No abstract

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.

Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.

The recent solution of the crystal structure of a fragment of the vesicular stomatitis virus matrix (M) protein suggested that amino acids 121 to 124, located on a solvent-exposed loop of the protein, are important for M protein self-association and association with membranes. These residues were mutated from the hydrophobic AVLA sequence to the polar sequence DKQQ. Expression and purification of this mutant from bacteria showed that it was structurally stable and that the mutant M protein had self-association kinetics similar to those of the wild-type M protein. Virus assembly is a complex process involving a number of important steps. For enveloped viruses, the viral genome must be packaged into a nucleoprotein (nucleocapsid), which must be brought to the host membrane. This nucleocapsid must acquire a lipid bilayer by budding from the host membrane (for a review, see reference 14). For negative-strand RNA viruses of the order Mononegavirales and for the families Orthomyxoviridae and Retroviridae, these assembly functions appear to be carried out largely by viral matrix, or M, proteins (28,32). This multifunctional nature of the M protein makes it a critical player in virus assembly, which has led to a great deal of interest in determining how M proteins carry out such different functions. M proteins from different viruses have very different sequences and structures (32), a fact that has led to few initial clues about how they carry out their multiple functions and has highlighted the importance of studying individual M proteins to shed light on the wider class of functional analogs.The M protein of vesicular stomatitis virus (VSV) is a particularly well-studied example of this class of proteins. VSV M protein has been shown to play several roles in the virus assembly pathway. VSV M is responsible for condensing the viral nucleocapsid, causing the collapse of the loosely coiled, flexible nucleocapsid into the characteristic tightly coiled, bullet-like shape found in assembled virions (23,25,26). Mutagenesis and proteolysis studies have shown that the interaction of M protein with nucleocapsid requires an N-terminal sequence of the protein (2, 17). The N-terminal sequence (amino acids 1 to 50) is protease accessible in assembled nucleocapsid-M (NCM) complexes (17) and, by using a photoactivatable membrane probe, has been shown to associate with cellular membranes (20). The sequence PPPY contained within the N-terminal sequence is involved in a late stage of budding, allowing the release of budded virions from the membrane (15).In addition to the N-terminal sequence, several different regions in the middle of the protein sequence have been implicated in virus assembly, but these studies have yet to be extended to an understanding of whether these sequences cooperate and how they contribute to viral assembly and budding in an intact virus. In particular, proteolysis studies have suggested the contribution of a 4-amino-acid (aa) region at aa 121 to 124 in membrane association (9).A detailed understanding of why t...

In addition to its role in virus assembly, the matrix (M) protein of vesicular stomatitis virus (VSV) is involved in virus-induced cell rounding and inhibition of host-directed gene expression. Previous experiments have shown that two M protein mutants genetically dissociate the ability of M protein to inhibit host-directed gene expression from its function in virus assembly. M protein from tsO82 virus is fully functional in virus assembly but defective in the inhibition of host-directed gene expression, while the MN1 deletion mutant, which lacks amino acids 4-21, inhibits host-directed gene expression but cannot function in virus assembly. Experiments presented here compared cell rounding induced by these two mutant M proteins to that of wt M protein. BHK cells were transfected with M protein mRNA transcribed in vitro, and the extent of cell rounding was evaluated at 24 hr posttransfection. The MN1 protein was nearly as effective as wt M protein in the induction of cell rounding, while tsO82 M protein expressed from transfected RNA was not able to induce cell rounding above that observed in negative controls without M protein, although it did cause BHK cells to have a less elongated shape. These results indicate that the ability of MN1 and tsO82 M proteins to induce cell rounding is not correlated with their virus assembly function. Instead the cell rounding activity of these mutants is correlated with their ability to inhibit host-directed gene expression. Previous data suggesting that these two cytopathic activities could be dissociated can be readily accounted for by quantitative differences in M protein expression required. Infection of either BHK cells or L cells with tsO82 virus induced cell rounding, although cell rounding was delayed relative to that following infection with wt VSV, suggesting that tsO82 M protein retains some cytopathic activity. The distribution of actin, vimentin, and tubulin in transfected cells was determined by fluorescence microscopy. In cells transfected with tsO82 M mRNA, these cytoskeletal elements were indistinguishable from those of negative control transfected cells. In cells rounded as a result of transfection with wt M or MN1 mRNA, actin-containing filaments were reorganized into a thick perinuclear ring but were not depolymerized. In contrast, tubulin and vimentin appeared to be diffusely distributed throughout the cytoplasm of rounded cells. These results support the idea that cell rounding induced by M protein results from the depolymerization of microtubules and/or intermediate filaments.