Global mRNA Degradation during Lytic Gammaherpesvirus Infection Contributes to Establishment of Viral Latency

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3′ end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment

Development of Live-Attenuated Arenavirus Vaccines

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2015.The significance of arenaviruses in human health and biodefense readiness, together with the limited existing armamentarium to combat them, highlight the importance of developing effective vaccines to prevent arenavirus infections. Plasmid-based reverse genetics have allowed for the generation of recombinant (r) arenaviruses, however viruses recovered from the rodent cell substrates do not meet FDA standards for vaccine manufacturing. Given the species-specific restrictions of the murine RNA polymerase I (Pol-I) promoter driving vRNA expression in the current reverse genetic systems, we evaluated the potential of using the human Pol-I promoter in FDA-approved Vero cells. Using the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we were able to rescue rLCM viruses in our redesigned system with high reproducibility. Furthermore, we were able to generate an attenuated Junín vaccine strain (Candid#1), demonstrating the possibility of rescuing live-attenuated arenavirus strains in FDAapproved cells with our system. Taking advantage of the codon usage bias that exists within viruses and their hosts, we recoded the LCMV nucleoprotein (NP) and glycoprotein (GP) open reading frames (ORFs) in a way that perfectly preserved the amino acid sequences but created a suboptimal utilization of codons to generate rLCM viruses containing either codon deoptimized NP or GP (rLCMV/NPCD and rLCMV/GPCD, respectively). rLCMV/NPCD and rLCMV/GPCD were attenuated in vivo and a single immunization with either virus conferred complete protection against a subsequent lethal challenge with WT LCMV. Importantly, serial passaging of these attenuated LCM viruses did not lead to reversion to virulence, demonstrating codon deoptimization as an effective strategy for the development of safe, stable and protective live-attenuated arenavirus vaccines. The arenavirus genome is uniquely organized such that specific cis-acting regions regulate expression of viral genes. Preliminary results demonstrated that each cis-acting element contributed to varying degrees in gene expression. Knowledge from these studies allowed us to generate a highly attenuated rLCM containing translocated S ORFs (rLCMV/TransS). As with the CD strategy, rLCMV/TransS was safe in vivo and conferred complete protection against a lethal challenge with rLCMV/WT. Interestingly, virus attenuation was contributed by a reduction in NP expression, demonstrating the rational design for the future development of live-attenuated arenavirus vaccines

Global mRNA degradation during lytic gammaherpesvirus infection contributes to establishment of viral latency.

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment