Regulation Of Viral RNA Synthesis Research Articles

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-2α 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.

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

Dissociation of rabies virus matrix protein functions in regulation of viral RNA synthesis and virus assembly.

Recently, we have shown that the rabies virus (RV) matrix (M) protein regulates the balance of virus RNA synthesis by shifting synthesis activity from transcription to replication (S. Finke, R. Mueller-Waldeck, and K. K. Conzelmann, J. Gen. Virol. 84:1613-1621, 2003). Here we describe the identification of an M residue critical for regulation of RV RNA synthesis. By analyzing the phenotype of heterotypic RV M proteins with respect to RNA synthesis of RV SAD L16, we identified the M proteins of the RV ERA and PV strains as deficient. Comparison of M sequences suggested that a single residue, arginine 58, was critical. A recombinant virus having this amino acid exchanged with a glycine, SAD M(R58G), has lost the abilities to downregulate RV transcription and to stimulate replication. This resulted in an increase in the transcription rate of more than 15-fold, as previously observed for M deletion mutants. Most importantly, the efficiencies of virus assembly and budding were equal for wild-type M and M(R58G), as determined in assays studying the transient complementation of an M- and G-deficient RV construct, NPgrL. In addition, virus particle density, protein composition, and specific infectivity of SAD L16 and SAD M(R58G) viruses were identical. Thus, we have identified mutations that affect the function of M only in regulation of RNA synthesis, but not in assembly and budding, providing evidence that these functions are genetically separable.

Naturally occurring substitutions in the P/V gene convert the noncytopathic paramyxovirus simian virus 5 into a virus that induces alpha/beta interferon synthesis and cell death.

The V protein of the paramyxovirus simian virus 5 (SV5) is responsible for targeted degradation of STAT1 and the block in alpha/beta interferon (IFN-alpha/beta) signaling that occurs after SV5 infection of human cells. We have analyzed the growth properties of a recombinant SV5 that was engineered to be defective in targeting STAT1 degradation. A recombinant SV5 (rSV5-P/V-CPI-) was engineered to contain six naturally occurring P/V protein mutations, three of which have been shown in previous transfection experiments to disrupt the V-mediated block in IFN-alpha/beta signaling. In contrast to wild-type (WT) SV5, human cells infected with rSV5-P/V-CPI- had STAT1 levels similar to those in mock-infected cells. Cells infected with rSV5-P/V-CPI- were found to express higher-than-WT levels of viral proteins and mRNA, suggesting that the P/V mutations had disrupted the regulation of viral RNA synthesis. Despite the inability to target STAT1 for degradation, single-step growth assays showed that the rSV5-P/V-CPI- mutant virus grew better than WT SV5 in all cell lines tested. Unexpectedly, cells infected with rSV5-P/V-CPI- but not WT SV5 showed an activation of a reporter gene that was under control of the IFN-beta promoter. The secretion of IFN from cells infected with rSV5-P/V-CPI- but not WT SV5 was confirmed by a bioassay for IFN. The rSV5-P/V-CPI- mutant grew to higher titers than did WT rSV5 at early times in multistep growth assays. However, rSV5-P/V-CPI- growth quickly reached a final plateau while WT rSV5 continued to grow and produced a final titer higher than that of rSV5-P/V-CPI- by late times postinfection. In contrast to WT rSV5, infection of a variety of cell lines with rSV5-P/V-CPI- induced cell death pathways with characteristics of apoptosis. Our results confirm a role for the SV5 V protein in blocking IFN signaling but also suggest new roles for the P/V gene products in controlling viral gene expression, the induction of IFN-alpha/beta synthesis, and virus-induced apoptosis.

A Potyvirus Polymerase Interacts with the Viral Coat Protein and VPg in Yeast Cells

The two-hybrid system was used to test for pairwise interactions between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb–CP interaction but not the NIb–NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb–NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of viral RNA synthesis in infected cells. We also conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells.

Replicative events in hepatitis A virus-infected MRC-5 cells

Replication of hepatitis A virus (HAV) in MRC-5 cells was studied under one-step growth conditions. Viral replication neither interfered detestably with cellular DNA, RNA, and protein synthesis, nor could cytopathologic changes be recorded over a prolonged period of incubation. Synthesis of mature, infectious HAV particles could be detected as early as 2–4 days p.i. and occurred at a maximal rate around 8 days p.i., shortly before infectivity titers reached a plateau. Synthesis of total viral RNA, of positive-strand genomic RNA, of viral mRNA, as well as of negative-strand RNA followed the same pattern. By Day 14 p.i., when active HAV replication had developed into persistent infection, synthesis of viral RNA declined to background levels. The mechanism(s) guiding active HAV replication into a state of persistent infection could not be positively defined. Yet there exists the possibility that this is brought about by down regulation of viral RNA synthesis. Whether this is related to the appearance of a subgenomic viral RNA molecule about 2000 nucleotides in length and detected in association with ribosomes on Days 7 and 10 p.i. remains to be shown.

Temporal regulation of RNA synthesis of bovine coronavirus.

It has been shown with mouse hepatitis virus (MHV) and avian infectious bronchitis virus that all of the subgenomic mRNAs and genomic RNA are synthesized at the same rate throughout the viral replication cycle (Leibowitz et al., 1981; Stern and Kennedy, 1980). The viral genomic RNA gradually accumulates late in infection. However, no clear switching from transcription of subgenomic mRNAs to replication of genomic-sized RNA is seen. Thus, it is not known whether there is a temporal regulation of viral RNA synthesis in coronavirus-infected cells.

Dominance of the CPE(+) phenotype in hybrid Aedes albopictus cells infected with Sindbis virus

The effect of Sindbis virus (SV) infection was analyzed in hybrid Aedes albopictus cells formed by fusing ouabain-resistant CPE(+) cells to CPE(−) a-amanitin resistant cells. Although the 24-h yields of virus from the parental CPE(+) and CPE(−) clones were similar, the rates of viral RNA synthesis and virus release at early times post-infection were higher in the CPE(+) cells. In all eight hybrid clones studied, the CPE(+) phenotype was dominant. In addition, the kinetics of viral RNA synthesis and virus release in the hybrids closely resembled what was observed in the CPE(+) parent clone. These data indicate that both in the parental and in the hybrid cells, expression of SV-induced CPE is associated with a high level of viral RNA synthesis and a rapid production of virus during the early period after infection. It is suggested that CPE(+) cells contain some factor or activity which is lacking or less abundant in the CPE(−) cells and that this activity is important in the regulation of viral RNA synthesis.

Herpes simplex virus immediate early infected-cell polypeptide 4 binds to DNA and promotes transcription.

In herpes simplex virus (HSV)-infected cells, there is a sequential expression of viral genes. In vivo experiments have implicated the Mr 175,000 immediate early protein ICP4 (infected-cell polypeptide 4) in the regulation of viral RNA synthesis, but the mechanism whereby ICP4 regulates transcription of viral genes is at present unknown. In this report we describe experiments with an in vitro transcription system and a purified preparation of ICP4 (estimated 5% of total protein). Using DNA from the HSV glycoprotein D gene (gD) as the template, we have observed that specific binding occurs between ICP4 and DNA sequences adjacent to the gD gene promoter and ICP4 stimulates initiation of transcription from the gD gene. The degree of stimulation depends on the amount of ICP4 present in the incubation. The kinetics of RNA synthesis demonstrate that the protein acts at the initiation step of transcription. These results identify ICP4 as a viral transcription factor whose presence on DNA facilitates the formation of transcription complexes.

An alfalfa mosaic virus RNA 2 mutant, which does not induce a hypersensitive reaction in cowpea plants, is multiplied to a high concentration in cowpea protoplasts

A mutant of alfalfa mosaic virus (AMV), which in contrast to wild type (wt) can invade cowpea plants systemically, is replicated more efficiently in cowpea protoplasts than the wt. Mutant preparations isolated from infected cowpea protoplasts contained a higher amount of middle component (M, containing RNA 2) than wt preparations. Both in cowpea plants and in cowpea protoplasts a wt phenotype is obtained upon addition of wt M to this mutant, suggesting a correlation between the type of plant reaction evoked by the virus infection and the regulation of viral RNA synthesis.