Sean Whelan, Ph.D.
Associate Professor of Microbiology and Molecular Genetics
Harvard Medical School
200 Longwood Ave.
Armenisi Bldg., Room 512
Boston, MA 02115
5 Postdoctoral fellows; 4 graduate student
We study the biology of the negative-sense (NS) RNA viruses. These viruses include significant human pathogens such as rabies, Nipah, measles, respiratory syncytial, Ebola and Marburg viruses. As a model for the biology of these viruses we study vesicular stomatitis virus (VSV). The advantages of VSV as a model include: (i) safety, VSV is not a significant human pathogen; (ii) abundant replication in a wide variety of cells in culture yielding up to 10,000 infectious particles per cell within 8 hours of inoculation; (iii) ability to employ genomic approaches to dissect the virus-host relationship in genetically tractable hosts, including Saccharomyces cerevisae, Caenorhabditis elegans, and Drosophila melanogaster; (iv) a robust reverse genetic system that permits generation of mutant viruses; and, (v) an in vitro system to study viral mRNA synthesis. For these reasons, studies on VSV have frequently provided novel insight into the biology of the less tractable NNS RNA viruses. Current areas of focus include:-
Viral entry, fusion and uncoating.
We are determining the mechanism by which VSV enters cells. Following attachment of VSV to the cell surface the virus is internalized through clathrin mediated endocytosis. The viral attachment protein G undergoes a pH triggered conformational change that mediates fusion of the viral and cellular membranes and delivers the viral ribonucleoprotein core into the cytoplasm of the cell. We are asking the following questions: Where does the viral core gain access to the cytoplasm of the cell? What are the consequences for altering this site? What host factors are essential for the virus to enter and uncoat? We have employed viral and host genetic approaches to study these questions combined with high resolution microscopy. We have developed recombinant VSV that allows us to dissect the viral entry pathway independent of gene expression. This permits a detailed analysis of the viral determinants of entry, and we have also utilized a genome wide RNAi approach to determine the host components that affect viral entry.
Viral gene expression.
We are determining the mechanism by which each of the following four steps of gene expression are regulated. First, the template for RNA synthesis is not naked RNA, rather a complex in which the RNA is tightly encapsidated by N protein. The structure of this N-RNA complex demonstrates that the N protein must be transiently remodeled or displaced to allow polymerase to gain access to the bases of the RNA, in a manner in which the RNA remains completely resistant to nuclease attack. Second, the mechanism by which mRNA’s acquire their 5’ cap and 3’ poly A tail involves a series of unusual capping and poly A enzymes provided by the L protein. Third, viral mRNAs are efficiently translated in the face of a shut down of cellular translation. Fourth, polymerase activity is regulated between two synthetic events of mRNA synthesis and genomic replication. We developed a system to reconstitute mRNA synthesis in vitro from purified recombinant components. We have used this system to define the mRNA capping activities within the polymerase, and demonstrated that these activities require highly specific cis-acting signals in the RNA. Using a genome wide RNAi approach we have also obtained insight into how viral mRNAs are translated. Using viral genetic approaches we have also gained insight into how the processes of mRNA synthesis and genome replication are governed. We have extended our studies into the filoviruses Ebola and Marburg and have now obtained insight into how those viruses modify their mRNA.
- Cureton DK, Massol RH, Saffarian S, Kirchhausen TL, Whelan SP. Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization. PLoS Pathog. 2009 Apr;5(4):e1000394. Epub 2009 Apr 24.
- Li J, Rahmeh A, Morelli M, Whelan SP A conserved motif in region V of the large polymerase proteins of nonsegmented negative-sense RNA viruses that is essential for mRNA capping. J Virol. 2008 Jan;82(2):775-84.
- Li J, Wang JT, Whelan SP. A unique strategy of mRNA cap methylation used by vesicular stomatitis virus. Proc Natl Acad Sci U S A 2006: 103(22):8493-8498
- Schott D, Cureton DK, Whelan SP, Hunter C. An antiviral role for the RNA interference machinery in Caenorhabditis elegans. Proc Natl Acad Sci U S A 2005: 102(51):18420-18424.
- Chandran K, Sullivan NJ, Felbor U, Whelan SP, Cunningham JM. Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection. Science. 2005;308(5728):1643-5.
Virology webpage updated 12/02/2009