Post-Transcriptional Regulation of Parvovirus Capsid Gene Expression

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DESCRIPTION (provided by applicant): The parvoviruses AMDV and B19 are unique among animal DNA viruses in that they have a single promoter, and so their genetic diversity is controlled exclusively by post-transcriptional mechanisms. A single class of pre-mRNA molecules generated by these viruses undergoes extensive alternative splicing and polyadenylation that generates sub-genomic mRNA molecules which program this diversity. Control of the synthesis of the capsid proteins is one of the key regulators of B19 tissue tropism, and governs the ability of AMDV to remain persistent in infected host animals. B19 permissive infection is characterized by a switch to increased capsid production, while persistence of AMDV infection in animal systems is characterized by, and likely requires, controlled low levels of capsid protein synthesis. All other parvoviruses have an internal promoter that regulates production of capsid-encoding mRNA during the synthesis phase of viral replication, however, this mechanism is not available to B19 and AMDV. Control of expression of the AMDV and B19 capsid-coding genes by post-transcriptional mechanisms is the focus of this application. Both viruses have an efficient polyA site in the center of the genome. Use of this site precludes inclusion of the capsid coding ORFs into mRNA, and an understanding of how the choice is made to either polyadenylate, or read-through this site, is the topic of Specific Aim 2 and 3. We propose to identify both the cis sequence and trans-factors that control these events, the mechanisms that govern their selective use, and for B19, exam how this choice is differently made in cells permissive or nonpermissive for replication. We have recently discovered that a single spliced AMDV mRNA encodes both capsid proteins VP1 and VP1, as well as an essential small non-structural protein NS2 at its 5'end. Thus, in addition to alternative splicing and polyadenylation, features of alternative translation initiation are critical for governing the proper expression of the AMDV capsid proteins. In Specific Aim 1, we will characterize how the cis-element in the NS2 gene affects translation of the capsid proteins, both genetically and biochemically. How these post-transcriptional processes generate appropriate levels of the capsid proteins from the single pre-mRNA molecules encoded by AMDV and B19, is critical to our understanding of parvovirus gene expression, and the biology of parvovirus infection. However, in addition, these viral systems provide very tractable models with which to learn much about these basic cellular mechanisms in general.