Poliovirus Infection and Poly(A) Sequences of Cytoplasmic Cellular RNA

The influence of polio infection on Poly(A) sequences of cellular cytoplasmic RNA was investigated. In the presence of guanidine, cellular protein synthesis was still shut off after poliovirus infection, although there was no viral RNA synthesis. The Poly(A) of these cells was unchanged with respect to quantity, size, and linkage to cellular cytoplasmic RNA. This finding strongly suggests that the shut off of cellular protein synthesis is not caused by a change of the Poly(A) sequences of cellular mRNA.     

Regulation of hepatitis C virus replication by the core protein through its interaction with viral RNA polymerase

The hepatitis C virus (HCV) core protein is a structural component of the nucleocapsid and has been shown to modulate cellular signaling pathways by interaction with various cellular proteins. In the present study, we investigated the role of HCV core protein in viral RNA replication. Immunoprecipitation experiments demonstrated that the core protein binds to the amino-terminal region of RNA-dependent RNA polymerase (RdRp), which encompasses the finger and palm domains. Direct interaction between HCV RdRp and core protein led to inhibition of RdRp RNA synthesis activity of in vitro. Furthermore, over-expression of core protein, but not its derivatives lacking the RdRp-interacting domain, suppressed HCV replication in a hepatoma cell line harboring an HCV subgenomic replicon RNA. Collectively, our results suggest that the core protein, through binding to RdRp and inhibiting its RNA synthesis activity, is a viral regulator of HCV RNA replication.     

Internal translation initiation of picornaviruses and hepatitis C virus

Picornaviruses and other positive-strand RNA viruses like hepatitis C virus (HCV) enter the cell with a single RNA genome that directly serves as the template for translation. Accordingly, the viral RNA genome needs to recruit the cellular translation machinery for viral protein synthesis. By the use of internal ribosome entry site (IRES) elements in their genomic RNAs, these viruses bypass translation competition with the bulk of capped cellular mRNAs and, moreover, establish the option to largely shut-down cellular protein synthesis. In this review, I discuss the structure and function of viral IRES elements, focusing on the recruitment of the cellular translation machinery by the IRES and on factors that may contribute to viral tissue tropism on the level of translation.     

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

ABSTRACT: BACKGROUND: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. RESULTS: Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3[PRIME] poly(A) sequence identifying the 3[PRIME] end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. CONCLUSIONS: NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.     

Mechanism of Mengo Virus-Induced Cell Injury in L Cells: Use of Inhibitors of Protein Synthesis to Dissociate Virus-Specific Events

L cells were infected with Mengo virus in the presence of varying concentrations of protein synthesis inhibitors (azetidine-2-carboxylic acid, p-fluorophenylalanine, puromycin), and examined with respect to the effects of the inhibitors on several features of virus-induced cell injury. The virus-specific events in the cells could be dissociated into three groups, based on their sensitivity to the inhibitors: (i) viral ribonucleic acid (RNA) synthesis, bulk viral protein synthesis, and infectious particle production, all of which were prevented by low inhibitor concentrations; (ii) the cytopathic effect (CPE) and stimulation of phosphatidylcholine synthesis, which were sensitive to intermediate concentrations of the inhibitors; and (iii) the virus-induced inhibitions of host RNA and protein synthesis, which were resistart to the inhibitors of protein synthesis except at very high concentrations. It is concluded from this that the virus-induced CPE and stimulation of phosphatidylcholine synthesis are not consequences of the inhibition of cellular RNA or protein synthesis. Analysis of the virus-specific protein and RNA synthesized at several concentrations of azetidine and puromycin suggests that the CPE may be induced by a viral protein precursor. Virus-induced inhibition of host RNA and protein synthesis occurred at azetidine concentrations which blocked the synthesis of over 99.7% of the total viral RNA and over 99% of the viral double-stranded RNA (dsRNA). Calculations show that this would correspond to less than 150 dsRNA molecules per infected cell, resulting in a dsRNA-polysome ratio of less than 1:1,000; this indicates that host protein synthesis cannot be inhibited by an irreversible binding of dsRNA to polysomes.     

Hepatitis C Virus Exploitation of Processing Bodies

During infection, positive-strand RNA viruses subvert cellular machinery involved in RNA metabolism to translate viral proteins and replicate viral genomes to avoid or disable the host defense mechanisms. Cytoplasmic RNA granules modulate the stabilities of cellular and viral RNAs. Understanding how hepatitis C virus and other flaviviruses interact with the host machinery required for protein synthesis, localization, and degradation of mRNAs is important for elucidating how these processes occur in both virus-infected and uninfected cells.     

Encephalomyocarditis Virus Infection of Mouse Plasmacytoma Cells I. Inhibition of Cellular Protein Synthesis

Mouse plasmacytoma ascites tumor cells (MOPC 460) were efficiently infected with encephalomyocarditis virus. Inhibition of host protein synthesis was evident after 2 h and complete by 4 h postinfection. The mechanism by which virus infection results in inhibition of host cell protein synthesis was studied in vitro. Cell-free protein-synthesizing systems, prepared from uninfected and infected cells, were found to be equally active with respect to their abilities to translate cellular and viral mRNAs. The plasmacytoma cell-free system was also shown to be insensitive to the addition of double-stranded viral RNA. Host cellular mRNA was isolated from uninfected and infected cells. No difference in the amount or size distribution of the mRNA was detected. However, the mRNA from infected cells was translated only 46 to 49% as actively as that from uninfected cells. mRNA isolated from cells in which initiation of protein synthesis was inhibited with pactamycin was similarly inactivated. Simultaneous addition of viral RNA and cellular mRNA to the plasmacytoma cell-free system resulted in a complete suppression of the translation of the cellular message, whereas viral RNA was translated normally.     

Effect of Inactivation by Hydroxylamine on Early Functions of Poliovirus

Evidence is presented that poliovirus particles with a single lethal hit by hydroxylamine do not induce in host cells either inhibition of cellular protein synthesis or viral ribonucleic acid (RNA) replication. The RNA of these viruses is not replicated even if the cells are simultaneously infected with both active and inactivated viruses. The damaged viral RNA seems to have lost both its template function and its function in the translation of normal viral proteins.     

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non‐structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1‐4 does not block cellular protein synthesis in BHK cells. Trans‐complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co‐expression of nsP1‐4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T‐cell intracellular antigen and polypyrimidine tract‐binding protein is clearly detected in SINV‐infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut‐off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR^?/? mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.     

Effect of Viral Double-Stranded RNA on Protein Synthesis in Intact Cells

The addition of purified, noninfectious, double-stranded RNA of bovine enterovirus, a picornavirus, to intact cells in culture results in a rapid cessation of cellular polypeptide synthesis. This inhibition is specific for host cell protein synthesis since the translation of picornavirus-specific proteins is not affected by the double-stranded viral RNA.     

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.     

The target reaction in the antiviral action of mouse interferon against vesicular stomatitis virus multiplication

Treatment of mouse L929 cells with mouse interferon (IFN) lowered the yield of vesicular stomatitis virus (VSV) in a dose-dependent manner. Accumulation of viral proteins was severely inhibited in IFN-treated cells, whereas cellular protein synthesis was not, indicating that the virus-induced shutoff of cellular protein synthesis was prevented by IFN. In order to identify the major target of IFN action precisely, the effect of IFN treatment on the synthesis of viral RNAs and proteins at various stages during the course of viral replication was examined. Accumulation of viral RNAs late in infection was inhibited, as was the case with viral proteins, but the synthesis of leader RNA and mRNAs early in infection was not significantly inhibited by treatment with a moderate dose of IFN. On the other hand, viral protein synthesis at an early stage of infection was strongly inhibited by IFN. The results indicate that the major target reaction of antiviral action of IFN against VSV multiplication is the translation of viral mRNA.     

Characterization of a Nodavirus Replicase Revealed a de Novo Initiation Mechanism of RNA Synthesis and Terminal Nucleotidyltransferase Activity

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3′ initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3′-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.     

Analysis of VSV mutant with attenuated cytopathogenicity: mutation in viral function, P, for inhibition of protein synthesis.

T1026, a ts mutant of VSV which is much less cytopathogenic than its parent, HR, and which can establish persistent infection under certain conditions, is a double mutant. In addition to its ts mutation in the virion RNA polymerase, T1026 has a second non-ts mutation in a viral function termed "P". This function is responsible for the inhibition of total protein synthesis in infected cells and acts chiefly at the level of translational initiation. In some cell systems, the inhibition of protein synthesis produced by P appears to be selective for cellular protein synthesis, whereas in other cell systems, both cellular and viral protein synthesis are inhibited. T1026 and its ts revertants are phenotypically P- -that is, cells infected with them show total protein synthesis rates equal to or greater than uninfected cells, while synthesizing viral proteins at the same or even greater rates than HR-infected cells. The P- mutation is correlated with failure to increase plaque size after 2-3 days of incubation. Since viral mutants obtained from persistently infected cultures in a variety of systems appear to be double mutants with a ts mutation in the virion RNA polymerase and a small plaque marker, we suggest that T1026 could represent a model for such mutants.     

Rotavirus Nonstructural Protein NSP3 is not required for viral protein synthesis

Initiation is the rate-limiting step in protein synthesis and therefore an important target for regulation. For the initiation of translation of most cellular mRNAs, the cap structure at the 5' end is bound by the translation factor eukaryotic initiation factor 4E (eIF4E), while the poly(A) tail, at the 3' end, is recognized by the poly(A)-binding protein (PABP). eIF4G is a scaffold protein that brings together eIF4E and PABP, causing the circularization of the mRNA that is thought to be important for an efficient initiation of translation. Early in infection, rotaviruses take over the host translation machinery, causing a severe shutoff of cell protein synthesis. Rotavirus mRNAs lack a poly(A) tail but have instead a consensus sequence at their 3' ends that is bound by the viral nonstructural protein NSP3, which also interacts with eIF4GI, using the same region employed by PABP. It is widely believed that these interactions lead to the translation of rotaviral mRNAs, impairing at the same time the translation of cellular mRNAs. In this work, the expression of NSP3 in infected cells was knocked down using RNA interference. Unexpectedly, under these conditions the synthesis of viral proteins was not decreased, while the cellular protein synthesis was restored. Also, the yield of viral progeny increased, which correlated with an increased synthesis of viral RNA. Silencing the expression of eIF4GI further confirmed that the interaction between eIF4GI and NSP3 is not required for viral protein synthesis. These results indicate that NSP3 is neither required for the translation of viral mRNAs nor essential for virus replication in cell culture.