Effects of Poliovirus 2A^pro on Vaccinia Virus Gene Expression

Abstract

The effects of transient expression of poliovirus 2Ap"' ori 11220 cleavage in COS cclls h:tve bccn analyzed. When 2AP"' was cloned in plasmid pTMl and transiently expressed in COS cclls, efficient cleavage of p220 occurred after infection of thesc cells with ii rccombinant vaccinia virus hearing phage T7 R N A polymerase. High numbers of COS cells were transfectcd with pTMf-2A, as ,judged hy 11220 cleavage, thereby allowing an analysis of the effects of poliovirus 2AI"" on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2Rl"" was observed ten hours post infcction and clcavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus, Profound inhibition of vaccinia virus protein synthesis was dctectable ten hours post infcction and was maximnl 20-25 hours post infection. This inhibition reriulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mKNAs present in cells that synthesize poliovirus 2A'"". Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthcsis of cellular proteins. Under these conditions, 2APrc* is expressed and blocks cellular translation. Finally, p220 cleavagc by 2APr" did not inhibit the translation of a mKNA encoding poliovirus protein 2C, as dirccted by the 5' leader sequences of encephalomiocarditis virus. Therefore, thcse findings show a corrclation between p220 cleavage and inhibition of translation from newly made mRNAs. Our rcsulis are discussed in the light of present knowledge of p220 function. and new approaches are considered that might pi-ovide furlher insights into the function(s) of initiation factor elI;-4F. Kc.yword.7: protease 2A : poliovirus; p220 cleavagc ; vaccinia virus.

A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2A pro : Effects on Cellular and Viral Gene Expression

“…By contrast, vaccinia virus replication was powerfully inhibited in 2A7d-On cells, in good agreement with the result showing that poliovirus 2A pro interferes with vaccinia virus translation (19). Although picornavirus translation in 2A7d cells was allowed, there was no stimulation of the total amount of viral protein synthesized, resulting in levels of virus production similar to those in 2A7d-Off cells.…”

“…Immunoblotting was carried out as described previously (6). The anti-2C (55), -2A and -3C (8), -eIF4GI (19), and -eIF4GII (23) sera were obtained and used as described previously. The anti-eIF4FG-2 antiserum was a generous gift from N. Sonenberg (McGill University).…”

“…Transient expression of poliovirus 2A pro has been achieved by infection with recombinant vaccinia virus that expresses the T7 RNA polymerase (2,3,19). Poliovirus 2A pro strongly inhibits vaccinia virus gene expression at the translational level (19). Microinjection of mRNA encoding coxsackievirus 2A pro in Xenopus oocytes leads to eIF4G cleavage and to strong inhibition of translation of luciferase mRNA, while ongoing cellular translation is much less affected (15).…”

“…Attempts to express 2A pro from recombinant vaccinia viruses have been hampered by 2A pro toxicity (30,62). Transient expression of poliovirus 2A pro has been achieved by infection with recombinant vaccinia virus that expresses the T7 RNA polymerase (2,3,19). Poliovirus 2A pro strongly inhibits vaccinia virus gene expression at the translational level (19).…”

Mutational Analysis of Poliovirus 2Apro

“…Further work analyzing the action of this protease on vaccinia virus gene expression indicated that 2A pro profoundly interferes with vaccinia virus replication, such that no viral protein synthesis is detected at late times of infection (48). Therefore, the action of the different 2A…”