Development of replicons to study flavivirus replication and assembly: Applications in antiviral compound screening

Chinmay G Patkar, Purdue University

Abstract

The flavivirus genus includes more than 70 members of enveloped, positive strand RNA viruses, including yellow fever virus (YFV), which cause severe human disease with complex pathologies. The flavivirus genome codes for three structural proteins (C, prM, and E) that comprise the virions and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that are responsible for genome replication and virus assembly. To facilitate study of these processes, replicons of YFV, expressing reporter genes in a replication-dependent manner, were constructed by deletion of the structural protein coding region. Using YFV replicons, a trans-complementation system was established for identification of cis- and trans-acting signals in flavivirus genome replication. While trans-complementation of NS1 and NS3 occurred efficiently, the levels of trans-complementation NS5 were significantly less, suggesting NS5 was required in cis for efficient genome replication. Additionally, using similar assays, it was shown that preventing cleavage within NS3 by mutation of the internal cleavage sequence abolished genome replication, although the helicase and ATPase activities were not affected. Surprisingly, replication could be rescued by expression of the smaller NS3 cleavage fragment in cis, suggesting a role for the NS3 cleavage fragments in genome replication. A trans-packaging system was established for packaging YFV replicons into pseudo-infectious particles by supplying the YFV structural proteins in trans. With the aim of identifying the functional elements within the C protein necessary for virus assembly, various N- and C-terminal truncations, internal deletions and point mutations were analyzed in the packaging assay. It was shown that the C protein is extremely flexible and tolerant to large deletions for its function. Some of these deletions and point mutations were also analyzed in the context of YFV virus and the results were analogous to those obtained using the trans-packaging system. The involvement of non-structural proteins in virion assembly was also investigated, and revealed an unexpected role for NS3 in virion assembly. Mutation of the conserved tryptophan at position 349 in the helicase domain of NS3 did not affect viral protein expression or genome replication but blocked production of infectious virus. However, this assembly defect could be complemented by supplying wild-type NS3 protein in trans. It was further shown that substitutions with phenylalanine, tyrosine or histidine were tolerated, suggesting the requirement of an aromatic residue at position 349 in NS3. Furthermore, trans-complemented replicons containing lethal deletions within NS 1 and NS3 were packaged into pseudo-infectious particles, indicating that full-length NS 1 or NS3 were not required in cis for virus assembly. Finally, using these replicons and modified YFV genome, high- and medium-throughput assays have been developed for screening inhibitors of flavivirus replication.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Microbiology

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