AAV Gene Augmentation of Truncated Complement Factor H Differentially Rescues Ocular Complement Dysregulation in a Mouse Model.

Purpose: Complement dysregulation in the eye has been implicated in the pathogenesis of age-related macular degeneration (AMD), and genetic variants of complement factor H (CFH) are strongly associated with AMD risk. We therefore aimed to untangle the role of CFH and its splice variant, factor H-like 1 (FHL-1), in ocular complement regulation derived from local versus circulating sources. We assessed the therapeutic efficacy of adeno-associated viruses (AAVs) expressing human FHL-1 and a truncated version of CFH (tCFH), which retains the functional N- and C-terminal ends of the CFH protein, in restoring the alternative complement pathway in Cfh-/- mouse eyes and plasma. Methods: Using Cfh-/- mice as a model of complement dysregulation, AAV vectors expressing tCFH or FHL-1 were injected subretinally or via tail vein, and the efficacy of the constructs was evaluated. Results: Following subretinal injections, tCFH expression rescued factor B (FB) retention in the eye, but FHL-1 expression did not. By contrast, both constructs restored FB detection in plasma following tail vein injections. Both tCFH and FHL-1 proteins accumulated in the posterior eyecup from the circulation following liver transduction; however, neither was able to significantly regulate local ocular complement. Conclusions: Our findings demonstrate that the C-terminus of human CFH is necessary for complement regulation in the murine eye. Furthermore, exogenous CFH must be synthesized locally to maximize complement regulation in the retina. These findings establish a critical foundation for development of CFH augmentation-based gene therapies for the eye.

Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice.

Human gamma herpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are capable of inducing tumors, particularly in in immune-compromised individuals. Due to the stringent host tropism, rodents are resistant to infection by human gamma herpesviruses, creating a significant barrier for the in vivo study of viral genes that contribute to tumorigenesis. The closely-related murine gamma herpesvirus 68 (γHV68) efficiently infects laboratory mouse strains and establishes robust persistent infection without causing apparent disease. Here, we report that a recombinant γHV68 carrying the KSHV G protein-coupled receptor (kGPCR) in place of its murine counterpart induces angiogenic tumors in infected mice. Although viral GPCRs are conserved in all gamma herpesviruses, kGPCR potently activated downstream signaling and induced tumor formation in nude mouse, whereas γHV68 GPCR failed to do so. Recombinant γHV68 carrying kGPCR demonstrated more robust lytic replication ex vivo than wild-type γHV68, although both viruses underwent similar acute and latent infection in vivo. Infection of immunosuppressed mice with γHV68 carrying kGPCR, but not wild-type γHV68, induced tumors in mice that exhibited angiogenic and inflammatory features shared with human Kaposi's sarcoma. Immunohistochemistry staining identified abundant latently-infected cells and a small number of cells supporting lytic replication in tumor tissue. Thus, mouse infection with a recombinant γHV68 carrying kGPCR provides a useful small animal model for tumorigenesis induced by a human gamma herpesvirus gene in the setting of a natural course of infection.

Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice.

Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.

Enteric bacteria promote human and mouse norovirus infection of B cells.

The cell tropism of human noroviruses and the development of an in vitro infection model remain elusive. Although susceptibility to individual human norovirus strains correlates with an individual's histo-blood group antigen (HBGA) profile, the biological basis of this restriction is unknown. We demonstrate that human and mouse noroviruses infected B cells in vitro and likely in vivo. Human norovirus infection of B cells required the presence of HBGA-expressing enteric bacteria. Furthermore, mouse norovirus replication was reduced in vivo when the intestinal microbiota was depleted by means of oral antibiotic administration. Thus, we have identified B cells as a cellular target of noroviruses and enteric bacteria as a stimulatory factor for norovirus infection, leading to the development of an in vitro infection model for human noroviruses.

A gammaherpesvirus Bcl-2 ortholog blocks B cell receptor-mediated apoptosis and promotes the survival of developing B cells in vivo.

Gammaherpesviruses such as Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) establish lifelong latency in their hosts and are associated with the development of several types of malignancies, including a subset of B cell lymphomas. These viruses are thought to co-opt the process of B cell differentiation to latently infect a fraction of circulating memory B cells, resulting in the establishment of a stable latency setpoint. However, little is known about how this infected memory B cell compartment is maintained throughout the life of the host. We have previously demonstrated that immature and transitional B cells are long-term latency reservoirs for murine gammaherpesvirus 68 (MHV68), suggesting that infection of developing B cells contributes to the maintenance of lifelong latency. During hematopoiesis, immature and transitional B cells are subject to B cell receptor (BCR)-mediated negative selection, which results in the clonal deletion of autoreactive B cells. Interestingly, numerous gammaherpesviruses encode homologs of the anti-apoptotic protein Bcl-2, suggesting that virus inhibition of apoptosis could subvert clonal deletion. To test this, we quantified latency establishment in mice inoculated with MHV68 vBcl-2 mutants. vBcl-2 mutant viruses displayed a marked decrease in the frequency of immature and transitional B cells harboring viral genome, but this attenuation could be rescued by increased host Bcl-2 expression. Conversely, vBcl-2 mutant virus latency in early B cells and mature B cells, which are not targets of negative selection, was remarkably similar to wild-type virus. Finally, in vivo depletion of developing B cells during chronic infection resulted in decreased mature B cell latency, demonstrating a key role for developing B cells in the maintenance of lifelong latency. Collectively, these findings support a model in which gammaherpesvirus latency in circulating mature B cells is sustained in part through the recurrent infection and vBcl-2-mediated survival of developing B cells.

Cyclophilin A is required for efficient human cytomegalovirus DNA replication and reactivation.

Human cytomegalovirus (HCMV) is a large DNA virus belonging to the subfamily Betaherpesvirinae. Haematopoietic cells of the myeloid lineage have been shown to harbour latent HCMV. However, following terminal differentiation of these cells, virus is reactivated, and in an immunocompromised host acute infection can occur. It is currently unknown which viral and cellular factors are involved in regulating the switch between lytic and latent infections. Cyclophilin A (CyPA) is a cellular protein that acts as a major factor in virus replication and/or virion maturation for a number of different viruses, including human immunodeficiency virus, hepatitis C virus, murine cytomegalovirus, influenza A virus and vaccinia virus. This study investigated the role of CyPA during HCMV infection. CyPA expression was silenced in human foreskin fibroblast (HF) and THP-1 cells using small interfering RNA (siRNA) technology, or the cells were treated with cyclosporin A (CsA) to inhibit CyPA activity. Silencing CyPA in HF cells with siRNA resulted in an overall reduction in virus production characterized by delayed expression of immediate-early (IE) proteins, decreased viral DNA loads and reduced titres. Furthermore, silencing of CyPA in THP-1 cells pre- and post-differentiation prevented IE protein expression and virus reactivation from a non-productive state. Interestingly, it was observed that treatment of THP-1 cells with CsA prevented the cells from establishing a fully latent infection. In summary, these results demonstrate that CyPA expression is an important factor in HCMV IE protein expression and virus production in lytically infected HF cells, and is a major component in virus reactivation from infected THP-1 cells.

HCMV protein LUNA is required for viral reactivation from latently infected primary CD14⁺ cells.

Human cytomegalovirus (HCMV) is a member of the Herpesviridae family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors in vivo. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.

Human cytomegalovirus latency-associated protein LUNA is expressed during HCMV infections in vivo.

Human cytomegalovirus (HCMV) latency is poorly understood. We previously described a novel HCMV latency-associated transcript, UL81-82ast, coding for a protein designated LUNA (latency unique natural antigen). The aim of this study was to confirm the presence of LUNA in HCMV-seropositive donors. Standard co-immunoprecipitation and ELISA assays were used to detect antibodies against the LUNA protein in the sera of HCMV-seropositive donors. Specific antibodies against LUNA were detected in all HCMV-seropositive donors but in none of the seronegative donors. These data confirm that LUNA is expressed during in vivo infections and is capable of eliciting an immune response.