Department of Microbiology and Immunobiology
Harvard Medical School
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The Whelan laboratory studies the biology of negative-strand (NS) RNA viruses. These viruses have genomes that are complementary to message sense RNA and must therefore carry into the cell the necessary machinery to copy their genomic RNA into mRNA. The genomes of the viruses can be non-segmented (ns) or segmented (s). We use vesicular stomatitis virus (VSV) as a model for the nsNS RNA viruses. A schematic of the VSV particle (Figure 1) and the viral replication cycle are shown (Figure 2). The nsNS RNA viruses include some of the most significant human pathogens, as well as a number of highly lethal emerging infections (Table 1). The work of the laboratory is divided between two major areas of focus: studies of viral gene expression and studies on viral-host cell interactions.

VSV gene expression

The nsNS RNA viruses have an unusual strategy of gene expression (Figure 3). The negative-sense genomic RNA is completely encapsidated by the viral nucleocapsid protein (N). It is this N-RNA complex that is recognized by the viral polymerase during RNA synthesis. For VSV the viral components of the polymerase are the phosphoprotein (P) and the large polymerase protein (L).

Polymerase recognizes a promoter and initiates mRNA synthesis at the beginning of the N-gene. At the end of the N-gene the polymerase encounters a sequence that signals termination and polyadenylation of the N mRNA. These events are essential for initiation of mRNA synthesis at the P gene-start sequence, i.e. the polymerase must STOP at the end of the upstream gene before STARTING at the beginning of the downstream gene. The polymerase continues with this stop-and-start mode of mRNA synthesis to produce the 5 viral mRNA’s. At each gene junction a highly localized transcriptional attenuation occurs, such that 30% of the polymerase fails to transcribe the downstream gene. This results in a gradient of mRNA abundance which is reflected in viral protein synthesis (Figure 3).

For some nsNS RNA viruses additional viral proteins are part of the polymerase complex. However, the mechanism of mRNA synthesis is essentially the same for each of these viruses. Because of the ability of VSV to replicate in a broad range of cell types, the ease with which precisely defined mutations can be introduced into the viral genome (Figure 4), and the availability of robust in vitro systems to study mRNA synthesis, it is an attractive model in which to study viral gene expression.

Messenger RNA processing, the Achilles heel of nsNS RNA viruses? The mRNA processing reactions of nsNS RNA viruses are unusual both in the formation of the 5’ mRNA cap-structure (Figure 5) and the 3’ poly (A) tail. These reactions are attractive targets for development of broadly active antiviral therapeutics because they are highly conserved and distinct from those of the host. We are studying in mechanistic detail each of the mRNA processing reactions. We are pursuing complementary approaches to dissect the domains of the viral polymerase responsible for these reactions, and defining the RNA signals that each of these enzymes recognize.

Virus-host cell interactions

Viruses are obligate intracellular parasites with complex and dynamic interactions with their hosts. Viruses must appropriate host-cell resources to support their replication, the host cell responds to infection with antiviral defense measures that in turn necessitate viral countermeasures to these defense mechanisms. Few systematic analyses of the virus-host interaction have been performed. The broad host range of VSV facilitated the application of genomic approaches to study the interaction of this model nsNS RNA virus with its host. We generated recombinant VSV expressing the reporter gene, green fluorescent protein (GFP) or firefly luciferase (LUC) (Figure 6). In collaboration with the group of Norbert Perrimon, the host genes that modulate VSV replication were identified through the use of a genome wide RNAi screen in Drosophila cells. Genes putatively involved in all aspects of the VSV replication cycle were identified. The role of these host genes in viral replication is currently being assessed using our well established assays for viral entry, uncoating, replication and assembly.