Vesicular stomatitis virus (VSV) uses an unconventional mechanism for translation of its mRNA. All viruses are entirely dependent on their hosts to carry out protein synthesis, and VSV is no exception. To provide itself access to the cellular translational machinery, VSV transcribes its negative-sense genome using its own polymerase into 5? capped, methylated, and polyadenylated mRNAs, making them indistinguishable from cellular RNAs. Thus, without any additional regulatory elements, they should also be subject to regulation of translation and protein synthesis by the host. However, despite the fact that VSV infection causes host translational shutdown, viral translation continues robustly. VSV infection results in the sequestration of eIF4E, which normally recognizes and binds to capped mRNAs as the rate-limiting step in initiating translation. Yet VSV translation is completely insensitive to the presence of eIF4E. How the capped viral RNAs continue to be translated under these circumstances remains unknown. However, recent studies have elucidated new mechanisms of translational control in the cell, allowing the possible reinterpretation of previous work done on VSV. It was established in the 1970s that, in addition to a 5? methylated mRNA cap, VSV mRNAs also contained an N6-methyladenosine (m6A) modification at the first transcribed nucleotide. More than 80% of each individual viral mRNA was shown to contain the 5? structure 7mGpppm6AmAm. The functional significance of this structure was unknown at the time. However, m6A has become a significant focus of research in the past few years, and has been directly implicated in translational control. When present in the 5? end of cellular mRNAs, it can serve as a site for direct binding of eIF3, another component of the translation complex. This allows it to act as a cap-independent internal ribosome entry site (IRES) even though the mRNAs are capped. Previous work in our lab has also shown that VSV is hypersensitive to depletion of eIF3 subunits by siRNA, while cellular translation is not. Based on this evidence, I propose that VSV mRNA is translated through an m6A and eIF3-dependent mechanism. The experiments outlined for this project will address the following gaps in our understanding of VSV translation: 1) Does eIF3 preferentially control VSV translation (as opposed to cellular translation), and what is the mechanism behind this control? 2) Do m6A and its associated machinery impact VSV translation? Viruses have often been used as a model to study basic cellular mechanisms, such as IRESes. The role of m6A in cellular translation has only recently been characterized, and represents a significant area of growth in the field of translation. This study will expand our understanding of the basic cellular mechanisms of translation used in the cell, and the methods used by viruses to subvert and usurp those mechanisms.
The proposed project will provide novel insights into the mechanism of Vesicular Stomatitis virus translation, which currently remains unknown. Identifying this pathway will elucidate a novel mechanism of viral subversion of host translation, and lead to a greater understanding of these underlying pathways that control cellular translation.