The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion.

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.

Lab Protocol Paper: Use of a High-throughput, Multiplex Reverse-transcription Quantitative Polymerase Chain Reaction Assay for Detection of Sabin Oral Polio Vaccine in Fecal Samples.

Background: Global polio eradication efforts rely in part on molecular methods of detecting polioviruses, both wild and vaccine strains, from human and environmental samples. Previous assays used for detection of Sabin oral polio vaccine (OPV) in fecal samples have been labor and time intensive and vary in their sensitivity and specificity. Methods: We developed a high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay able to detect all 3 OPV strains in fecal samples. The assay used a KingFisher Duo Prime system for viral RNA isolation and extraction. Positive samples were retested and Sanger sequenced for verification of Sabin serotype identity. Results: The 95% lower limit of detection was determined to be 3 copies per reaction for Sabin 1 and 3 and 4 copies per reaction for Sabin 2, with no cross-reactivity between the 3 serotypes and their primers. A total of 554 samples (3.6%) were positive, with 304 positive samples (54.9%) containing >1 serotype. Of the positive samples, 476 (85.9%) contained enough RNA to be sequenced, and of these all sequences were Sabin serotypes. The previous assay we used could process 48 samples in a 10-hour period, whereas the new assay processed >100 samples in 6 hours. Conclusions: The new high-throughput, multiplex reverse-transcription quantitative polymerase chain reaction assay allowed for sensitive and specific detection of OPV serotypes while greatly decreasing sample handling and processing time. We were able to sequence 72.4% of the 210 positive samples in the cycle threshold range of 35-37.

Validation of a High-throughput, Multiplex, Real-time Qualitative Polymerase Chain Reaction Assay for the Detection of Sabin Oral Polio Vaccine in Environmental Samples.

Background: Currently, the primary mechanism for poliovirus detection is acute flaccid paralysis (AFP) surveillance, with environmental sampling serving as a complement. However, as AFP cases drop, environmental surveillance will become increasingly critical for poliovirus detection. Mexico provides a natural environment to study oral polio vaccine (OPV) transmission, as it provides routine injected polio vaccine immunization and biannual OPV campaigns in February and May. Methods: As part of a study of OPV transmission in which 155 children were vaccinated with OPV, monthly sewage samples were collected from rivers leading from 3 indigenous Mexican villages (Capoluca, Campo Grande, and Tuxpanguillo) from February to May 2015. Samples were also collected from October 2015 to October 2017, during which time there were standard OPV campaigns. Samples were analyzed for the presence of OPV serotypes, using a real-time qualitative polymerase chain reaction assay capable of detecting as few as 9, 12, and 10 copies/100 µL of viral ribonucleic acid for OPV serotypes 1, 2, and 3 (OPV-1, -2, and -3), respectively. Included here are 54 samples, taken up to November 2016. Results: Of the 54 samples, 13 (24%) were positive for OPV. After the vaccination of 155 children in February 2015, OPV was found 2 months after vaccination. After unrestricted OPV administration in February 2016, OPV was detected in sewage up to 8 months after vaccination. OPV-3 was found in 11 of the 13 positive samples (85%), OPV-2 was found in 3 positive samples (23%), and OPV-1 was found in 1 sample (8%). Conclusions: OPV can be detected even when small amounts of the vaccine are introduced into a community, as shown by OPV-positive sewage samples even when only 155 children were vaccinated. When OPV vaccination was unrestricted, sewage samples were positive up to 8 months after vaccination, implying community OPV circulation for at least 8 months. OPV-3 was the serotype most found in these samples, indicating prolonged transmission of OPV-3 when compared to the other serotypes. Future work could compare the phylogenetic variance of OPV isolates from sewage after OPV vaccinations.

Characterization of Household and Community Shedding and Transmission of Oral Polio Vaccine in Mexican Communities With Varying Vaccination Coverage.

Background: The World Health Assembly 2012 Polio Eradication and Endgame Strategic Plan calls for the eventual cessation of all oral polio vaccines (OPVs), to be replaced with inactivated polio vaccine (IPV); however, IPV induces less robust mucosal immunity than OPV. This study characterized household and community OPV shedding and transmission after OPV vaccination within primarily IPV-vaccinated communities. Methods: Households in 3 IPV-vaccinated Mexican communities were randomized to receive 3 levels of OPV vaccination coverage (70%, 30%, or 10%). Ten stool samples were collected from all household members over 71 days. Analysis compared vaccinated subjects, household contacts of vaccinated subjects, and subjects in unvaccinated households. Logistic and Cox regression models were fitted to characterize transmission of OPV by coverage and household vaccination status. Results: Among 148 vaccinated children, 380 household contacts, and 1124 unvaccinated community contacts, 78%, 18%, and 7%, respectively, shed OPV. Community and household contacts showed no differences in transmission (odds ratio [OR], 0.67; 95% confidence interval [CI], .37-1.20), in shedding trajectory (OR, 0.61; 95% CI, .35-1.07), or in time to shedding (hazard ratio, 0.68; 95% CI, .39-1.19). Transmission began as quickly as 1 day after vaccination and persisted as long as 71 days after vaccination. Transmission within unvaccinated households differed significantly across vaccination coverage communities, with the 70% community experiencing the most transmissions (15%), and the 10% community experiencing the least (4%). These trends persisted over time and in the time to first shedding analyses. Conclusions: Transmission did not differ between household contacts of vaccinees and unvaccinated households. Understanding poliovirus transmission dynamics is important for postcertification control.

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis.

Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

Identification of a hydrophobic domain in varicella-zoster virus ORF61 necessary for ORF61 self-interaction, viral replication, and skin pathogenesis.

The varicella-zoster virus (VZV) ORF61 protein is necessary for normal replication in vitro and virulence in human skin xenografts in the severe combined immunodeficiency mouse model in vivo. These experiments identify a hydrophobic domain that mediates ORF61 self-interaction. While not needed to inhibit host cell defenses, disruption of this domain (residues 250 to 320) severely impairs VZV growth, transactivation of the immediate early 63 and glycoprotein E genes, and the pathogenesis of VZV skin infection in vivo.

ORF11 protein interacts with the ORF9 essential tegument protein in varicella-zoster virus infection.

The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin.

Structure-function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism.

Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an α-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential (724)CXXC(727) motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.

An Sp1/Sp3 site in the downstream region of varicella-zoster virus (VZV) oriS influences origin-dependent DNA replication and flanking gene transcription and is important for VZV replication in vitro and in human skin.

The distribution and orientation of origin-binding protein (OBP) sites are the main architectural contrasts between varicella-zoster virus (VZV) and herpes simplex virus (HSV) origins of DNA replication (oriS). One important difference is the absence of a downstream OBP site in VZV, raising the possibility that an alternative cis element may replace its function. Our previous work established that Sp1, Sp3, and YY1 bind to specific sites within the downstream region of VZV oriS; we hypothesize that one or both of these sites may be the alternative cis element(s). Here, we show that the mutation of the Sp1/Sp3 site decreases DNA replication and transcription from the adjacent ORF62 and ORF63 promoters following superinfection with VZV. In contrast, in the absence of DNA replication or in transfection experiments with ORF62, only ORF63 transcription is affected. YY1 site mutations had no significant effect on either process. Recombinant viruses containing these mutations were then constructed. The Sp1/Sp3 site mutant exhibited a significant decrease in virus growth in MeWo cells and in human skin xenografts, while the YY1 site mutant virus grew as well as the wild type in MeWo cells, even showing a late increase in VZV replication in skin xenografts following infection. These results suggest that the Sp1/Sp3 site plays an important role in both VZV origin-dependent DNA replication and ORF62 and ORF63 transcription and that, in contrast to HSV, these events are linked during virus replication.

Disruption of PML nuclear bodies is mediated by ORF61 SUMO-interacting motifs and required for varicella-zoster virus pathogenesis in skin.

Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier.

Entrapment of viral capsids in nuclear PML cages is an intrinsic antiviral host defense against varicella-zoster virus.

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection.

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-DeltaP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-DeltaY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.

Mutagenesis of varicella-zoster virus glycoprotein I (gI) identifies a cysteine residue critical for gE/gI heterodimer formation, gI structure, and virulence in skin cells.

Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ΔgI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and Δ105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ΔgI virus. The Δ105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the Δ105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.

Varicella zoster virus ORF25 gene product: an essential hub protein linking encapsidation proteins and the nuclear egress complex.

Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.

Herpes simplex virus-1 induces expression of a novel MxA isoform that enhances viral replication.

MxA is an antiviral protein induced by interferon (IFN)-α/ß that is known to inhibit the replication of many RNA viruses. In these experiments, the 76-kDa MxA protein expressed in IFN-α-treated cells was shown to have antiviral activity against herpes simplex virus-1 (HSV-1), a human DNA virus. However, MxA was expressed as a 56-kDa protein in HSV-1-infected cells in the absence of IFN-α. This previously unrecognized MxA isoform was produced from an alternatively spliced MxA transcript that had a deletion of Exons 14-16 and a frame shift altering the C-terminus. The variant MxA (varMxA) isoform was associated with HSV-1 regulatory proteins and virions in nuclear replication compartments. varMxA expression enhanced HSV-1 infection as shown by a reduction in infectious virus titers from cells in which MxA had been inhibited by RNA interference and by an increase in HSV-1 titers when the 56-kDa varMxA was expressed constitutively. Thus, the human MxA gene encodes two MxA isoforms, which are expressed differentially depending on whether the stimulus is IFN-α or HSV-1. These findings show that alternative splicing of cellular mRNA can result in expression of a novel isoform of a host defense gene that supports instead of restricting viral infection.

Varicella-zoster virus immediate-early protein 62 blocks interferon regulatory factor 3 (IRF3) phosphorylation at key serine residues: a novel mechanism of IRF3 inhibition among herpesviruses.

Varicella-zoster virus (VZV) is an alphaherpesvirus that is restricted to humans. VZV infection of differentiated cells within the host and establishment of latency likely require evasion of innate immunity and limited secretion of antiviral cytokines. Since interferons (IFNs) severely limit VZV replication, we examined the ability of VZV to modulate the induction of the type I IFN response in primary human embryonic lung fibroblasts (HELF). IFN-beta production was not detected, and transcription of two interferon response factor 3 (IRF3)-dependent interferon-stimulated genes (ISGs), ISG54 and ISG56, in response to poly(I:C) stimulation was downregulated in VZV-infected HELF. Inhibition of IRF3 function did not require VZV replication; the viral immediate-early protein 62 (IE62) alone was sufficient to produce this effect. IE62 blocked TBK1-mediated IFN-beta secretion and IRF3 function, as shown in an IFN-stimulated response element (ISRE)-luciferase reporter assay. However, IRF3 function was preserved if constitutively active IRF3 (IRF3-5D) was expressed in VZV-infected or IE62-transfected cells, indicating that VZV interferes with IRF3 phosphorylation. IE62-mediated inhibition was mapped to blocking phosphorylation of at least three serine residues on IRF3. However, IE62 binding to TBK1 or IRF3 was not detected and IE62 did not perturb TBK1-IRF3 complex formation. IE62-mediated inhibition of IRF3 function was maintained even if IE62 transactivator activity was disrupted. Thus, IE62 has two critical but discrete roles following VZV entry: to induce expression of VZV genes and to disarm the IFN-dependent antiviral defense through a novel mechanism that prevents IRF3 phosphorylation.

Varicella-zoster virus T cell tropism and the pathogenesis of skin infection.

Varicella-zoster virus (VZV) is a medically important human alphaherpesvirus that causes varicella and zoster. VZV initiates primary infection by inoculation of the respiratory mucosa. In the course of primary infection, VZV establishes a life-long persistence in sensory ganglia; VZV reactivation from latency may result in zoster in healthy and immunocompromised patients. The VZV genome has at least 70 known or predicted open reading frames (ORFs), but understanding how these gene products function in virulence is difficult because VZV is a highly human-specific pathogen. We have addressed this obstacle by investigating VZV infection of human tissue xenografts in the severe combined immunodeficiency mouse model. In studies relevant to the pathogenesis of primary VZV infection, we have examined VZV infection of human T cell (thymus/liver) and skin xenografts. This work supports a new paradigm for VZV pathogenesis in which VZV T cell tropism provides a mechanism for delivering the virus to skin. We have also shown that VZV-infected T cells transfer VZV to neurons in sensory ganglia. The construction of infectious VZV recombinants that have deletions or targeted mutations of viral genes or their promoters and the evaluation of VZV mutants in T cell and skin xenografts has revealed determinants of VZV virulence that are important for T cell and skin tropism in vivo.

Analysis of the functions of glycoproteins E and I and their promoters during VZV replication in vitro and in skin and T-cell xenografts in the SCID mouse model of VZV pathogenesis.

The two VZV glycoproteins, gE and gI, are encoded by genes that are designated open reading frames, ORF67 and ORF68, located in the short unique region of the VZV genome. These proteins have homologs in the other alphaherpesviruses. Like their homologues, VZV gE and gI exhibit prominent co-localization in infected cells and form heterodimers. However, VZV gE is much larger than its homologues because it has a unique N-terminal domain, consisting of 188 amino acids that are not present in these other gene products. VZV gE also differs from the related gE proteins, in that it is essential for viral replication. Targeted mutations of gE that are compatible with VZV replication in cultured cells have varying phenotypes in skin and T-cell xenografts in the SCID mouse model of VZV pathogenesis in vivo. While gI is dispensable for growth in cultured cells in vitro, this glycoprotein is essential for VZV infection of differentiated human skin and T cells in vivo. The promoter regions of gE and gI are regulated by the cellular transactivator, specificity protein factor 1 (Sp1) in combination with the major VZV transactivator in reporter construct experiments and some Sp1 promoter elements are important for VZV virulence in vivo. Further analysis of VZV gE and gI functions and their interactions with other viral and host cell proteins are important areas for studies of VZV replication and pathogenesis.

Regulation of the ORF61 promoter and ORF61 functions in varicella-zoster virus replication and pathogenesis.

Varicella-zoster virus (VZV) open reading frame 61 (ORF61) encodes a protein that transactivates viral and cellular promoters in transient-transfection assays and is the ortholog of herpes simplex virus ICP0. In this report, we mapped the ORF61 promoter and investigated its regulation by viral and cellular proteins in transient-expression experiments and by mutagenesis of the VZV genome (parent Oka strain). The 5' boundary of the minimal ORF61 promoter required for IE62 transactivation was mapped to position -95 relative to the mRNA start site, and three noncanonical GT-rich Sp1-binding sites were documented to occur within the region comprising positions -95 to -45. Contributions of the three Sp1-binding-site motifs, designated Sp1a, Sp1b, and Sp1c, to ORF61 expression and viral replication were varied despite their similar sequences. Two sites, Sp1a and Sp1c, functioned synergistically. When both sites were mutated in the pOka genome to produce pOka-61proDeltaSp1ac, the mutant virus expressed significantly less ORF61 protein. Using this mutant to investigate ORF61 functions resulted in reductions in the expression levels of IE proteins, viral kinases ORF47 and ORF66, and the major glycoprotein gE, with the most impact on gE. Virion morphogenesis appeared to be intact despite minimal ORF61 expression. Pretreating melanoma cells with sodium butyrate enhanced titers of pOka-61proDeltaSp1ac but not pOka, suggesting that ORF61 has a role in histone deacetylase inhibition. Growth of pOka-61proDeltaSp1ac was impaired in SCIDhu skin xenografts, indicating that the regulation of the ORF61 promoter by Sp1 family proteins is important for ORF61 expression in vivo and that ORF61 contributes to VZV virulence at skin sites of replication.