Genome-Wide Mutagenesis Reveals That ORF7 Is a Novel VZV Skin-Tropic Factor

The Varicella Zoster Virus (VZV) is a ubiquitous human alpha-herpesvirus that is the causative agent of chicken pox and shingles. Although an attenuated VZV vaccine (v-Oka) has been widely used in children in the United States, chicken pox outbreaks are still seen, and the shingles vaccine only reduces the risk of shingles by 50%. Therefore, VZV still remains an important public health concern. Knowledge of VZV replication and pathogenesis remains limited due to its highly cell-associated nature in cultured cells, the difficulty of generating recombinant viruses, and VZV's almost exclusive tropism for human cells and tissues. In order to circumvent these hurdles, we cloned the entire VZV (p-Oka) genome into a bacterial artificial chromosome that included a dual-reporter system (GFP and luciferase reporter genes). We used PCR-based mutagenesis and the homologous recombination system in the E. coli to individually delete each of the genome's 70 unique ORFs. The collection of viral mutants obtained was systematically examined both in MeWo cells and in cultured human fetal skin organ samples. We use our genome-wide deletion library to provide novel functional annotations to 51% of the VZV proteome. We found 44 out of 70 VZV ORFs to be essential for viral replication. Among the 26 non-essential ORF deletion mutants, eight have discernable growth defects in MeWo. Interestingly, four ORFs were found to be required for viral replication in skin organ cultures, but not in MeWo cells, suggesting their potential roles as skin tropism factors. One of the genes (ORF7) has never been described as a skin tropic factor. The global profiling of the VZV genome gives further insights into the replication and pathogenesis of this virus, which can lead to improved prevention and therapy of chicken pox and shingles

New intranasal and injectable gene therapy for healthy life extension

AbstractAs the global elderly population grows, it is socioeconomically and medically critical to have diverse and effective means of mitigating the impact of aging on human health. Previous studies showed that adenovirus-associated virus (AAV) vector induced overexpression of certain proteins can suppress or reverse the effects of aging in animal models. Here, we sought to determine whether the high-capacity cytomegalovirus vector can be an effective and safe gene delivery method for two such-protective factors: telomerase reverse transcriptase (TERT) and follistatin (FST). We found that the mouse cytomegalovirus (MCMV) carrying exogenous TERT or FST (MCMVTERT or MCMVFST) extended median lifespan by 41.4% and 32.5%, respectively. This is the first report of CMV being used successfully as both an intranasal and injectable gene therapy system to extend longevity. Treatment significantly improved glucose tolerance, physical performance, and prevented loss of body mass and alopecia. Telomere shortening seen with aging was ameliorated by TERT, and mitochondrial structure deterioration was halted in both treatments. Intranasal and injectable preparations performed equally well in safely and efficiently delivering gene therapy to multiple organs, with long-lasting benefits and without carcinogenicity or unwanted side effects. Translating this research to humans could have significant benefits associated with increased health span.</jats:p

ORF7 of Varicella-Zoster Virus Is a Neurotropic Factor

Research Center for Minority Institutes (RCMI) Program [2G12RR003050-24]; American Cancer Society grant [RSG-090289-01-MPC]; NIH/NCRR [U54RR022762]Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens

Development of a skin- and neuro-attenuated live vaccine for varicella

AbstractVaricella caused by the primary infection of varicella-zoster virus (VZV) exerts a considerable disease burden globally. Current varicella vaccines consisting of the live-attenuated vOka strain of VZV are generally safe and effective. However, vOka retains full neurovirulence and can establish latency and reactivate to cause herpes zoster in vaccine recipients, raising safety concerns. Here, we rationally design a live-attenuated varicella vaccine candidate, v7D. This virus replicates like wild-type virus in MRC-5 fibroblasts and human PBMCs, the carrier for VZV dissemination, but is severely impaired for infection of human skin and neuronal cells. Meanwhile, v7D shows immunogenicity comparable to vOka both in vitro and in multiple small animal species. Finally, v7D is proven well-tolerated and immunogenic in nonhuman primates. Our preclinical data suggest that v7D is a promising candidate as a safer live varicella vaccine with reduced risk of vaccine-related complications, and could inform the design of other herpes virus vaccines.</jats:p

ORF7 of Varicella-Zoster Virus Is a Neurotropic Factor

ABSTRACT Varicella-zoster virus (VZV) is the causative agent of chickenpox and herpes zoster (shingles). After the primary infection, the virus remains latent in sensory ganglia and reactivates upon weakening of the cellular immune system due to various conditions, erupting from sensory neurons and infecting the corresponding skin tissue. The current varicella vaccine is highly attenuated in the skin and yet retains its neurovirulence and may reactivate and damage sensory neurons. The factors involved in neuronal invasion and establishment of latency are still elusive. Previously, we constructed a library of whole-gene deletion mutants carrying a bacterial artificial chromosome sequence and a luciferase marker in order to perform a comprehensive VZV genome functional analysis. Here, screening of dispensable gene deletion mutants in differentiated neuronal cells led to the identification of ORF7 as the first known, likely a main, VZV neurotropic factor. ORF7 is a virion component localized to the Golgi compartment in infected cells, whose deletion causes loss of polykaryon formation in epithelial cell culture. Interestingly, ORF7 deletion completely abolishes viral spread in human nervous tissue ex vivo and in an in vivo mouse model. This finding adds to our previous report that ORF7 is also a skin-tropic factor. The results of our investigation will not only lead to a better understanding of VZV neurotropism but could also contribute to the development of a neuroattenuated vaccine candidate against shingles or a vector for delivery of other antigens. </jats:p