HIV-1 transcripts use IRES-initiation under conditions where Cap-dependent translation is restricted by poliovirus 2A protease.

Raquel Amorim,Sara Mesquita Costa,Luciana Jesus Da Costa,Edson Elias Da Silva,Nathalia Pereira Cavaleiro,Jean-Luc Eph Darlix

doi:10.1371/journal.pone.0088619

Raquel Amorim, Sara Mesquita Costa + Show 4 more

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https://doi.org/10.1371/journal.pone.0088619

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Abstract

The 30 different species of mRNAs synthesized during the HIV-1 replication cycle are all capped and polyadenilated. Internal ribosome entry sites have been recognized in the 5′ untranslated region of some mRNA species of HIV-1, which would contribute to an alternative mechanism of initiation of mRNA translation. However, the Cap-dependent translation is assumed to be the main mechanism driving the initiation of HIV-1 protein synthesis. In this work, we describe a cell system in which lower to higher levels of transient expression of the poliovirus 2A protease strongly inhibited cellular Cap-dependent translation with no toxic effect to the cells during a 72-hour time frame. In this system, the synthesis of HIV-1 proteins was inhibited in a temporal dose-dependent way. Higher levels of 2A protease expression severely inhibited HIV-1 protein synthesis during the first 24 hours of infection consequently inhibiting viral production and infectivity. Intermediate to lower levels of 2A Protease expression caused the inhibition of viral protein synthesis only during the first 48 hours of viral replication. After this period both protein synthesis and viral release were recovered to the control levels. However, the infectivity of viral progeny was still partially inhibited. These results indicate that two mechanisms of mRNA translation initiation contribute to the synthesis of HIV-1 proteins; during the first 24–48 hours of viral replication HIV-1 protein synthesis is strongly dependent on Cap-initiation, while at later time points IRES-driven translation initiation is sufficient to produce high amounts of viral particles.

Highlights

Translation in eukaryotic cells is mainly initiated via two mechanisms: one involves the recognition and association of several eukaryotic initiation factors to the Cap structure present at the 59 end of all eukaryotic messenger RNAs; the second mechanism does not rely on the recognition of Cap but on the association of a limited number of eIFs to specific regions of highly structured 59 untranslated regions (UTR) of mRNAs, called internal ribosome entry sites (IRES)
Since we demonstrated that transfection of Poliovirus 2A protease (2APro) leads to the inhibition of de novo protein synthesis in HeLa cells, the presence of eIF4G-I was evaluated in these cells up to 72 hours posttransfection of 2APro
It was less pronounced when compared to cells co-expressing 2APro. These results demonstrate that the recovery in HIV-1 protein synthesis at later times of 2APro expression occurred even with the maintenance of the inhibitory effect imposed on the cellular capdepended mRNA translation

Summary

Introduction

Translation in eukaryotic cells is mainly initiated via two mechanisms: one involves the recognition and association of several eukaryotic initiation factors (eIFs) to the Cap structure present at the 59 end of all eukaryotic messenger RNAs (mRNAs); the second mechanism does not rely on the recognition of Cap but on the association of a limited number of eIFs to specific regions of highly structured 59 untranslated regions (UTR) of mRNAs, called internal ribosome entry sites (IRES). IRES-dependent translation occurs for certain mammalian mRNAs under certain metabolic conditions [1]. Viruses must use the cellular machinery to synthesize their own proteins, as this process is highly complex and involves several components that are not encoded by the viral genomes. Especially for highly cytolytic RNA viruses, viral and host mRNAs compete for the translation machinery components. Animal viruses have evolved sophisticated mechanisms to maximize the selective translation of their own mRNAs [2]. As initiation of mRNA translation is critical to ensure the synthesis of all eukaryotic proteins, and is a tightly regulated step, in order to ensure synthesis of their own proteins, viruses frequently target this step [3]

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