Preferential translation of Vesicular Stomatitis Virus (VSV) mRNAs

Source:

Whitlow ZW, Connor JH, Lyles DS. New mRNAs are preferentially translated

during vesicular stomatitis virus infection. J Virol. 2008 Mar;82(5):2286-94.

Epub 2007 Dec 19. PubMed PMID: 18094194; PubMed Central PMCID: PMC2258916.

 

 

Introduction:

Vesicular Stomatitis Virus (VSV) is a virus that belongs to the same family of viruses to which Rabies virus belong, i.e. Rhabdoviridae. The infections occur in two steps, cytolytic infections of the mammals and transmission by insects. It is a negative sense single stranded RNA virus. The virus replicates inside the cytoplasm of infected cells and viral mRNAs are transcribed from the viral genome by viral RNA dependent RNA polymerase (RDRP). These mRNAs are quite similar in structure to host mRNAs. The translation of viral mRNA is dependent on the host cell machinery. Most of the viruses have developed mechanisms that inhibit host protein synthesis while viral mRNAs are preferentially translated. A lot of research has been going on to understand the mechanisms behind preferential translation of viral mRNAs. Knowledge of these mechanisms is essential for understanding viral replication.

Earlier studies on VSV have shown that the matrix (M) protein inhibits host gene expression at multiple levels. These include transcription, transport of mRNA into the cytoplasm, and translation. Studies have also shown that in cells infected with VSV, viral protein synthesis increases while host protein synthesis decreases. Thus, in the present study, the authors sought to determine why viral mRNAs are translated during the time that translation of host mRNAs is inhibited.

Results:

• mRNA transfected during infection is resistant VSV-induced translation inhibition, while mRNA transfected before infection is translationally inhibited by VSV : As it is known that during VSV infection, there is inhibition of host transcription and mRNA transport, which prevents new host mRNAs from reaching the cytoplasm and viral transcription is the primary source of mRNA in the cytoplasm. At the same time, there is inhibition of host translation and the predominance of viral translation. Based on these observations the authors speculated that the timing of transcription may be involved in the control of translation in VSV infected cells. They speculated that most recently synthesized mRNA might be translated preferentially. Thus to evaluate this hypothesis, the authors examined whether the time of appearance of mRNA in the cytoplasm relative to the time of infection was involved in controlling translation by trasfecting HELA cells with in-vitro transcribed EGFP reporter mRNAs at different time points relative to time of infection. Thus, HELA cells were transfected with SP6-EGFP mRNA and translation rates at various time points after infection were determined by pulse labeling with S35methionine. EGFP synthesis was determined by immunoprecipitation and measurement of labeled EGFP by SDS-PAGE and phosphorimaging. They observed that the minimum time required for transfected SP6-EGFP mRNA to be translated optimally is around 4h. In the next set of experiments, HeLa cells were trasfected with SP6-EGFP mRNA 22hrs before infection, 4h before infection or 1h after infection with a recombinant VSV expressing a wild type M protein. They observed that in recombinant wild type virus infected cells that were transfected 22 or 4h before infection, EGFP was synthesized at much lower rates in comparison to mock-infected cells. In cells in which SP6-EFGP mRNA was transfected at 1h post infection, EGFP was synthesized at much higher rates than in mock infected cells. These results clearly show that mRNA transfected during infection is resistant to VSV-induced inhibition, while mRNA transfected before infection is translationally inhibited by VSV.
  
• Transfection has no effect on viral protein synthesis: The authors also analyzed total cellular protein synthesis by SDS-PAGE and phosphorimaging in total cell lysates of cells that were transfected 22h before infection or of untrasfected cells that were subsequently mock infected or infected with rwt virus. Transfection of SP6-EGFP mRNA slightly reduced levels of total protein synthesis in comparison to control and this reduction was independent of time of infection. They observed that Inhibition of total protein synthesis by rwt virus was similar for all three times of transfection.
  
• mRNAs introduced into the cytoplasm during infection or close to the time of infection are resistant to VSV induced inhibition of translation: Furthermore, rates of SP6-EGFP mRNA protein synthesis and total host protein synthesis relative to those in mock infected cells were determined. When cells were transfected 22h before infection, the production of SP6-EGFP was reduced to less than half of that in mock-infected cells. There was a similar reduction in total host protein synthesis. When cells were transfected 4h before infection, the translation was inhibited but not as much as observed in case of cells transfected 22h before infection. In contrast, when cells were transfected 1h after infection, EGFP was translated almost two fold better in cells infected with rwt virus than in mock-infected cells. Also, the translation rate of EGFP relative to mock infected cells was significantly different from translation rate of total host protein synthesis in this case.
  
• The ability of VSV to inhibit host translation is separated from its ability to promote translation of viral mRNAs: To prove this point the authors determined translation efficiencies of host-derived and virus-derived EGFP mRNAs at different time points after infection with virus containing wild type or mutant M protein (not able to inhibit host protein synthesis). For this they infected HeLA-EGFP (host derived mRNA) with mock or rwt or M51R-M virus and HeLa cells with mock or rwt-EGFP or M51R-M-EGFP virus (virus-derived mRNA). EGFP was analyzed at 4, 8 or 12h post infection. The data obtained from these experiments showed that EGFP translation from HeLa-EGFP cells was inhibited to a greater extent following infection with rwt virus than with rM51R-M virus. In case of HeLa cells, infection with rwt-EFGP virus caused increase in EGFP synthesis from 4 to 8h but decrease by 12h post infection. Infection with rM51R-M-EGFP caused EGFP synthesis similar to that observed in HeLa cells infected with rwt-EGFP virus, although EGFP remained elevated at 12h post infection. The authors further determined the mRNA levels by Northern Blotting. In HeLa-EGFP cells infected with rwt virus, the level of EGFP mRNA was not significantly different from the level observed in mock infected cells until 12h post infection. After 12h, EGFP mRNA level in rwt-infected cells was reduced to 0.28 relative to mock infected cells. In HeLa-EGFP cells infected with rM51R-M virus, no difference in EGFP mRNA levels was observed in comparison to control at any time points. In HeLa cells infected with rwt-EGFP virus, the EGFP mRNA levels were 10 to 12 times to that observed in mock infected cells and were consistent throughout the time course. Translation efficiencies were determined by dividing the EGFP translation rates by the EGFP mRNA levels. Infection of HeLa-EGFP cells by rwt virus caused dramatic reduction in translation efficiency in comparison t controls 12 h post infection. Infection with rM51R-M also reduced translation efficiency, although to a lesser extent in these cells. In contrast, infection with of HeLa cells with rwt-EGFP and rM51R-M-EGFP, translation efficiency was found to be increased in comparison to controls. These results indicate that inhibition of host translation does not affect the translation efficiency of viral mRNAs before 8 h post infection and thus the ability of VSV to inhibit host translation is separated from its ability to promote viral translation.
  

Important Findings:

1. The authors show that newly appearing mRNAs are resistant to VSV induced inhibition of translation
   
2. Efficient translation of viral mRNAs or other newly appearing mRNAs is not dependent on inhibition of host translation
   
3. The increase in translational efficiency of viral mRNAs between 4 to 8h post infection is not due to inherent changes in mRNA translatability over time
   

Future Aspects: It seems that VSV and perhaps other viruses exploit the phenomena of preference of newly synthesized mRNA by translation machinery. The mechanisms that are responsible for the preferential translation of newly transcribed mRNAs in VSV infected cells are not known. During certain type of stress responses, preexisting mRNAs are shuttled into stress granules, while newly synthesized mRNAs are preferentially translated. These similarities can be exploited to understand the mechanisms responsible for such preferential translation.