Inhibition of nucleo-cytoplasmic transport by rhinovirus

The most prevalent form of viral respiratory illness is the common cold, which is itself most often associated with infection by rhinovirus. Viral respiratory infections are the most common acute illness in the US and children are the most frequent victims, with infants aged less than one year experiencing greater than 6 infections a year. Analysis of the economic burden imposed by viral respiratory infections estimates $40 billion in direct and indirect costs every year in the U.S. Rhinoviruses are positive-stranded RNA viruses that replicate entirely within the cytoplasm of host cells. Despite replication occurring in the cytoplasm, host nuclear factors have been
implicated in replication and shown to relocalize to the cytoplasm during infection. Recently, rhinovirus infection was shown to result in an inhibition of nuclear import and the degradation of nuclear pore complex (NPC) proteins. Inhibition of nuclear import could account for the accumulation of host nuclear factors in the cytoplasm of rhinovirus-infected cells and is predicted to contribute to efficient viral replication and pathogenesis. The overall goal of this proposal is to identify the molecular mechanisms responsible for inhibition of nuclear import and to determine how inhibition of nuclear import contributes to rhinovirus replication. The first aim of this proposal examines the role of viral proteases in degradation of NPC proteins and inhibition of nuclear import. Using purified proteases cleavage sites within NPC proteins will be mapped and mutants generated that are resistant to cleavage. Using these mutant NPC proteins we will examine the consequence of preventing degradation on viral replication.
In the second aim, viral proteins responsible for relocalization of nuclear proteins, degradation of NPC components and inhibition of nuclear import will be identified by expressing individual viral proteins in the context of uninfected cells. Mutants of identified proteins will be generated and analyzed and ultimately introduced into an infectious rhinovirus cDNA to determine their effect upon replication. The third aim will determine the alterations to the NPC in infected cells using a biochemical fractionation procedure to purify all NPC proteins from infected cells. Proteins will be identified by mass spectroscopy and compared with those isolated from uninfected cells. Immunoelectron microscopy will be used to determine structural changes that are a consequence of viral infection.