The 3′ Untranslated Region of Sindbis Virus Represses Deadenylation of Viral Transcripts in Mosquito and Mammalian Cells

Abstract

The positive-sense transcripts of Sindbis virus (SINV) resemble cellular mRNAs in that they possess a 5 cap and a 3 poly(A) tail. It is likely, therefore, that SINV RNAs must successfully overcome the cytoplasmic mRNA decay machinery of the cell in order to establish an efficient, productive infection. In this study, we have taken advantage of a temperature-sensitive polymerase to shut off viral transcription, and we demonstrate that SINV RNAs are subject to decay during a viral infection in both C6/36 (Aedes albopictus) and baby hamster kidney cells. Interestingly, in contrast to most cellular mRNAs, the decay of SINV RNAs was not initiated by poly(A) tail shortening in either cell line except when most of the 3 untranslated region (UTR) was deleted from the virus. This block in deadenylation of viral transcripts was recapitulated in vitro using C6/36 mosquito cell cytoplasmic extracts. Two distinct regions of the 319-base SINV 3 UTR, the repeat sequence elements and a U-rich domain, were shown to be responsible for mediating the repression of deadenylation of viral mRNAs. Through competition studies performed in parallel with UV cross-linking and functional assays, mosquito cell factors-including a 38-kDa protein-were implicated in the repression of deadenylation mediated by the SINV 3 UTR. This same 38-kDa protein was also implicated in mediating the repression of deadenylation by the 3 UTR of another alphavirus, Venezuelan equine encephalitis virus. In summary, these data provide clear evidence that SINV transcripts do indeed interface with the cellular mRNA decay machinery during an infection and that the virus has evolved a way to avoid the major deadenylation-dependent pathway of mRNA decay.The Alphavirus genus of the Togaviridae family consists primarily of a group of viruses with single-stranded, positive-sense RNA genomes that are transmitted by arthropods and replicate exclusively within the cytoplasm of infected cells (45, 51). Sindbis virus (SINV), the prototypic virus of the genus, encodes genomic and subgenomic RNAs that are capped on the 5Ј terminus, polyadenylated at the 3Ј end, and contain 5Ј and 3Ј untranslated regions (UTRs) similar to cellular mRNAs. The viral genomic RNA, in fact, functions as an mRNA immediately upon infection. This strategy of mimicking cellular mRNAs has potential advantages and disadvantages for the virus. While this strategy allows viral RNAs to be effectively translated, viral transcripts may also be fully subject to the activity of the mRNA decay machinery within the cytoplasm of the host cell. Aspects of this anticipated interplay between SINV transcripts and cellular mRNA decay enzymes were investigated in this study.

Noncapped Alphavirus Genomic RNAs and Their Role during Infection

“…Previous characterizations of viral RNA half-lives have used either temperature-sensitive mutants or chemical inhibitors of transcription (27,28). Either of these options is likely to have effects on the host cell environment that may prove detrimental to the determination of the viral RNA half-life.…”

“…The unbound RNAs were then phenol extracted and ethanol precipitated prior to use as a template for cDNA synthesis via RT with Random Hexamer. The resulting cDNAs were assayed via qRT-PCR to determine the relative abundances of the incoming SINV genomic RNAs normalized to the cellular 18S rRNA as previously described (27,28). RNA half-lives were calculated via nonlinear regression.…”

Poliovirus 2A Pro Increases Viral mRNA and Polysome Stability Coordinately in Time with Cleavage of eIF4G

“…We and others (11) postulate that positive-strand RNA viruses likely evolved mechanisms to uncouple the translation of their viral mRNA from host mRNA turnover pathways be- cause the viral mRNA goes on to become the template for RNA replication (34). Degradation of viral mRNA by host mRNA turnover machinery would preclude subsequent viral RNA replication.…”

“…If mRNAs with 5Ј-3Ј interactions mediated by eIF4E, eIF4G NH , and PABP are specifically recognized by components of host mRNA turnover machinery, then perhaps the strategy of HCV to translate in an eIF4E-and eIF4G-independent manner might exist, in part, to evade 5Ј exonuclease. Intriguingly, even 5Ј capped and polyadenylated positive-strand RNA viruses appear to encode mechanisms to evade host mRNA turnover machinery (11). Elucidating the alternate mechanisms by which positive-strand RNA viruses evade host mRNA turnover may help reveal how normal cellular mRNA turnover is regulated.…”

ZAP-mediated mRNA degradation

“…The mechanisms by which viruses protect their mRNAs from general RNA decay have been previously reviewed. [18][19][20] For viruses to escape ZAP-specific viral mRNA degradation, one intriguing possibility is that viruses might encode factors that either inactivate ZAP or block ZAP-mediated RNA degradation. An analogous example is that many viruses encode suppressors of RNA silencing (SRS) 21 to protect the virus from inhibition by host RNA silencing.…”