited both viral translation and RNA synthesis. These results show that the 3'CS and the 3'SL domains regulate DEN translation and RNA synthesis and further demonstrate that P-PMOs are potentially useful as antiviral agents.     

Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.     

An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC(50) values lower than 1 microM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC(50) = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA.     

Role of RNA structures present at the 3'UTR of dengue virus on translation, RNA synthesis, and viral replication

We have developed a dengue virus replicon system that can be used to discriminate between translation and RNA replication. Using this system, we analyzed the functional role of well-defined RNA elements present at the 3'UTR of dengue virus in mammalian and mosquito cells. Our results show that deletion of individual domains of the 3'UTR did not significantly affect translation of the input RNA but seriously compromised or abolished RNA synthesis. We demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification. We also characterized the vaccine candidate rDEN2Delta30 in the replicon system and found that viral attenuation is caused by inefficient RNA synthesis. Furthermore, using both the replicon system and recombinant viruses, we identified an RNA region of the 3'UTR that enhances dengue virus replication in BHK cells while is dispensable in mosquito cells.     

Antibodies to the Giardia lamblia double-stranded RNA virus major protein can block the viral infection

The double-stranded RNA virus-like particles, found among several independent isolates and cloned strains of Giardia lamblia, have previously been reported to be spheres of 35 nm with a genome of 7 kilobase pairs and a major protein of 100 kDa. The virus is capable of infecting certain virus-free isolates of G. lamblia. Antisera raised in mice against the intact virus did not react with the double-stranded RNA, but reacted strongly with the 100 kDa protein in Western blots. Preincubation of the virus with antisera abolished viral infectivity, whereas the antisera against double-stranded RNA showed only a weak blocking effect. Inclusion of the antiviral sera in the cultures of virus-infected G. lamblia at 10³-fold dilution resulted in elimination of the virus from the protozoa. Apparently, the 100 kDa protein is necessary for the initiation of viral infection and possibly subsequent assembly or replication of viral progeny particles.     

Characterization of RNA synthesis,replication mechanism,and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus

In vitro RNA-dependent RNA polymerase assays revealed that the JEV replication complex (RC) synthesized viral RNA utilizing a semiconservative and asymmetric mechanism. Peak viral replicase activity and levels of viral RNA observed 15-18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. Among divalent cations, Mg(2+) was essential and exhibited cooperative binding for its two replicase-binding sites. Mn(2+), despite sixfold higher affinity for the replicase, elicited only 70% of the maximum Mg(2+)-dependent activity, and deficit of either cation led to synthesis of incomplete RNA products. We also determined as a first instance for a flavivirus RC, kinetic parameters using cytoplasmic "virus-induced heavy membranes" after depleting endogenous nucleotides. Exhaustive trypsin treatment, which degraded the bulk of NS3 and NS5, had no effect on replicase activity, suggesting that the active flaviviral RC resides behind a membrane barrier and recruits minuscule proportions of the replicase proteins.     

RNA chaperone activity of the tombusviral p33 replication protein facilitates initiation of RNA synthesis by the viral RdRp in vitro

Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 replication protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity.     

Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication

Despite ongoing research investigating mechanisms of coronavirus replication, functions of many viral nonstructural proteins (nsps) remain unknown. In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. The results demonstrated that the carboxy-terminal half of nsp1 (residues K(124) through L(241)) was dispensable for virus replication in culture but was required for efficient proteolytic cleavage of nsp1 from the gene 1 polyprotein and for optimal viral replication. Furthermore, whereas deletion of nsp1 residues amino-terminal to K(124) failed to produce infectious virus, point mutagenesis of the nsp1 amino-terminus allowed recovery of several mutants with altered replication and RNA synthesis. This study identifies nsp1 residues important for protein processing, viral RNA synthesis, and viral replication.     

Classical swine fever virus NS5A protein interacts with 3'-untranslated region and regulates viral RNA synthesis

To investigate the function of classical swine fever virus (CSFV) NS5A protein, the experiments for viral RNA synthesis and viral replication were performed in the co-presence of NS5A and NS5B. Results showed that small concentrations of NS5A stimulated, large concentrations of NS5A inhibited, viral RNA synthesis and viral replication. Affinity chromatography experiments and UV-crosslinking assays revealed that CSFV NS5A and NS5B bound its cognate 3′UTR and that NS5A had higher affinity than NS5B protein in binding to 3′UTR. 200 ng of NS5A inhibited NS5B-3′UTR complex formation by about 95%. CSFV 3′UTR was found to contain two NS5A-binding sites, located in 3′UTRSL-1 (nt 161-231) and 3′UTRSL-2 (nt 90-160), respectively, a NS5B-binding site, also located in 3′UTRSL-1. The 3′UTRSL-1 is the common binding site for NS5A and NS5B. Furthermore, competitive electrophoretic mobility shift assays indicated that binding of CSFV NS5A to 3′UTRSL-1 is more efficiently than to 3′UTRSL-2. These results suggested that the different concentrations of NS5A, the different binding activities of NS5A and NS5B to 3′UTR and binding of NS5A to different regions of 3′UTR might contribute at least partially to modulation of CSFV replication.     

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.     

Heterologous RNA replication enhancer stimulates in vitro RNA synthesis and template-switching by the carmovirus, but not by the tombusvirus, RNA-dependent RNA polymerase: implication for modular evolution of RNA viruses

The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- (Cucumber necrosis virus, CNV), carmovirus (Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and post-recombinational amplification of new recombinant viruses.     

Phosphorylation of the RNA polymerase II carboxyl-terminal domain in human cytomegalovirus-infected cells and in vitro by the viral UL97 protein kinase

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) ordinarily exists in electrophoretically distinct hypophosphorylated and hyperphosphorylated forms. Human cytomegalovirus infection induced forms of this subunit whose electrophoretic mobilities were intermediate without decreases in abundance of the original forms. Phosphatase treatment nearly eliminated the intermediate migrating forms. In vitro, the viral protein kinase, UL97, phosphorylated this subunit, a recombinant protein containing the CTD, and peptides containing the CTD consensus sequence, YSPTSPS. Phosphorylation occurred predominantly on serine 5 and was substantially reduced when either serine 2 or 5 was already phosphorylated. The abundance of the intermediate and hypophosphorylated forms was reduced at most twofold during infections in which UL97 was genetically or pharmacologically inhibited. These results identify a new pattern of RNA polymerase II modification induced by virus infection and a viral enzyme that phosphorylates the CTD in vitro, but only possibly in vivo.     

Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein

The SR-domain protein kinase (SRPK) 1 and 2 are two important kinases involved in cellular RNA splicing. Recently, it was suggested that these two kinases, which could bind to the hepatitis B virus (HBV) core protein, might be the major cellular kinases that phosphorylate the core protein to regulate HBV replication. In this report, we tested the role of SRPK1 and SRPK2 in HBV replication and found that both of them could suppress HBV replication by reducing the packaging efficiency of the pgRNA without affecting the formation of the viral core particles. This suppressive effect of SRPK1 and SRPK2 on HBV replication cannot be explained by their phosphorylation activities on the HBV core protein as the over-expression of these two kinases had no detectable effects on HBV core protein phosphorylation in vivo and their mutants that lacked the kinase activity could still suppress HBV DNA replication. Thus, these findings demonstrate a negative role of SRPK1 and SRPK2 in the regulation of HBV replication through a mechanism not involving the phosphorylation of the core protein.     

Replication of mouse mammary tumor virus in tissue culture. II. Kinetics of virus production and the effect of RNA and protein inhibitors on viral synthesis.

The incorporation of virus-specific RNA into mouse mammary tumor virus (MuMTV) was studied using an established mouse mammary tumor cell line (MuMT-73). Two species of RNA (70 and 35 S) were found to be virus specific by molecular hybridization using radioactive MuMTV-complementary DNA as a probe. The time between the addition of [³H]uridine and the initial release of virions continued to increase RNA was found to be 2 to 4 hr. The rate of release of labeled virions continued to increase for 20–30 hr before a steady state was reached. Actinomycin D treatment of the cells completely inhibited viral RNA synthesis within 1 hr; however, synthesis of the viral proteins and the release of mature virions continued for at least 10 hr. The morphology and protein composition of the virus particles produced by the MuMT-73 cells with and without actinomycin D treatment were identical. Exposure of cells to puromycin or cycloheximide inhibited the replication of MuMTV, but such treatments induced the production of an endogenous type-C virus.