Poliovirus mutant that does not selectively inhibit host cell protein synthesis.

Abstract

A poliovirus type I (Mahoney strain) mutant was obtained by inserting three base pairs into an infectious cDNA clone. The extra amino acid encoded by the insertion was in the amino-terminal (protein 8) portion of the P2 segment of the polyprotein. The mutant virus makes small plaques on HeLa and monkey kidney (CV-1) cells at all temperatures. It lost the ability to mediate the selective inhibition of host cell translation which ordinarily occurs in the first few hours after infection. As an apparent consequence, the mutant synthesizes far less protein than does wild-type virus. In mutant-infected CV-1 cells enough protein was produced to permit a normal course of RNA replication, but the yield of progeny virus was very low. In mutant-infected HeLa cells there was a premature cessation of both cellular and viral protein synthesis followed by a premature halt of viral RNA synthesis. This nonspecific translational inhibition was distinguishable from wild-type-mediated inhibition and did not appear to be part of an interferon or heat shock response. Because the mutant is recessive, our results imply that (at least in HeLa cells) wild-type poliovirus not only actively inhibits translation of cellular mRNAs, but also avoids early inhibition of its own protein synthesis. Cleavage of the cap-binding complex protein P220, which has been associated with the selective inhibition of capped mRNA translation, did not occur in mutant-infected cells. This result supports the hypothesis that cleavage of P220 plays an important role in normal poliovirus-mediated translational inhibition.It has long been recognized that infection of cells with poliovirus results in a selective inhibition of host cell protein synthesis (for a review, see reference 2). Translation of cellular mRNA steadily declines as virus-specific translation increases; by 2 to 3 h after infection viral RNA, which unlike most other translated eucaryotic RNAs lacks a 5' m7Gp-ppN(m) cap structure (8,28), is translated almost exclusively. Presumably, a viral product mediates this inhibition, but it has not been identified.Results of early studies have shown that translation of cellular mRNA is blocked at the initiation step (19). More specifically, the defective initiation factor appears to be a salt-stable complex composed of a cap-binding protein (CBP) and associated proteins. This complex is thought to be crucial for the attachment of capped mRNAs to 40S ribosomal subunits (for a review, see reference 37). Infected cell extracts do not support the translation of capped RNAs (7,34) or their attachment to the CBP complex (16), but both activities can be restored by the addition of CBP complex from uninfected cells (17, 41). In vivo, the decline in host protein synthesis is preceded by specific cleavage of a 220,000-dalton protein (P220) which is part of the CBP complex (6). It has been proposed that the complex is inactivated by this cleavage (6). A viral product is thought to carry out or promote the cleavage of P220, but the known viral protease (protein 7c) is pr...

Regulation of a double-stranded RNA modification activity in human cells.

“…This observation suggests that, far from being a component of the antiviral response in mammalian cells, the dsRNA modifier has a normal cellular function that is inhibited during the antiviral response. Consistent with this idea, the dsRNA modifier was also found to be inhibited during infection with poliovirus, a single-stranded RNA virus whose infection can lead to the accumulation of intracellular dsRNA (6,7,29). The dsRNA modifier was significantly less inhibited during infection with adenovirus, a virus whose virus-associated (VA) RNA products are known to subvert the cellular antiviral response (36).…”

“…Others have shown that some posttranslational components of the cellular response to poly(I -C) are stimulated during poliovirus infection, suggesting that sufficient dsRNA to activate these responses must be present. For 4 example, in HeLa cells infected with a poliovirus mutant defective in the inhibition of cellular translation, viral translation was found to be inhibited eventually by the cell (6). Furthermore, the activation and destabilization of the DAI has been observed during poliovirus infection (7,29).…”

Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex

“…The mutant virus, 3A-2, contains a single amino acid insertion in the 3A coding region (Bernstein et al, 1985). Mutant 3A-2 protein, when expressed in isolation, causes a reduced inhibition of ERto-Golgi traffic compared with wild-type 3A protein (Doedens et al, 1997) and cells infected with 3A-2 mutant virus secrete much higher levels of several cytokines than wild-type-infected cells (Dodd et al, 2001).…”

Mutational Analysis of Poliovirus 2Apro

“…In addition, 17 point mutations in 2A pro were obtained, some of them by site-directed mutagenesis (Y88S, Y88P, and Y88L) and the rest using a recently described genetic system to select cytotoxic-deficient 2A pro mutants in yeast cells (40). Mutant 2A-1 contains a leucine insertion at position 103 as described (44 (Fig. 2).…”