Nontranslated cellular mRNAs are associated with the cytoskeletal framework in influenza virus or adenovirus infected cells

In an effort to understand the molecular mechanisms underlying the selective shutoff of host protein synthesis in influenza virus and adenovirus infected cells, we analyzed the subcellular location of representative cellular and viral mRNAs. Earlier work has shown that the majority of cellular mRNAs remain polysome associated after infection by either virus and that both the initiation and elongation steps of host protein synthesis were blocked in infected cells (M. G. Katze, D. DeCorato, and R. M. Krug, J. Virol., 60, 1027-1039, 1986). The present study was undertaken to test whether these cellular mRNAs were rendered nontranslatable during infection as a result of their dissociation from the cytoskeleton framework. HeLa cells were fractionated into subcellular components by first gently disrupting the cells with Triton X-100 yielding the soluble fraction (SOL); the cytoskeleton (CSK) fraction was obtained from the Triton insoluble material by the double detergent treatment of Tween-40 and sodium deoxycholate. In uninfected cells the majority of host mRNAs were associated with polysomes which were exclusively bound to the CSK as would be expected of actively translated mRNAs. The cellular mRNAs also remained almost totally associated with the cytoskeleton in adenovirus and influenza virus infected cells despite the fact that these mRNAs are not translated during infection. Indeed, the host mRNAs and the efficiently translated viral mRNAs were CSK associated to the same extent. In contrast to the adenovirus and influenza systems, significant amounts of cellular mRNAs were dissociated from the CSK and found in the SOL fraction of poliovirus infected cells as others have reported. In accordance with the biochemical analysis, morphological studies utilizing electron microscopy revealed that the cytoskeleton remained relatively intact during adenovirus and influenza infection but was substantially reorganized in poliovirus infected cells. We conclude that translational regulatory events are likely different in the poliovirus system and that cytoskeletal association of mRNAs may be required but is not sufficient for efficient mRNA translation during adenovirus or influenza virus infection.     

Functional impairment of eIF4A and eIF4G factors correlates with inhibition of influenza virus mRNA translation

Influenza virus mRNAs contain a 5′-cap structure followed by short cell-derived heterogeneous oligonucleotides and they are polyadenylated. However, selective translation of viral mRNAs occurs upon infection. Thus, we have studied whether differential requirements for the eIF4F components on viral and cellular translation could mediate this selectivity. We have previously reported that influenza virus infection proceeds efficiently upon functional impairment of the cap-binding factor eIF4E. Now, the requirements for the eIF4A helicase and the eIF4G scaffolding factor have been examined. The two proteins are essential for viral translation both in in vivo and in vitro analysis. Consequently, viral mRNAs do not contain cis-acting signals that could mediate eIF4A and eIF4G independence and trans-acting viral proteins do not replace their function. Thus, eIF4A and eIF4G proteins are not responsible for the selective translation of viral mRNAs and the translational shut-off of cellular protein synthesis observed in influenza virus infected cells.     

A protein synthesizing system from interferon-treated cells that discriminates between cellular and viral messenger RNAs

The effect of treating Krebs II ascites tumor cells with interferon on the ability of extracts prepared from them to catalyze the translation of a variety of messenger RNAs was investigated. The following results were obtained: (1) Extracts of untreated cells translated endogenous mRNA, as well as exogenously added synthetic mRNA [poly(U)], cellular mRNA (Krebs cell and L cell), and viral mRNA (reovirus and vaccinia virus). By contrast, extracts of Krebs cells treated in the ascitic form with interferon translated endogenous mRNA, exogenously added cellular mRNA and poly(U) as efficiently as extracts of untreated cells, but they translated viral mRNAs very poorly (less than 10% as efficiently as extracts of untreated cells). Thus, the ability to discriminate between cellular and viral mRNAs that is characteristic of whole cells was also exhibited by these cell-free extracts. (2) Virus infection was not required for the development of the interferon-induced inhibition of viral mRNA translation. (3) Mixing experiments indicated that the inability of extracts of interferon-treated cells to catalyze the translation of viral mRNAs was due to the presence of an inhibitory factor (s) rather than to the absence of a required factor (s). (4) The inhibitory factor (s) was associated with ribosomes, and could be dissociated from them by washing with KC1. (5) Polyacrylamide gel electrophoresis revealed the presence of a 48,000 dalton polypeptide in the 0.3–0.6 M KC1 wash fraction of ribosomes prepared from interferon-treated cells that was not detectable in the corresponding wash of ribosomes prepared from untreated cells. This fraction inhibited the translation of viral mRNAs in cell-free extracts of untreated cells more than any other salt wash fraction.These results suggest that the antiviral activity of interferon is mediated, at least in part, by a ribosome-associated polypeptide that permits discrimination between cellular and viral mRNAs.     

Influenza virus polymerase confers independence of the cellular cap-binding factor eIF4E for viral mRNA translation

The influenza virus mRNAs are structurally similar to cellular mRNAs nevertheless; the virus promotes selective translation of viral mRNAs despite the inhibition of host cell protein synthesis. The infection proceeds normally upon functional impairment of eIF4E cap-binding protein, but requires functional eIF4A helicase and eIF4G factor. Here, we have studied whether the presence of cis elements in viral mRNAs or the action of viral proteins is responsible for this eIF4E-independence. The eIF4E protein is required for viral mRNA translation in vitro, indicating that cis-acting RNA sequences are not involved in this process. We also show that PB2 viral polymerase subunit interacts with the eIF4G protein. In addition, a chimeric mRNA containing viral UTR sequences transcribed by the viral polymerase out of the infection is successfully translated independently of an impaired eIF4E factor. These data support that the viral polymerase is responsible for the eIF4E independence of influenza virus mRNA translation.     

Phosphorylation and dephosphorylation events that regulate viral mRNA translation

As they are completely dependent upon the protein synthesis machinery resident in the cells of their host to translate their mRNAs, it is imperative that viruses are able to effectively manipulate the elaborate cellular regulatory network that controls translation. Indeed, this exquisite dependence on host functions has made viral models attractive systems to explore translational regulatory mechanisms operative in eukaryotic cells. Central among these are an intricate array of phosphorylation and dephosphorylation events that have far reaching consequences on the activity of cellular translation factors. Not only do these modulate the activity of a given factor, but they can also determine if the translation of host proteins persists in infected cells, the efficiency with which viral mRNAs are translated and the outcome of a systemic host anti-viral response. In this review, we discuss how various viruses manipulate the phosphorylation state of key cellular translation factors, illustrating the critical nature these interactions play in virus replication, pathogenesis and innate host defense.     

Poliovirus protease 2A is required for interference with vesicular stomatitis virus-specified protein synthesis

Summary A subunit of eukaryotic initiation factor-4F (eIF-4F) which is a component of the protein complex which binds to the methylated cap structure at the 5 end of most cellular mRNAs, is proteolytically cleaved in poliovirus-infected cells resulting in the shutoff of cellular protein synthesis. Poliovirus mRNA is selectively translated in infected cells, in part, because translation of the uncapped viral mRNA does not require an intact cap binding protein complex. Wild-type poliovirus also inhibits the translation of vesicular stomatitis virus (VSV) mRNAs in coinfected cells, however, it has been unclear whether similar mechanisms are employed by poliovirus to interfere with cellular and VSV protein synthesis. Degradation of eIF-4F appears to be an indirect function of the poliovirus-encoded protease 2A. A poliovirus mutant in 2A failed to mediate eIF-4F cleavage and selectively terminate translation of capped cellular mRNAs. Unlike wild-type poliovirus, 2A-1 does not interfere with VSV-specified protein synthesis. These results indicate that the same viral protein, 2A protease, is required not only to effectively terminate host protein synthesis, but also to interfere with expression of a heterologous virus, VSV. In addition, 2A-1 specifies a function, heretofore undescribed for poliovirus, which interferes with VSV-induced shutoff of protein synthesis.     

Biochemical characterization of vesicular stomatitis virus-infected Aedes albopictus cells deprived of methionine

Aedes albopictus cells infected with vesicular stomatitis virus (VSV) and maintained in medium lacking methionine produced 1000-fold less infectious virus than cultures maintained in complete medium. Analysis of viral macromolecular synthesis in cells maintained in the presence of varying concentrations of methionine showed that the reduction in virus yield was directly correlated with a reduction in viral RNA and protein synthesis. Of the viral mRNA which was made in methionine-starved cells the majority was not polysome associated. In contrast, virtually all of the virus mRNA in cells maintained in complete medium was polysome associated. In vitro translation of those mRNAs from methionine-starved cells, which were not polysome associated, indicated that they could be translated in vitro as efficiently as polysome-associated virus mRNA but only if the methylation inhibitor, S-adenosylhomocysteine, was not present. These results strongly suggest that methionine starvation of A. albopictus cells inhibits VSV replication by preventing cap methylation of the viral mRNAs, and thus reducing the efficiency with which they are translanted.     

Polyadenylated RNA sequences produced in vaccinia virus-infected cells under aberrant conditions inhibit protein synthesis in vitro.

We have previously demonstrated that small nontranslated polyadenylated RNAs (POLADS) produced in vaccinia virus (VV)-infected cells inhibit the translation of cellular mRNAs, but minimally affect the translation of VV mRNAs in a cell-free protein synthesizing system. Infection of HeLa cells with ultraviolet-irradiated vaccinia virus or infection in the presence of actinomycin D (ACD) amplifies the synthesis of POLADS compared to the amount produced in cells infected under normal conditions. The effect of these POLADS on translation was studied in the reticulocyte lysate system. Polyadenylated RNAs isolated from cells infected with wild-type virus (V-POLADS) had a greater inhibitory effect on HeLa cell protein synthesis than on VV protein synthesis. Polyadenylated sequences obtained from cells infected with ultraviolet-irradiated virus (UV-POLADS) or from cells infected in the presence of ACD (ACD-POLADS), however, inhibited translation of both HeLa and viral mRNAs. Ultraviolet-POLADS and ACD-POLADS were found to possess, on average, longer poly(A) tails than V-POLADS. The inhibition of translation of both host and viral mRNAs effected by V-POLADS, UV-POLADS, and ACD-POLADS was reversed by poly(A) binding protein.     

On the regulation of protein synthesis in vaccinia virus infected cells.

All eukaryotic mRNA species show a characteristic individual translational efficiency under conditions of restricted polypeptide chain initiation caused by an increase in the osmolarity of the growth medium. In vaccinia virus infected L cells or HeLa cells virus mRNAs can be grouped into classes on the basis of their relative labelling under standard and hypertonic conditions. Under the latter conditions, most of the "early" mRNAs possess very high translational efficiencies, most of the "intermediate" mRNAs show an intermediate efficiency and the most prominent "late" mRNAs show a translational efficiency which is lower than that of other virus mRNAs but still higher than the average cellular mRNA. Late in the infection cycle virus mRNAs with a relative low translational efficiency are preferentially translated under standard growth conditions whereas "early" virus mRNAs which are still present and which show a higher translational resistance to hypertonic conditions are not translated. These results indicate a unique translational control operating late in the growth cycle of vaccinia virus.     

Nuclear export of influenza viral ribonucleoprotein is temperature-dependently inhibited by dissociation of viral matrix protein

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.     

The relative translation efficiencies of reovirus messenger RNAs

The relative translation efficiencies of reovirus messenger RNAs were measured by infecting BHK cells with serotype 1, 2, or 3 virus and measuring the rate of formation of each viral protein species and the amount of each viral single-stranded (ss) RNA species present in the infected cells throughout the multiplication cycle. The translation efficiency of each ssRNA species was then calculated as a percentage of the species that was translated most efficiently. The relative translation efficiencies of the various ssRNA species did not change significantly during the multiplication cycle and cognate mRNA species of serotype 1, 2, and 3 virus were generally translated with similar efficiencies. However, the relative translation efficiencies of the individual ssRNA species differed greatly. The most efficiently translated ssRNA species was usually species s4, followed by species m2 which, on average, was translated about two-thirds as efficiently as species s4. Species s2 and s3 were translated slightly less than one-half as efficiently as species s4; species m3, l2, and l3 one-quarter to one-third as efficiently; and species s1 one-twentieth to one-tenth as efficiently. Finally, species l1 and m1 were translated very inefficiently under all conditions; their translation efficiencies were usually no more than 1% of that of species s4.     

Positive and negative effects on translation of the hepatitis C virus 3' untranslated region

OBJECTIVES: To determine the role of the hepatitis C virus 3' untranslated region in viral mRNA translation in transfected cells and in cell extracts. STUDY DESIGN/METHODS: Noninfectious hepatitis C virus mini-genome RNAs with various deletions in the viral 3' untranslated region were transfected into cells or translated in vitro, and the translation efficiency was determined. RESULTS: We have found that the presence of the hepatitis C virus 3' untranslated region modestly increases mRNA translation. The positive effect correlated with the binding of a 45-kDa cytoplasmic factor to the hepatitis C virus 3' untranslated region. Furthermore, the U-rich sequence in the hepatitis C virus 3' untranslated region inhibits translation of capped and polyadenylated mRNAs as a result of the hybridization. CONCLUSIONS: The modest effect of the hepatitis C virus 3' untranslated region on translation suggests that it does not play a major role in mRNA translation. The inhibitory effect of the hepatitis C virus 3' untranslated region on translation of polyadenylated mRNAs supports the notion that translation of hepatitis C virus mRNAs occurs independently of a polyA tail.