Mechanisms involved in the host range restriction of parainfluenza

Abstract

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Microbiology and Immunology, 2009.An essential means to understanding virus-host interaction is to determine what makes a specific cell type or host permissive or non-permissive for virus replication. Many viruses are able to productively infect only a limited number of host species. The molecular basis behind this restricted host range involves the most fundamental aspects of the virus-host interaction. With the recent emergence of highly pathogenic animal pathogens, which have evolved the ability to infect human hosts, it has become extremely important to understand the mechanism behind host range restriction. As a model to study host range, the paramyxoviruses Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are ideal since they are highly homologous and structurally similar, but maintain distinct host ranges, murine versus human. The gene that is likely to determine the host range of parainfluenza viruses is the P/C gene. It expresses accessory proteins, which are known to counteract the IFN activities of host cells. In addition to the C proteins, SeV, but not hPIV1, expresses V protein from the gene. To evaluate the role and specificity of the anti-IFN activities of the P/C gene products in host range restriction, we determined the infectivity and anti-IFN activities of the viruses in human and murine cultured lung cells. SeV and hPIV1 infection were able to block the Jak/STAT pathway in both murine and human cells, through the inhibition of STAT1 nuclear localization. However, the viruses could not overcome antiviral activities induced by IFN pre-treatment in their non-native hosts, suggesting specificity in anti-IFN activity. We further characterized the specificity of the P/C gene products in antagonizing the signaling pathway that leads to IFN production, using a recombinant SeV, rSeVhP, whose P/C gene was replaced with that of hPIV1. Our data indicate that the ability of the P/C gene products to inhibit IFN induction is species specific, and the SeV V protein plays a central role in the antagonism of IFN induction in murine cells. We also found that the hPIV1 P/C gene product cannot prevent infected murine cells from apoptotic cell death, and the SeV V protein is required for this protection. Our study indicates specificity in the activity of hPIV1 and SeV P/C gene products in antagonizing IFN induction and apoptosis, which is likely to be one of the factors that affect the host range of these viruses. In addition to anti-innate activity, C protein has been suggested to have a role in viral assembly. We found that production of hPIV1 or rSeVhP from murine cells was limited, and large aggregates of viral nucleocapsids (vRNP) were detected in infected cells. Upon addition of the SeV C proteins, vRNP distribution was restored similar to SeV, suggesting interplay of the C proteins with specific host cellular machinery to mediate transport of vRNPs and virus assembly. It is not known, however, how vRNPs are transported from areas of synthesis to sites of assembly at the plasma membrane. We created a recombinant SeV, rSeVLeGFP, which expresses the L protein fused to enhanced green fluorescent protein (eGFP), and visualized trafficking of vRNP in live infected cells, using time-lapse digital video microscopy. We captured directional, saltatory movement suggestive of cargo transport along the microtubule network. Disruption of microtubules by nocodazole-treatment inhibited vRNP movement, and reduced the progeny virion production without affecting viral protein synthesis. Additional study using electron microscopy and membrane flotation assays revealed vRNP association with vesicles present in the infected cells. Our results suggest that SeV utilize cellular vesicles for transport of vRNPs on microtubules to plasma membrane budding sites. The factors involved in viral host range restriction are multi-faceted. Here we have shown specificity of the P/C gene products in counteracting the anti-viral response of host cells. These gene products also play a role in viral assembly and production of progeny virions. Thus, the interactions of the P/C gene products with host cellular machinery are an essential determinant in host range restriction