Sindbis virus interaction with cells
Sindbis virus is the prototype of Togaviridae family, Alphavirus genus. In nature, alphaviruses can infect and grow in both mammals and insects. They can also reproduce themselves in many laboratory cell cultures. Understanding the virus infection process is fundamental for drug discovery and vaccine development. Two scenarios of alphavirus infection have been proposed. One proposed mechanism involves uptake of virion into endocytic vesicles followed by low-pH dependent fusion and genome release. This infection pathway was first discovered in influenza and proposed later to be the entry mechanism for most enveloped viruses. The other mechanism proposes direct penetration at the cell surface and neutral pH, supported by both electron microscopy and biochemical studies. We studied the ability of Sindbis virus to infect Baby Hamster Kidney (BHK) in the absence of endocytosis. Instead of using drugs or cellular mutations, we used low temperature to inhibit endocytosis. We performed an antibody escape experiment at different temperatures combined with a plaque assay. Our results showed that Sindbis virus can infect cells in the absence of endocytosis at both 5 and 15 oC. To confirm these results, we infected BHK cells at low temperatures with Sindbis virus containing Green Fluorescent Protein (GFP) gene as a reporter. The result shows that Sindbis virus is capable of injecting its genome into host cell and initiating an infection under conditions which have been established to inhibit endocytosis and membrane fusion.
Sindbis virus has a host derived membrane bilayer. E1 and E2 are the two structural proteins that are anchored in this membrane by their transmembrane (TM) domains. Studies on chimeric alphavirus composed of Ross River E1 and Sindbis E2 proteins revealed a role of E1-E2 TM domain interaction in affecting virus stability (Strauss, Lenches, and Strauss, 2002). The importance and characteristics of different amino acids in E2 TM domain was also studied by making deletions in the region (Hernandez et al., 2003; Whitehurst et al., 2006). We studied the impacts of making deletions in E1 TM domain on virus life cycle. Although short deletions at different positions result in diverse infectivity, virus assembly is normal for all the short deletion mutants we constructed. We also found that large deletions in E1 TM domain are lethal to virus production by severely inhibiting the virus budding process, while structural protein synthesis and nucleocapsid assembly are not impaired. We also constructed a combined double deletion mutant in which short deletions were made in both E1 and E2 TM domains. The combined double deletion mutant produced no infectious viruses. These results provided important information about E1-E2 TM domain interaction.
Advisor:Keith Weninger; Jason A. Osborne; Paul Wollenzien; Stuart Maxwell; Dennis Brown
School:North Carolina State University
School Location:USA - North Carolina
Source Type:Master's Thesis
Date of Publication:07/11/2008