Antivirals and Engineering of RNA Viruses: A Molecular Approach

RNA viruses outnumber DNA viruses in higher organisms and cause numerous diseases in humans, animals, and plants. Recent world events have heightened fears that viruses could be used as bioterrorist weapons. Most RNA viruses encode only a handful of genes whose expression can have varied effects on their host cells ranging from total destruction to inapparent infections. The deadly Ebola virus for example encodes only eight genes. Ebola is a member of a large group of viruses with negative sense, single-stranded RNA genomes (genome complementary to mRNA) which includes measles, rabies, mumps, respiratory syncytial, parainfluenza, and vesicular stomatitis virus (VSV). The latter virus is a favorite model for laboratory studies because it is not pathogenic for humans but can be grown easily and in large amounts. VSV has been the subject of our NIH-funded studies for the past twenty five years.

In the past five years, it has become possible to engineer mutations and/or additional genes into the VSV genome and recover highly infectious recombinant virus. VSV is now considered a very promising vector for vaccine production (grafting other virus genes onto the VSV genome) and is currently being explored as a possible HIV vaccine. Moreover, VSV shows great promise for some gene therapy applications. 

Recent developments in my laboratory raise the possibility of engineering VSV as a platform to develop a new class of antiviral drugs for agents such as Ebola and measles. Our basic studies have long focused on the virally-encoded RNA-dependent RNA polymerase. The VSV polymerase is a multifunctional protein that also functions as a mRNA capping enzyme as well as a cap methylase enzyme. Methylation of the cap structure at the ends of mRNAs is essential for proper recognition by cellular ribosomes and translation of viral proteins. We have mapped the domain of the VSV polymerase that encodes the methylase and have engineered several mutations in this domain. We have developed assays that will permit us to screen drugs that inhibit virus growth by inhibiting the viral methylase. We also plan to map and engineer mutations in the viral cap function as an alternative target for antivirals.

We have also recently developed a novel approach for studying cellular antiviral defenses against VSV and related viruses. Many viruses encode proteins that specifically antagonize cellular antiviral functions and we have grafted some of these genes into a defective VSV genome.  Using a defective VSV genome here is crucial since we cannot rule out the possibility that "new" infectious chimeric viruses coud possess increased pathogenicity and/or a wider host range. We have so far engineered defective chimeric VSV recombinants that encode influenza, vaccinia, and Sendai virus genes. These chimeric viruses have allowed us to show that double-stranded RNA signalling is very important in cellular antiviral defense mechanisms prior to engagement of the well known interferon signalling pathway.  

9/24/02

Representative recent publications
 

Canter, D. M., Jackson, R. L., and Perrault, J. (1993). Faithful and efficient in vitro reconstitution of vesicular stomatitis virus transcription using plasmid-encoded L and P proteins. Virology 194, 518-529.

Jackson, R. L., Spadafora, D., and Perrault, J. (1995). Hierarchal constitutive phosphorylation of the vesicular stomatitis virus P protein and lack of effect on P1 to P2 conversion. Virology 214, 189-197.

Canter, D. M, and Perrault, J. (1996). Stabilization of vesicular stomatitis virus L polymerase protein by P protein binding: a small deletion in the C-terminal domain of L abrogates binding. Virology 219, 376-386.

Spadafora, D., Canter, D. M., Jackson, R. L. and Perrault, J. (1996). Constitutive phosphorylation of the vesicular stomatitis virus P protein modulates polymerase complex formation but is not essential for transcription or replication. J. Virol. 70, 4538-4548.

Chuang, J., and Perrault, J. (1997). Initiation of vesicular stomatitis virus mutant polR1 transcription internally at the N gene in vitro. J. Virol. 71, 1466-1475.

Chuang, J., Jackson, R. L. and Perrault, J. (1997). Isolation and characterization of vesicular stomatitis virus polR revertants: Polymerase readthrough of the leader-N gene junction is linked to an ATP-dependent function. Virology 229, 57-67.

Chu, W-M., Ostertag, D., Li, Z-W., Chang, L., Hu, Y., Williams, B. Perrault, J, and Karin, M. (1999). JNK2 and IKKb are required for activating the innate response to viral infection. Immunity 11, 721-731.

Spadafora, D., and Perrault, J. (2002). Both RNA cap methyl transferases of vesicular stomatitis virus utilize the same SAM-binding site in the viral polymerase protein.  To be submitted. 

Canter, D. M., Spadafora, D, and Perrault, J. (2002). SAM-binding site in the vesicular stomatitis virus L polymerase protein modulates a switch between transcriptase and replicase activity. To be submitted.

Spadafora, D., and Perrault, J. (2002). Mutation of SAM-binding site in vesicular stomatitis virus L polymerase protein enhances defective interfering particle virus production in vivo. To be submitted.

Clizbe, D., Hayden, J, Nogales, J., and Perrault, J. (2002). Constitutive phosphorylation of P protein of vesicular stomatitis virus is dispensable for growth in cell culture. To be submitted. 
 

Ph.D. students: Derek Ostertag