Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.     

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.     

Regulation of Protein Synthesis in HeLa Cells: III. Inhibition During Poliovirus Infection

The mechanism of inhibition of host cell protein synthesis by poliovirus has been studied by examining the behavior of host polyribosomes and polyribosome-associated messenger ribonucleic acid (mRNA). Virus infection appears to result in a specific inhibition of the initiation of translation of host cell mRNA. The site of inhibition does not appear to be mRNA itself. Human cells respond to virus infection by producing a factor antagonistic to the virus inhibition which promotes the initiation of host mRNA translation. The production of the host factor is sensitive to actinomycin; however, the behavior of the host cell factor and that of host mRNA appear distinctly different.     

Inhibition of protein synthesis in vesicular stomatitis virus infected Chinese hamster ovary cells: role of virus mRNA-ribonucleoprotein particle

Although host protein synthesis is preferentially inhibited, there is a steady decline in the ability of Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) to synthesize both host and viral proteins. We previously reported finding an mRNA-ribonucleoprotein particle (mRNP) that contained all five VSV mRNAs and viral N protein exclusively. This particle apparently regulates translation by sequestering a majority of the VSV mRNA made late in infection and thus rendering it unavailable for protein synthesis. In the present investigation the mRNP was also shown to inhibit in vitro protein synthesis in rabbit reticulocyte and wheat germ lysates programmed with mRNA isolated from VSV-infected cells. The synthesis of eIF-2 X GTP X Met-tRNA (ternary) complex, the first step in initiation of protein synthesis, was markedly inhibited by the mRNP. The inhibition was partially reversed by addition of purified eIF-2 to the inhibited lysate or ternary complex formation reaction. These results indicate a dual role of the mRNP in regulating protein synthesis during infection. Nucleocapsid also inhibited in vitro protein synthesis, although this inhibition was not reversed by eIF-2. Nucleocapsid did not inhibit ternary complex formation in vitro. Consequently, nucleocapsid may also regulate in vivo protein synthesis, but by a mechanism different from the mRNP.     

La Crosse virions contain a primer-stimulated RNA polymerase and a methylated cap-dependent endonuclease.

Uncapped reovirus mRNA extracted at late times from infected L-cells is preferentially translated in extracts from infected L-cells. However, translation of this uncapped, late, reovirus mRNA in extracts from infected cells is sensitive to inhibition by the cap analog m7GTP . These results imply that reovirus infection does not induce a transition from cap-dependent to cap-independent translation. Nevertheless, the results of in vitro translational competition experiments between L-cell mRNA and late viral mRNA were consistent with the view that reovirus does induce an alteration in the cap-dependent translational apparatus of L-cells. The reduced efficiency of translation of a variety of capped mRNAs in extracts from infected cells is also consistent with this notion. We further conclude that a factor exists in reovirus-infected L-cells that specifically stimulates translation of uncapped reovirus mRNAs.     

Host-Dependent Restriction of Mengovirus Replication

Mengovirus infection of a restrictive cell line, Maden's bovine kidney (MDBK), results in a virus yield 1,000-fold less than that obtained from productively infected cell lines such as L cells or Ehrlich ascites tumor cells (EAT). Cells of both types of host systems are infected with comparable efficiencies and are completely killed as a consequence of infection. Infective center assays, coupled with the observation of total cell killing, suggest that comparable numbers of cells synthesize viral antigen and release virus in both types of host system. Viral-specific ribonucleic acid (RNA) synthesis is initiated and proceeds in an identical fashion for approximately 4 hr after the infection of MDBK, EAT, or L-cells. At this time, viral RNA synthesis in MDBK ceases, whereas viral RNA synthesis in EAT and L-cells continues at a linear rate. These results indicate that none of the early viral events leading to the initiation of viral-specific RNA synthesis constitutes the primary site of mengovirus restriction in MDBK. Rather it appears that the cessation of viral RNA synthesis in restrictive cells constitutes the primary limiting event. Based on its delayed interaction with mengovirus RNA synthesis, it appears that the host-related restrictive agent is initially compartmentalized and then released as a consequence of infection subsequent to those early events in mengovirus infection leading to the initiation and continued synthesis of viral RNA.     

Adenovirus inhibition of cellular protein synthesis involves inactivation of cap-binding protein.

Adenovirus (Ad) infection results in a marked inhibition of cellular protein synthesis that initiates during the late phase of the viral infectious cycle. We show that the mechanism used for suppression of cellular protein synthesis during cell cycle progression is exploited by Ad to repress host and enhance late viral mRNA translation. Discrimination between cellular and late Ad mRNAs and inhibition of host protein synthesis are shown to involve viral-mediated underphosphorylation of cap-binding protein (CBP) and subsequent inactivation of CBP complex, a large enzymatic complex required for cap-dependent mRNA translation. Late Ad mRNAs, like those of poliovirus, possess the unique ability to translate independent of a normal cap recognition process and do not require the activity of CBP complex. Inhibition of cellular translation by these two viruses is quite similar, except that whereas CBP complex is proteolytically degraded by poliovirus, it is functionally inactivated by Ad.     

Inhibition of Host Protein Synthesis in Type 5 Adenovirus-infected Cells

The effect of type 5 adenovirus infection on the synthesis of host-cell proteins by suspension cultures of KB cells was investigated. Although total protein synthesis continued at a constant rate for approximately 36 hr, net synthesis of five host enzymes (lactic dehydrogenase, acid phosphatase, deoxyribonuclease, fumarase, and phosphoglucose isomerase) was found to stop 16 to 20 hr after infection. The synthesis of alkaline phosphatase stopped 9 to 12 hr after infection. The inhibition of host protein synthesis occurred shortly after the synthesis of viral antigens had begun, accounting for the continued synthesis of total protein. An investigation of the relationship between synthesis of viral antigens and inhibition of host protein synthesis yielded results which suggest that the two processes are in some way coupled.     

Human rhinovirus 14 infection of HeLa cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mRNA translation in vitro.

One of the characteristics of picornavirus infection of cells in tissue culture is a specific inhibition of utilization of host cell mRNA for protein synthesis. In this study we show that human rhinovirus 14 is similar to poliovirus in that the inhibition of host cell translation that occurs during infection correlates with the proteolytic cleavage of an Mr 220,000 subunit of the cap-binding protein complex.     

Inhibition of host cell protein synthesis by UV-inactivated poliovirus.

The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.     

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells

The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.