Publications by authors named "Nandini Sen"

23 Publications

  • Page 1 of 1

The Use of Single Cell Mass Cytometry to Define the Molecular Mechanisms of Varicella-Zoster Virus Lymphotropism.

Front Microbiol 2020 26;11:1224. Epub 2020 Jun 26.

Department of Pediatrics, Stanford University, Stanford, CA, United States.

Unraveling the heterogeneity in biological systems provides the key to understanding of the fundamental dynamics that regulate host pathogen relationships at the single cell level. While most studies have determined virus-host cell interactions using cultured cells in bulk, recent advances in deep protein profiling from single cells enable the understanding of the dynamic response equilibrium of single cells even within the same cell types. Mass cytometry allows the simultaneous detection of multiple proteins in single cells, which helps to evaluate alterations in multiple signaling networks that work in tandem in deciding the response of a cell to the presence of a pathogen or other stimulus. In applying this technique to studying varicella zoster virus (VZV), it was possible to better understand the molecular basis for lymphotropism of the virus and how virus-induced effects on T cells promoted skin tropism. While the ability of VZV to manifest itself in the skin is well established, how the virus is transported to the skin and causes the characteristic VZV skin lesions was not well elucidated. Through mass cytometry analysis of VZV-infected tonsil T cells, we were able to observe that VZV unleashes a "remodeling" program in the infected T cells that not only makes these T cells more skin tropic but also at the same time induces changes that make these T cells unlikely to respond to immune stimulation during the journey to the skin.
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http://dx.doi.org/10.3389/fmicb.2020.01224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333520PMC
June 2020

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

Elife 2020 05 26;9. Epub 2020 May 26.

Gladstone Institute of Virology and Immunology, San Francisco, United States.

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.
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http://dx.doi.org/10.7554/eLife.55487DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250576PMC
May 2020

Self-Assessment of Psychological and Mechanical Factors Affecting Oral Hygiene Among Indian College-Going Students: A Model-Guided Study.

Int Q Community Health Educ 2020 Jul 6;40(4):307-315. Epub 2019 Nov 6.

Department of Public Health Dentistry, Pacific Dental College and Hospital, Udaipur, India.

Good oral hygiene is the foundation for a healthy mouth. This study was aimed to determine the efficacy of oral health education based on an integrated model on oral hygiene attitude and behavior among the college students of Udaipur city. An intervention study was conducted among 156 college students in Udaipur city. The questionnaire based on the new integrated model was tested for validity and reliability. Paired test and multinomial regression analysis were employed for statistical analysis. Significant differences were observed regarding all the indicators of oral hygiene practices, perceived susceptibility, seriousness, benefits, barriers, self-efficacy, and external locus of control. Odds ratio was significantly greater among undergraduate regarding oral hygiene practices. Also odds ratio of perceived susceptibility and seriousness was more among male population. The educational intervention was successful in conveying the message regarding the importance of oral hygiene practices.
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http://dx.doi.org/10.1177/0272684X19885496DOI Listing
July 2020

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

Med Pharm Rep 2019 Apr 25;92(2):165-171. Epub 2019 Apr 25.

Oral and Maxillo-Facial Surgery Department, Jaipur Dental College, Jaipur, 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" 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.
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http://dx.doi.org/10.15386/mpr-978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510351PMC
April 2019

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

Afr Health Sci 2018 Dec;18(4):1046-1056

Dept. of Public Health Dentistry, Pacific Dental College and Hospital, Debari, Udaipur, Rajasthan, India.

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.
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http://dx.doi.org/10.4314/ahs.v18i4.26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354858PMC
December 2018

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

J Virol 2018 11 12;92(21). Epub 2018 Oct 12.

Department of Pediatrics, Stanford University, Stanford, California, USA

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. 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.
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http://dx.doi.org/10.1128/JVI.01151-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189497PMC
November 2018

Association of sugary foods and drinks consumption with behavioral risk and oral health status of 12- and 15-year-old Indian school children.

J Educ Health Promot 2018 9;7:19. Epub 2018 Feb 9.

Department of Conservative Dentistry and Endodontics, Aditya Dental College, Beed, Maharashtra, India.

Aim: This study aims to assess the association of sugary foods and drinks consumption with behavioral risk and oral health status of 12- and 15-year-old government school children in Udaipur.

Materials And Methods: A descriptive cross-sectional study was conducted among of 12- and 15-year-old government schoolchildren of Udaipur. A survey pro forma designed based on HBSC (Health behaviour in School-aged Children) study protocol and WHO Oral Health Assessment Form for Children (2013) was used. Chi-Square test, Independent Sample -test, and Multinomial Logistic Regression analysis were used with 95% confidence interval and 5% significance level.

Results: Out of 710 participants, 455 (64.1%) were males and 255 females (35.9%). Majority of 15 years age (57.3%) consumed more soft drinks than 12-year-old. Males showed a comparatively greater tendency to have sugar sweetened products than females. The decayed, missing, and filled teeth (dmft) and DMFT scores were relatively higher for subjects who consumed sugary substances more than once/day than who had less than once/day. Gingivitis was associated with high sugar diet.

Conclusion: Sugary foods and drinks consumption is significantly associated with behavioral habits of children and is a clear behavioral risk for oral health.
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http://dx.doi.org/10.4103/jehp.jehp_53_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5852983PMC
February 2018

Influence of sleep disturbance, fatigue, vitality on oral health and academic performance in indian dental students.

Clujul Med 2017 15;90(3):333-343. Epub 2017 Jul 15.

Public Health Dentistry, Pacific Dental College & Hospital, Udaipur, India.

Background: Oral health and academic performance are important contributing factors for a student's professional life. Countless factors affect both, among which sleep, vitality and fatigue are less explored areas that also have a strong impact.

Objective: The objective of the study was to assess the association of sleep disturbances, fatigue and vitality with self reported oral health status, oral hygiene habits and academic performance of dental students of Udaipur.

Methods: A descriptive cross-sectional study was conducted among undergraduate and postgraduate dental students of Udaipur. Self-administered structured questionnaire was used to assess the psychological factors, vitality, sleep quality, fatigue, self reported oral health status, habits and academic performance. Analysis of variance and stepwise multiple linear regression were utilized for statistical analysis with 95% confidence level and 5% level of significance.

Results: Of the 230 participants, 180 (78.3%) were undergraduates and 50 (21.7%) were postgraduates. Among them, females showed higher scores in disturbed sleep index (2.69±2.14) as compared to males (2.45±1.91). Respondents who had "Poor" dental health, scored more in disturbed sleep index (3.15±1.64) and fatigue scale (20.00±4.88). Subjects who flossed "everyday", were found to have good sleep and more energy (p=0.01) and those who assessed themselves as excellent students scored more in the Vitality Scale (p=0.01) and less in the Sleep index (p=0.01).

Conclusion: The present study confirms that disturbed sleep, aliveness and fatigue, all are interlinked with each other and are imperative factors having the potential to alter the oral health status, habits and academics of dental students.
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http://dx.doi.org/10.15386/cjmed-749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536213PMC
July 2017

Mass Cytometric Analysis of HIV Entry, Replication, and Remodeling in Tissue CD4+ T Cells.

Cell Rep 2017 07;20(4):984-998

Gladstone Institute of Virology and Immunology, San Francisco, CA 94158, USA; Department of Urology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.
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http://dx.doi.org/10.1016/j.celrep.2017.06.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560086PMC
July 2017

Varicella-Zoster Virus Activates CREB, and Inhibition of the pCREB-p300/CBP Interaction Inhibits Viral Replication In Vitro and Skin Pathogenesis In Vivo.

J Virol 2016 10 12;90(19):8686-97. Epub 2016 Sep 12.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

Unlabelled: Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella upon primary infection and zoster upon reactivation from latency in sensory ganglion neurons. The replication of herpesviruses requires manipulation of cell signaling pathways. Notably, CREB, a factor involved in the regulation of several cellular processes, is activated upon infection of T cells with VZV. Here, we report that VZV infection also induced CREB phosphorylation in fibroblasts and that XX-650-23, a newly identified inhibitor of the phosphorylated-CREB (pCREB) interaction with p300/CBP, restricted cell-cell spread of VZV in vitro CREB phosphorylation did not require the viral open reading frame 47 (ORF47) and ORF66 kinases encoded by VZV. Evaluating the biological relevance of these observations during VZV infection of human skin xenografts in the SCID mouse model of VZV pathogenesis showed both that pCREB was upregulated in infected skin and that treatment with XX-650-23 reduced infectious-virus production and limited lesion formation compared to treatment with a vehicle control. Thus, processes of CREB activation and p300/CBP binding are important for VZV skin infection and may be targeted for antiviral drug development.

Importance: Varicella-zoster virus (VZV) is a common pathogen that causes chicken pox and shingles. As with all herpesviruses, the infection is acquired for life, and the virus can periodically reactivate from latency. Although VZV infection is usually benign with few or no deleterious consequences, infection can be life threatening in immunocompromised patients. Otherwise healthy elderly individuals who develop zoster as a consequence of viral reactivation are at risk for postherpetic neuralgia (PHN), a painful and long-lasting complication. Current vaccines use a live attenuated virus that is usually safe but cannot be given to many immunodeficient patients and retains the capacity to establish latency and reactivate, causing zoster. Antiviral drugs are effective against severe VZV infections but have little impact on PHN. A better understanding of virus-host cell interactions is relevant for developing improved therapies to safely interfere with cellular processes that are crucial for VZV pathogenesis.
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http://dx.doi.org/10.1128/JVI.00920-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021407PMC
October 2016

mRNA and Protein levels of rat pancreas specific protein disulphide isomerase are downregulated during Hyperglycemia.

Indian J Exp Biol 2016 Feb;54(2):100-7

Diabetes (Type I and Type II) which affects nearly every organ in the body is a multi-factorial non-communicable disorder. Hyperglycemia is the most characteristic feature of this disease. Loss of beta cells is common in both types of diabetes whose detailed cellular and molecular mechanisms are yet to be elucidated. As this disease is complex, identification of specific biomarkers for its early detection, management and devising new therapies is challenging. Based on the fact that functionally defective proteins provide the biochemical basis for many diseases, in this study, we tried to identify differentially expressed proteins during hyperglycemia. For that, hyperglycemia was induced in overnight fasted rats by intra-peritoneal injection of streptozotocin (STZ). The pancreas was isolated from control and treated rats for subsequent analyses. The 2D-gel electrophoresis followed by MALDI-TOF-MS-MS analyses revealed several up- and down-regulated proteins in hyperglycemic rat pancreas including the downregulation of a pancreas specific isoform of protein disulphide isomerase a2 (Pdia2).This observation was validated by western blot. Quantitative PCR experiments showed that the level of Pdia2 mRNA is also proportionally reduced in hyperglycemic pancreas.
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February 2016

Dissecting the Molecular Mechanisms of the Tropism of Varicella-Zoster Virus for Human T Cells.

J Virol 2016 Jan 20;90(7):3284-7. Epub 2016 Jan 20.

Departments of Pediatrics and Microbiology & Immunology, Stanford University, Stanford, California, USA

Studies of varicella-zoster virus (VZV) tropism for T cells support their role in viral transport to the skin during primary infection. Multiparametric single-cell mass cytometry demonstrates that, instead of preferentially infecting skin-homing T cells, VZV alters cell signaling and remodels surface proteins to enhance T cell skin trafficking. Viral proteins dispensable in skin, such as that encoded by open reading frame 66, are necessary in T cells. Interference with VZV T cell tropism may offer novel strategies for drug and vaccine design.
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http://dx.doi.org/10.1128/JVI.03375-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794656PMC
January 2016

Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

Methods 2015 Nov 23;90:85-94. Epub 2015 Jul 23.

Departments of Pediatrics, Stanford University, Stanford, CA 94025, USA; Departments of Microbiology & Immunology, Stanford University, Stanford, CA 94025, USA. Electronic address:

The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis.
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http://dx.doi.org/10.1016/j.ymeth.2015.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655147PMC
November 2015

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

Cell Rep 2014 Jul 17;8(2):633-45. Epub 2014 Jul 17.

Department of Pediatrics, Stanford University, Stanford, CA 94025, USA; Department of Microbiology and Immunology, Stanford University, Stanford, CA 94025, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2014.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4127309PMC
July 2014

Molecular mechanisms of varicella zoster virus pathogenesis.

Nat Rev Microbiol 2014 Mar 10;12(3):197-210. Epub 2014 Feb 10.

Departments of Pediatrics and of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.

Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and zoster (shingles). Investigating VZV pathogenesis is challenging as VZV is a human-specific virus and infection does not occur, or is highly restricted, in other species. However, the use of human tissue xenografts in mice with severe combined immunodeficiency (SCID) enables the analysis of VZV infection in differentiated human cells in their typical tissue microenvironment. Xenografts of human skin, dorsal root ganglia or foetal thymus that contains T cells can be infected with mutant viruses or in the presence of inhibitors of viral or cellular functions to assess the molecular mechanisms of VZV-host interactions. In this Review, we discuss how these models have improved our understanding of VZV pathogenesis.
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http://dx.doi.org/10.1038/nrmicro3215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066823PMC
March 2014

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

Proc Natl Acad Sci U S A 2012 Jan 21;109(2):600-5. Epub 2011 Dec 21.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA.

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.
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http://dx.doi.org/10.1073/pnas.1114232109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258638PMC
January 2012

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

PLoS Pathog 2011 Feb 3;7(2):e1001266. Epub 2011 Feb 3.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

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.
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http://dx.doi.org/10.1371/journal.ppat.1001266DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033373PMC
February 2011

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.

J Virol 2010 Sep 14;84(18):9240-53. Epub 2010 Jul 14.

Department of Pediatrics, Stanford University School of Medicine, Room S356, Grant Building, 300 Pasteur Drive, Stanford, CA 94305-5208, USA.

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.
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http://dx.doi.org/10.1128/JVI.01147-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937611PMC
September 2010

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

J Virol 2008 Sep 9;82(18):9115-22. Epub 2008 Jul 9.

Molecular and Cellular Biology Graduate Program, SUNY at Stony Brook, Stony Brook, NY 11794, USA.

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.
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http://dx.doi.org/10.1128/JVI.00290-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2546897PMC
September 2008

The formation of viroplasm-like structures by the rotavirus NSP5 protein is calcium regulated and directed by a C-terminal helical domain.

J Virol 2007 Nov 15;81(21):11758-67. Epub 2007 Aug 15.

Department of Medicine, SUNY at Stony Brook, Stony Brook, NY 11794, USA.

The rotavirus NSP5 protein directs the formation of viroplasm-like structures (VLS) and is required for viroplasm formation within infected cells. In this report, we have defined signals within the C-terminal 21 amino acids of NSP5 that are required for VLS formation and that direct the insolubility and hyperphosphorylation of NSP5. Deleting C-terminal residues of NSP5 dramatically increased the solubility of N-terminally tagged NSP5 and prevented NSP5 hyperphosphorylation. Computer modeling and analysis of the NSP5 C terminus revealed the presence of an amphipathic alpha-helix spanning 21 C-terminal residues that is conserved among rotaviruses. Proline-scanning mutagenesis of the predicted helix revealed that single-amino-acid substitutions abolish NSP5 insolubility and hyperphosphorylation. Helix-disrupting NSP5 mutations also abolished localization of green fluorescent protein (GFP)-NSP5 fusions into VLS and directly correlate VLS formation with NSP5 insolubility. All mutations introduced into the hydrophobic face of the predicted NSP5 alpha-helix disrupted VLS formation, NSP5 insolubility, and the accumulation of hyperphosphorylated NSP5 isoforms. Some NSP5 mutants were highly soluble but still were hyperphosphorylated, indicating that NSP5 insolubility was not required for hyperphosphorylation. Expression of GFP containing the last 68 residues of NSP5 at its C terminus resulted in the formation of punctate VLS within cells. Interestingly, GFP-NSP5-C68 was diffusely dispersed in the cytoplasm when calcium was depleted from the medium, and after calcium resupplementation GFP-NSP5-C68 rapidly accumulated into punctate VLS. A potential calcium switch, formed by two tandem pseudo-EF-hand motifs (DxDxD), is present just upstream of the predicted alpha-helix. Mutagenesis of either DxDxD motif abolished the regulatory effect of calcium on VLS formation and resulted in the constitutive assembly of GFP-NSP5-C68 into punctate VLS. These results reveal specific residues within the NSP5 C-terminal domain that direct NSP5 hyperphosphorylation, insolubility, and VLS formation in addition to defining residues that constitute a calcium-dependent trigger of VLS formation. These studies identify functional determinants within the C terminus of NSP5 that regulate VLS formation and provide a target for inhibiting NSP5-directed VLS functions during rotavirus replication.
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http://dx.doi.org/10.1128/JVI.01124-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2168809PMC
November 2007

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

J Virol 2007 Apr 31;81(8):4323-30. Epub 2007 Jan 31.

Departments of Medicine, HSC T17, Rm. 60, SUNY at Stony Brook, Stony Brook, NY 11794, and Northport VA Medical Center, NY 11768, USA.

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.
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http://dx.doi.org/10.1128/JVI.02279-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866138PMC
April 2007

The pathogenic NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I- and TBK-1-directed interferon responses.

J Virol 2006 Oct;80(19):9676-86

Molecular and Cellular Biology Graduate Program, SUNY at Stony Brook, Stony Brook, NY 11794, USA.

Hantaviruses cause two diseases with prominent vascular permeability defects, hemorrhagic fever with renal syndrome and hantavirus pulmonary syndrome. All hantaviruses infect human endothelial cells, although it is unclear what differentiates pathogenic from nonpathogenic hantaviruses. We observed dramatic differences in interferon-specific transcriptional responses between pathogenic and nonpathogenic hantaviruses at 1 day postinfection, suggesting that hantavirus pathogenesis may in part be determined by viral regulation of cellular interferon responses. In contrast to pathogenic NY-1 virus (NY-1V) and Hantaan virus (HTNV), nonpathogenic Prospect Hill virus (PHV) elicits early interferon responses following infection of human endothelial cells. We determined that PHV replication is blocked in human endothelial cells and that RNA and protein synthesis by PHV, but not NY-1V or HTNV, is inhibited at 2 to 4 days postinfection. The addition of antibodies to beta interferon (IFN-beta) blocked interferon-directed MxA induction by >90% and demonstrated that hantavirus infection induces the secretion of IFN-beta from endothelial cells. Coinfecting endothelial cells with NY-1V and PHV resulted in a 60% decrease in the induction of interferon-responsive MxA transcripts by PHV and further suggested the potential for NY-1V to regulate early IFN responses. Expression of the NY-1V G1 cytoplasmic tail inhibited by >90% RIG-I- and downstream TBK-1-directed transcription from interferon-stimulated response elements or beta-interferon promoters in a dose-dependent manner. In contrast, expression of the NY-1V nucleocapsid or PHV G1 tail had no effect on RIG-I- or TBK-1-directed transcriptional responses. Further, neither the NY-1V nor PHV G1 tails inhibited transcriptional responses directed by a constitutively active form of interferon regulatory factor 3 (IRF-3 5D), and IRF-3 is a direct target of TBK-1 phosphorylation. These findings indicate that the pathogenic NY-1V G1 protein regulates cellular IFN responses upstream of IRF-3 phosphorylation at the level of the TBK-1 complex. These findings further suggest that the G1 cytoplasmic tail contains a virulence element which determines the ability of hantaviruses to bypass innate cellular immune responses and delineates a mechanism for pathogenic hantaviruses to successfully replicate within human endothelial cells.
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http://dx.doi.org/10.1128/JVI.00508-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1617216PMC
October 2006

Translation of duck hepatitis B virus reverse transcriptase by ribosomal shunting.

J Virol 2004 Nov;78(21):11751-7

Department of Molecular Microbiology and Immunology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA.

The duck hepatitis B virus (DHBV) polymerase (P) is translated by de novo initiation from a downstream open reading frame (ORF) that partially overlaps the core (C) ORF on the bicistronic pregenomic RNA (pgRNA). The DHBV P AUG is in a poor context for translational initiation and is preceded by 14 AUGs that could intercept scanning ribosomes, yet P translation is unanticipatedly rapid. Therefore, we assessed C and P translation in the context of the pgRNA. Mutating the upstream C ORF revealed that P translation was inversely related to C translation, primarily due to occlusion of P translation by ribosomes translating C. Translation of the pgRNA was found to be cap dependent, because inserting a stem-loop (BamHI-SL) that blocked >90% of scanning ribosomes at the 5' end of the pgRNA greatly inhibited C and P synthesis. Neither mutating AUGs between the C and P start sites in contexts similar to that of the P AUG nor blocking ribosomal scanning by inserting the BamHI-SL between the C and P start codons greatly altered P translation, indicating that most ribosomes that translate P do not scan through these sequences. Finally, optimizing the P AUG context did not increase P translation. Therefore, the majority of the ribosomes that translate P are shunted from a donor region near the 5' end of the pgRNA to an acceptor site at or near the P AUG, and the shunt acceptor sequences may augment initiation at the P AUG.
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http://dx.doi.org/10.1128/JVI.78.21.11751-11757.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC523253PMC
November 2004