Biology of alphavirus infection: Keys for the way in, tolls on the way out
Ooi, Yaw Shin
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Alphaviruses are plus-sense RNA viruses that are transmitted by mosquito vectors and can cause debilitating illness, severe arthritis, and encephalitis in humans. They include important and emerging human pathogens such as Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus. Important prototypical species of the virus that have been intensively studied in our laboratory are Semliki Forest virus (SFV) and Sindbis virus (SINV). Alphaviruses enter cells by clathrin-mediated endocytosis, and exit by budding from the plasma membrane. While there has been considerable progress in defining the structure and function of the viral proteins, little is known about the host factors involved in alphavirus infection.;In this dissertation, the interaction between alphaviruses and the human host was examined using various strategies, i.e. from classical molecular virology techniques to state-of-the-art high-throughput RNA interference (RNAi) screening. In Chapter 3, we utilized a genome-wide siRNA screen to identify novel host factors that promote or inhibit alphavirus infection in human cells. We then defined the mechanisms of three host proteins that promote alphavirus infection. ARCN1, the delta subunit of the COPI complex, was required for initial virus-cell binding. Fuzzy homologue (FUZ), a protein with reported roles in planar cell polarity and cilia biogenesis, was required for the clathrin-dependent internalization of both alphaviruses and the classical endocytic ligand transferrin. The tetraspanin membrane protein, TSPAN9, did not affect endocytic uptake or acidification, but was critical for the efficient fusion of the virus in the endosome. FUZ and TSPAN9 were broadly required for infection by the alphaviruses SINV, SFV, and CHIKV, but were not required by the structurally related flavivirus Dengue virus (DENY). Our results highlight the unanticipated functions of ARCNI, FUZ and TSPAN9 in alphavirus entry and suggest novel host proteins that may serve as targets for antiviral therapy.;Tetherin, or bone marrow stromal cell antigen 2, is an interferon-induced membrane protein that inhibits release of many enveloped viruses via direct tethering of budded particles to the cell surface. However, the role of this antiviral protein in restricting highly organized and compact viruses such as alphaviruses remains obscure. In Chapter 4, we examined the role of tetherin during alphavirus exit. We demonstrated that exogenously-expressed tetherin inhibited the release of infectious SFV and CHIKV from host cells without affecting virus entry or trafficking of viral envelope glycoproteins to the plasma membrane. On the other hand, SFV exit was only modestly sensitive to endogenously-expressed tetherin. Perhaps, the discrepancy between the overexpression system and the endogenous system can be simply explained by differential inhibitory effects caused by the distinct expression systems. Alternatively, the results also suggest that alphaviruses may be able to antagonize tetherin, as is the case for a number of other viruses. In this scenario, the lower level of endogenously-expressed tetherin may not be able to effectively saturate a counteractive mechanism of alphaviruses, resulting in a more modest inhibition of progeny virus release. Although Rubella virus (RuV) and DENY have structural similarities to alphaviruses, tetherin only attenuated release of RuV but not DENY. Notably, we found that two recently identified tetherin isoforms termed long-isoform and short-isoform, exhibit distinct capability in modulating the release of alphaviruses. SFV exit was sensitive to the long-isoform but not the short-isoform of tetherin, while either of the isoforms efficiently inhibited Vesicular stomatitis virus exit from the host cells. Together, our findings identified tetherin as a unique host protein that blocks the release of alphavirus from host cells. The discovery of tetherin as the first cellular protein that specifically interferes with alphavirus release has paved the way for further mechanistic studies, and may promote the understanding of the virus exit mechanism, which remains indeterminate.