Although poliovirus, the most well known enterovirus in the picornavirus family, is soon to be declared eradicated by the World Health Organization, there are 66 other distinct human enteroviruses that are still infecting populations worldwide. However, there are no vaccines or antiviral treatments available for any of the non-polio enteroviruses. The key to developing an effective antiviral agent is to gain an understanding of the molecular processes that govern the viral life cycle. Although considerable progress has been made towards understanding the function of the picornaviral proteins, little is known about the host cell factors that are required for viral gene amplification. Identification of host cell factors has been hampered by the absence of an easily amenable genetic system. Previous attempts to study animal virus replication in yeast have been thwarted by the fact that the picornaviruses initiate translation by a cap-independent mechanism, using an internal ribosome entry site (IRES), which does not function in yeast. However, the recent finding that an IRES from an insect viral genome functions in yeast opened the possibility to develop a system in yeast to identify host cell factors required for picornavirus replication. The properties of the cricket paralysis virus (CrPV) IRES are extraordinary, because this IRES can form 80S ribosomes from purified 40S and 60S subunits in the absence of any initiation factors, tRNAs or GTP. In the first specific aim, existing yeast mutants will be used to gain insight into the mechanism by which the CrPV IRES recruits ribosomes. In addition, yeast mRNAs will be identified that can initiate translation by a mechanism similar to the CrPV IRES. Specifically, yeast microarray analysis will be used to identify polysomal mRNAs in a Afun12 mutant yeast strain. This strain lacks the non-essential FUN12 gene encoding elF5B, which is involved in ribosomal subunit joining. As a consequence, fewer ribosomes are associated with mRNAs in the mutant strain. In the second specific aim, chimeric picornaviral RNA genomes, containing the CrPV IRES in place of their normal IRES, will be constructed and their replication in yeast will be examined. In addition, replicons will be constructed, containing the selectable yeast marker URA3 in place of the capsid proteins. Replicon-expressing cells will be isolated and mutant cells that are defective replicon amplification will be isolated. The functions of identified genes will then be examined both in yeast and in mammalian cells. Overall, these studies will contribute to our understanding on the functioning of viral IRESs and potentially lead to a novel way of studying host genes that are required for virus propagation. ? ?
| Deniz, Nilsa; Lenarcic, Erik M; Landry, Dori M et al. (2009) Translation initiation factors are not required for Dicistroviridae IRES function in vivo. RNA 15:932-46 |
