Modulation of Host Cell Signaling Pathways by Varicella-Zoster Virus.

Host-pathogen interactions involve complex inside-out and outside-in signal transmission through critical cellular networks that dictate disease outcomes. The phosphoinositide 3-kinase (PI3K)/Akt pathway is a pivotal junction that regulates several cell functions, and phospho-Akt (pAkt) is often found to be constitutively active in cancer cells, similar to phospho-STAT3. In this chapter, we discuss the regulation of PI3K/Akt pathway in VZV infected cells and of other pathways including p53 which, unlike pAkt and pSTAT3, directs cells towards apoptosis. The fine spatio-temporal balance of activation of pro- and anti-apoptotic factors during VZV infection likely provides an optimum environment for the virus to replicate and cause disease in the human host.

HIV efficiently infects T cells from the endometrium and remodels them to promote systemic viral spread.

The female reproductive tract (FRT) is the most common site of infection during HIV transmission to women, but viral remodeling complicates characterization of cells targeted for infection. Here, we report extensive phenotypic analyses of HIV-infected endometrial cells by CyTOF, and use a 'nearest neighbor' bioinformatics approach to trace cells to their original pre-infection phenotypes. Like in blood, HIV preferentially targets memory CD4+ T cells in the endometrium, but these cells exhibit unique phenotypes and sustain much higher levels of infection. Genital cell remodeling by HIV includes downregulating TCR complex components and modulating chemokine receptor expression to promote dissemination of infected cells to lymphoid follicles. HIV also upregulates the anti-apoptotic protein BIRC5, which when blocked promotes death of infected endometrial cells. These results suggest that HIV remodels genital T cells to prolong viability and promote viral dissemination and that interfering with these processes might reduce the likelihood of systemic viral spread.

Caries risk assessment using Cariogram model among smokeless tobacco users in India.

BACKGROUND: Smokeless tobacco forms are known to have fermentable sugar compounds which may strengthen the development of cariogenic microbes. In addition, cervical abrasion of teeth occur at the site of tobacco pouch placement. These components may assume an essential role in caries advancement in smokeless tobacco users. OBJECTIVE: The objective of the study was to assess caries risk among smokeless tobacco users using Cariogram model. METHODS: A descriptive cross sectional study was conducted among 50 smokeless tobacco users of Udaipur for 3 months. Caries risk assessment was done by employing a proforma survey based on the Cariogram Model. Statistical analysis included descriptive statistics, Chi-square test and Stepwise multiple linear regression with 95% confidence interval and 5% significance level. RESULTS: The majority of the smokeless tobacco users (46%) were found to be in the "Moderate" Streptococcus mutans count category and portrayed "Moderate" plaque amount score (82%). Smokeless tobacco users (34%) depicted a higher caries risk profile than the control group (6%) utilizing the Cariogram model. CONCLUSION: Cariogram model could be a useful tool to represent caries risk among smokeless tobacco users.

Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus.

Both type I and type II interferons (IFNs) have been implicated in the host defense against varicella-zoster virus (VZV), a common human herpesvirus that causes varicella and zoster. The purpose of this study was to compare their contributions to the control of VZV replication, to identify the signaling pathways that are critical for mediating their antiviral activity, and to define the mechanisms by which the virus counteracts their effects. Gamma interferon (IFN-γ) was much more potent than IFN-α in blocking VZV infection, which was associated with a differential induction of the interferon regulatory factor (IRF) proteins IRF1 and IRF9, respectively. These observations account for the clinical experience that while the formation of VZV skin lesions is initially controlled by local immunity, adaptive virus-specific T cell responses are required to prevent life-threatening VZV infections.IMPORTANCE While both type I and type II IFNs are involved in the control of herpesvirus infections in the human host, to our knowledge, their relative contributions to the restriction of viral replication and spread have not been assessed. We report that IFN-γ has more potent activity than IFN-α against VZV. Findings from this comparative analysis show that the IFN-α-IRF9 axis functions as a first line of defense to delay the onset of viral replication and spread, whereas the IFN-γ-IRF1 axis has the capacity to block the infectious process. Our findings underscore the importance of IRFs in IFN regulation of herpesvirus infection and account for the clinical experience of the initial control of VZV skin infection attributable to IFN-α production, together with the requirement for induction of adaptive IFN-γ-producing VZV-specific T cells to resolve the infection.

A comparative assessment of caries risk using cariogram among smokers and smokeless tobacco users in india - a cross-sectional study.

BACKGROUND: A dearth of literature exists concerning utilization of the unique cariogram model for caries risk assessment in tobacco users. OBJECTIVE: To assess & compare caries risk among smokers & smokeless tobacco users using Cariogram model. METHODS: A descriptive cross sectional study was conducted among smokers and smokeless tobacco users of Udaipur for 3 months. Caries risk assessment was done by employing a survey proforma based on the Cariogram model. Statistical analysis included descriptive statistics, Chi-square test followed by Marascuilo procedure and Stepwise multiple linear regression with 95% confidence interval and 5% significance level. RESULTS: Majority of the smokers (56%) portrayed high caries risk (less chance to avoid new caries) followed by smokeless Tobacco users (34%). Only 40% smokeless tobacco users had relatively high chances (>60%) of avoiding future new caries. The susceptibility sector of the cariogram model contributed primarily to caries risk in the study population. CONCLUSION: The study findings from the different cariogram elements converged to indicate that smokers were at maximum caries risk, followed by smokeless tobacco users and therefore Cariogram model could be a useful tool to represent caries risk among smokers and smokeless tobacco users.

Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus.

Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.

Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis.

Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by γ-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses.

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.

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.

The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1-TRAF3 complex formation.

Pathogenic hantaviruses replicate within human endothelial cells and cause two diseases, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. In order to replicate in endothelial cells pathogenic hantaviruses inhibit the early induction of beta interferon (IFN-beta). Expression of the cytoplasmic tail of the pathogenic NY-1 hantavirus Gn protein is sufficient to inhibit RIG-I- and TBK1-directed IFN responses. The formation of TBK1-TRAF3 complexes directs IRF-3 phosphorylation, and both IRF-3 and NF-kappaB activation are required for transcription from the IFN-beta promoter. Here we report that the NY-1 virus (NY-1V) Gn tail inhibits both TBK1-directed NF-kappaB activation and TBK1-directed transcription from promoters containing IFN-stimulated response elements. The NY-1V Gn tail coprecipitated TRAF3 from cellular lysates, and analysis of TRAF3 deletion mutants demonstrated that the TRAF3 N terminus is sufficient for interacting with the NY-1V Gn tail. In contrast, the Gn tail of the nonpathogenic hantavirus Prospect Hill virus (PHV) failed to coprecipitate TRAF3 or inhibit NF-kappaB or IFN-beta transcriptional responses. Further, expression of the NY-1V Gn tail blocked TBK1 coprecipitation of TRAF3 and infection by NY-1V, but not PHV, blocked the formation of TBK1-TRAF3 complexes. These findings indicate that the NY-1V Gn cytoplasmic tail forms a complex with TRAF3 which disrupts the formation of TBK1-TRAF3 complexes and downstream signaling responses required for IFN-beta transcription.

Degrons at the C terminus of the pathogenic but not the nonpathogenic hantavirus G1 tail direct proteasomal degradation.

Pathogenic hantaviruses cause two human diseases: hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). The hantavirus G1 protein contains a long, 142-amino-acid cytoplasmic tail, which in NY-1 virus (NY-1V) is ubiquitinated and proteasomally degraded (E. Geimonen, I. Fernandez, I. N. Gavrilovskaya, and E. R. Mackow, J. Virol. 77: 10760-10768, 2003). Here we report that the G1 cytoplasmic tails of pathogenic Andes (HPS) and Hantaan (HFRS) viruses are also degraded by the proteasome and that, in contrast, the G1 tail of nonpathogenic Prospect Hill virus (PHV) is stable and not proteasomally degraded. We determined that the signals which direct NY-1V G1 tail degradation are present in a hydrophobic region within the C-terminal 30 residues of the protein. In contrast to that of PHV, the NY-1V hydrophobic domain directs the proteasomal degradation of green fluorescent protein and constitutes an autonomous degradation signal, or "degron," within the NY-1V G1 tail. Replacing 4 noncontiguous residues of the NY-1V G1 tail with residues present in the stable PHV G1 tail resulted in a NY-1V G1 tail that was not degraded by the proteasome. In contrast, changing a different but overlapping set of 4 PHV residues to corresponding NY-1V residues directed proteasomal degradation of the PHV G1 tail. The G1 tails of pathogenic, but not nonpathogenic, hantaviruses contain intervening hydrophilic residues within the C-terminal hydrophobic domain, and amino acid substitutions that alter the stability or degradation of NY-1V or PHV G1 tails result from removing or adding intervening hydrophilic residues. Our results identify residues that selectively direct the proteasomal degradation of pathogenic hantavirus G1 tails. Although a role for the proteasomal degradation of the G1 tail in HPS or HFRS is unclear, these findings link G1 tail degradation to viral pathogenesis and suggest that degrons within hantavirus G1 tails are potential virulence determinants.