Publications by authors named "Jaya Rajamani"

21 Publications

  • Page 1 of 1

Correlation of ROS1 Immunohistochemistry With Fusion Status Determined by Fluorescence In Situ Hybridization.

Authors:
Richard S P Huang Derek Smith Catherine H Le Wen-Wei Liu Ellen Ordinario Chitra Manohar Michael Lee Jaya Rajamani Huan Truong Jing Li Cindy Choi Jingchuan Li Amrita Pati Lukas Bubendorf Reinhard Buettner Keith M Kerr Fernando Lopez-Rios Antonio Marchetti Ivonne Marondel Andrew G Nicholson Ayşim Büge Öz Patrick Pauwels Frederique Penault-Llorca Giulio Rossi Erik Thunnissen Amy Hanlon Newell Greg Pate Ina Menzl

Arch Pathol Lab Med 2020 06 11;144(6):735-741. Epub 2019 Sep 11.

From Roche Molecular Solutions, Pleasanton, California (Dr Huang, Mr Smith, Dr Le, Mr Liu, Dr Ordinario, Dr Manohar, Mr Lee, Mr Rajamani, Mr Truong, Dr Jing Li, Ms Choi, Dr Jingchuan Li, Dr Pati, Dr Hanlon Newell, Mr Pate, and Dr Menzl); Institute of Pathology, University Hospital Basel, Basel, Switzerland (Dr Bubendorf); Institute for Pathology, University Hospital, Cologne, Germany (Dr Buettner); the Department of Pathology, Aberdeen University Medical School & Aberdeen Royal Infirmary, Foresterhill, Aberdeen, United Kingdom (Dr Kerr); Laboratorio de Dianas Terapeuticas, HM Hospitales, Spain (Dr Lopez-Rios); Center of Predictive Molecular Medicine, CeSIMeT, University of Chieti-Pescara, Italy (Dr Marchetti); Pfizer Oncology, International Developed Markets, Berlin, Germany (Dr Marondel); the Department of Histopathology, Royal Brompton and Harefield Hospitals, and National Heart and Lung Division, Imperial College, London, United Kingdom (Dr Nicholson); the Pathology Department, Istanbul University, Cerrahpassa Medical Faculty, Istanbul, Turkey (Dr Öz); CORE, Antwerp University, and the Department of Pathology, Antwerp University Hospital, Antwerp, Belgium (Dr Pauwels); the Department of Biopathology, Centre Jean Perrin & INSERM U240 IMoST and Université Clermont Auvergne, Clermont-Ferrand, France (Dr Penault-Llorca); Operative Unit of Pathology, AUSL della Romagna, Hospital S. Maria delle Croci, Ravenna, Italy (Dr Rossi); and the Department of Pathology, VU University Medical Center, Amsterdam, the Netherlands (Dr Thunnissen).

Context.—: The ability to determine status has become mandatory for patients with lung adenocarcinoma, as many global authorities have approved crizotinib for patients with -positive lung adenocarcinoma.

Objective.—: To present analytical correlation of the VENTANA ROS1 (SP384) Rabbit Monoclonal Primary Antibody (ROS1 [SP384] antibody) with fluorescence in situ hybridization (FISH).

Design.—: The immunohistochemistry (IHC) and FISH analytical comparison was assessed by using 122 non-small cell lung cancer samples that had both FISH (46 positive and 76 negative cases) and IHC staining results available. In addition, reverse transcription-polymerase chain reaction (RT-PCR) as well as DNA and RNA next-generation sequencing (NGS) were used to further examine the ROS1 status in cases that were discrepant between FISH and IHC, based on staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive. Here, we define the consensus status as the most frequent result across the 5 different methods (IHC, FISH, RT-PCR, RNA NGS, and DNA NGS) we used to determine ROS1 status in these cases.

Results.—: Of the IHC scoring methods examined, staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells considered as IHC positive had the highest correlation with a FISH-positive status, reaching a positive percentage agreement of 97.8% and negative percentage agreement of 89.5%. A positive percentage agreement (100%) and negative percentage agreement (92.0%) was reached by comparing ROS1 (SP384) using a cutoff for staining in the cytoplasm of 2+ or above in more than 30% of total tumor cells to the consensus status.

Conclusions.—: Herein, we present a standardized staining protocol for ROS1 (SP384) and data that support the high correlation between ROS1 status and ROS1 (SP384) antibody.
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http://dx.doi.org/10.5858/arpa.2019-0085-OADOI Listing
June 2020

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

Authors:
Li Wang Jaya Rajamani Marvin Sommer Leigh Zerboni Ann M Arvin

J Virol 2013 Apr 23;87(7):4075-9. Epub 2013 Jan 23.

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

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.
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http://dx.doi.org/10.1128/JVI.02963-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624212PMC
April 2013

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

Authors:
Nandini Sen Xibing Che Jaya Rajamani Leigh Zerboni Phillip Sung Jason Ptacek Ann M Arvin

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

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

Authors:
Susan E Vleck Stefan L Oliver Jennifer J Brady Helen M Blau Jaya Rajamani Marvin H Sommer Ann M Arvin

Proc Natl Acad Sci U S A 2011 Nov 24;108(45):18412-7. Epub 2011 Oct 24.

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

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.
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http://dx.doi.org/10.1073/pnas.1111333108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215059PMC
November 2011

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

Authors:
Li Wang Stefan L Oliver Marvin Sommer Jaya Rajamani Mike Reichelt Ann M Arvin

PLoS Pathog 2011 Aug 25;7(8):e1002157. Epub 2011 Aug 25.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1002157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161977PMC
August 2011

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.

Authors:
Stefan L Oliver Marvin H Sommer Mike Reichelt Jaya Rajamani Leonssia Vlaycheva-Beisheim Shaye Stamatis Jason Cheng Carol Jones James Zehnder Ann M Arvin

J Virol 2011 May 23;85(9):4095-110. Epub 2011 Feb 23.

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

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.
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http://dx.doi.org/10.1128/JVI.02596-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126246PMC
May 2011

Identification and functional characterization of the Varicella zoster virus ORF11 gene product.

Authors:
Xibing Che Stefan L Oliver Marvin H Sommer Jaya Rajamani Mike Reichelt Ann M Arvin

Virology 2011 Mar 26;412(1):156-66. Epub 2011 Jan 26.

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

The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphaherpesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.
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http://dx.doi.org/10.1016/j.virol.2010.12.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068617PMC
March 2011

Varicella-zoster virus glycoprotein E is a critical determinant of virulence in the SCID mouse-human model of neuropathogenesis.

Authors:
Leigh Zerboni Barbara Berarducci Jaya Rajamani Carol D Jones James L Zehnder Ann Arvin

J Virol 2011 Jan 20;85(1):98-111. Epub 2010 Oct 20.

Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305, USA.

Varicella-zoster virus (VZV) is a neurotropic alphaherpesvirus. VZV infection of human dorsal root ganglion (DRG) xenografts in immunodeficient mice models the infection of sensory ganglia. We examined DRG infection with recombinant VZV (recombinant Oka [rOka]) and the following gE mutants: gEΔ27-90, gEΔCys, gE-AYRV, and gE-SSTT. gEΔ27-90, which lacks the gE domain that interacts with a putative receptor insulin-degrading enzyme (IDE), replicated as extensively as rOka, producing infectious virions and significant cytopathic effects within 14 days of inoculation. Since neural cells express IDE, the gE/IDE interaction was dispensable for VZV neurotropism. In contrast, gEΔCys, which lacks gE/gI heterodimer formation, was significantly impaired at early times postinfection; viral genome copy numbers increased slowly, and infectious virus production was not detected until day 28. Delayed replication was associated with impaired cell-cell spread in ganglia, similar to the phenotype of a gI deletion mutant (rOkaΔgI). However, at later time points, infection of satellite cells and other supportive nonneuronal cells resulted in extensive DRG tissue damage and cell loss such that cytopathic changes observed at day 70 were more severe than those for rOka-infected DRG. The replication of gE-AYRV, which is impaired for trans-Golgi network (TGN) localization, and the replication of gE-SSTT, which contains mutations in an acidic cluster, were equivalent to that of rOka, causing significant cytopathic effects and infectious virus production by day 14; genome copy numbers were equivalent to those of rOka. These experiments suggest that the gE interaction with cellular IDE, gE targeting to TGN sites of virion envelopment, and phosphorylation at SSTT are dispensable for VZV DRG infection, whereas the gE/gI interaction is critical for VZV neurovirulence.
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http://dx.doi.org/10.1128/JVI.01902-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3014186PMC
January 2011

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

Authors:
Chia-Chi Ku Xi-Bing Che Mike Reichelt Jaya Rajamani Anne Schaap-Nutt Ke-Jung Huang Marvin H Sommer Yi-Shun Chen Yi-Yuan Chen Ann M Arvin

Immunol Cell Biol 2011 Feb 6;89(2):173-82. Epub 2010 Jul 6.

The Graduate Institute of Immunology, College of Medicine, National Taiwan University, Taipei, Taiwan, ROC.

MxA is an antiviral protein induced by interferon (IFN)-α/β 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.
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http://dx.doi.org/10.1038/icb.2010.83DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520797PMC
February 2011

Expression of varicella-zoster virus immediate-early regulatory protein IE63 in neurons of latently infected human sensory ganglia.

Authors:
Leigh Zerboni Raymond A Sobel Vasavi Ramachandran Jaya Rajamani William Ruyechan Allison Abendroth Ann Arvin

J Virol 2010 Apr 27;84(7):3421-30. Epub 2010 Jan 27.

Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, S-356, 300 Pasteur Dr., Stanford, CA 94305, USA.

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory nerve ganglia, but the characteristics of VZV latency are not well defined. Immunohistochemical detection of the VZV immediate-early 63 (IE63) protein in ganglion neurons has been described, but there are significant discrepancies in estimates of the frequency of IE63-positive neurons, varying from a rare event to abundant expression. We examined IE63 expression in cadaver ganglia using a high-potency rabbit anti-IE63 antibody and corresponding preimmune serum. Using standard immunohistochemical techniques, we evaluated 10 ganglia that contained VZV DNA from seven individuals. These experiments showed that neuronal pigments were a confounding variable; however, by examining sections coded to prevent investigator bias and applying statistical analysis, we determined that IE63 protein, if present, is in a very small proportion of neurons (<2.8%). To refine estimates of IE63 protein abundance, we modified our protocol by incorporating a biological stain to exclude the pigment signal and evaluated 27 ganglia from 18 individuals. We identified IE63 protein in neurons within only one ganglion, in which VZV glycoprotein E and an immune cell infiltrate were also demonstrated. Antigen preservation was shown by detection of neuronal synaptophysin. These data provide evidence that the expression of IE63 protein, which has been referred to as a latency-associated protein, is rare. Refining estimates of VZV protein expression in neurons is important for developing a hypothesis about the mechanisms by which VZV latency may be maintained.
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http://dx.doi.org/10.1128/JVI.02416-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2838126PMC
April 2010

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

Authors:
Barbara Berarducci Jaya Rajamani Leigh Zerboni Xibing Che Marvin Sommer Ann M Arvin

Proc Natl Acad Sci U S A 2010 Jan 4;107(1):282-7. Epub 2009 Dec 4.

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

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.
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http://dx.doi.org/10.1073/pnas.0912373107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806775PMC
January 2010

Anti-glycoprotein H antibody impairs the pathogenicity of varicella-zoster virus in skin xenografts in the SCID mouse model.

Authors:
Susan E Vleck Stefan L Oliver Mike Reichelt Jaya Rajamani Leigh Zerboni Carol Jones James Zehnder Charles Grose Ann M Arvin

J Virol 2010 Jan;84(1):141-52

Stanford University, Stanford, CA 94305, USA.

Varicella-zoster virus (VZV) infection is usually mild in healthy individuals but can cause severe disease in immunocompromised patients. Prophylaxis with varicella-zoster immunoglobulin can reduce the severity of VZV if given shortly after exposure. Glycoprotein H (gH) is a highly conserved herpesvirus protein with functions in virus entry and cell-cell spread and is a target of neutralizing antibodies. The anti-gH monoclonal antibody (MAb) 206 neutralizes VZV in vitro. To determine the requirement for gH in VZV pathogenesis in vivo, MAb 206 was administered to SCID mice with human skin xenografts inoculated with VZV. Anti-gH antibody given at 6 h postinfection significantly reduced the frequency of skin xenograft infection by 42%. Virus titers, genome copies, and lesion size were decreased in xenografts that became infected. In contrast, administering anti-gH antibody at 4 days postinfection suppressed VZV replication but did not reduce the frequency of infection. The neutralizing anti-gH MAb 206 blocked virus entry, cell fusion, or both in skin in vivo. In vitro, MAb 206 bound to plasma membranes and to surface virus particles. Antibody was internalized into vacuoles within infected cells, associated with intracellular virus particles, and colocalized with markers for early endosomes and multivesicular bodies but not the trans-Golgi network. MAb 206 blocked spread, altered intracellular trafficking of gH, and bound to surface VZV particles, which might facilitate their uptake and targeting for degradation. As a consequence, antibody interference with gH function would likely prevent or significantly reduce VZV replication in skin during primary or recurrent infection.
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http://dx.doi.org/10.1128/JVI.01338-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798403PMC
January 2010

Mutagenesis of varicella-zoster virus glycoprotein B: putative fusion loop residues are essential for viral replication, and the furin cleavage motif contributes to pathogenesis in skin tissue in vivo.

Authors:
Stefan L Oliver Marvin Sommer Leigh Zerboni Jaya Rajamani Charles Grose Ann M Arvin

J Virol 2009 Aug 27;83(15):7495-506. Epub 2009 May 27.

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

Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Delta491RSRR494 and point mutation 491GSGG494 in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Delta491RSRR494 or 491GSGG494 mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBDelta491RSRR494 or pOka-gB491GSGG494 virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.
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http://dx.doi.org/10.1128/JVI.00400-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708640PMC
August 2009

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

Authors:
Li Wang Marvin Sommer Jaya Rajamani Ann M Arvin

J Virol 2009 Aug 20;83(15):7560-72. Epub 2009 May 20.

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

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.
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http://dx.doi.org/10.1128/JVI.00118-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708633PMC
August 2009

Deletion of the first cysteine-rich region of the varicella-zoster virus glycoprotein E ectodomain abolishes the gE and gI interaction and differentially affects cell-cell spread and viral entry.

Authors:
Barbara Berarducci Jaya Rajamani Mike Reichelt Marvin Sommer Leigh Zerboni Ann M Arvin

J Virol 2009 Jan 22;83(1):228-40. Epub 2008 Oct 22.

Institut Pasteur, Départment de Virologie, 25 rue du Dr Roux, 75015 Paris, France.

Varicella-zoster virus (VZV) glycoprotein E (gE) is the most abundant glycoprotein in infected cells and, in contrast to those of other alphaherpesviruses, is essential for viral replication. The gE ectodomain contains a unique N-terminal region required for viral replication, cell-cell spread, and secondary envelopment; this region also binds to the insulin-degrading enzyme (IDE), a proposed VZV receptor. To identify new functional domains of the gE ectodomain, the effect of mutagenesis of the first cysteine-rich region of the gE ectodomain (amino acids 208 to 236) was assessed using VZV cosmids. Deletion of this region was compatible with VZV replication in vitro, but cell-cell spread of the rOka-DeltaCys mutant was reduced significantly. Deletion of the cysteine-rich region abolished the binding of the mutant gE to gI but not to IDE. Preventing gE binding to gI altered the pattern of gE expression at the plasma membrane of infected cells and the posttranslational maturation of gI and its incorporation into viral particles. In contrast, deletion of the first cysteine-rich region did not affect viral entry into human tonsil T cells in vitro or into melanoma cells infected with cell-free VZV. These experiments demonstrate that gE/gI heterodimer formation is essential for efficient cell-cell spread and incorporation of gI into viral particles but that it is dispensable for infectious varicella-zoster virion formation and entry into target cells. Blocking gE binding to gI resulted in severe impairment of VZV infection of human skin xenografts in SCIDhu mice in vivo, documenting the importance of cell fusion mediated by this complex for VZV virulence in skin.
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http://dx.doi.org/10.1128/JVI.00913-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612333PMC
January 2009

Development of recombinant varicella-zoster viruses expressing luciferase fusion proteins for live in vivo imaging in human skin and dorsal root ganglia xenografts.

Authors:
Stefan L Oliver Leigh Zerboni Marvin Sommer Jaya Rajamani Ann M Arvin

J Virol Methods 2008 Dec 24;154(1-2):182-93. Epub 2008 Sep 24.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, United States.

Varicella-zoster virus (VZV) is a host specific human pathogen that has been studied using human xenografts in SCID mice. Live whole-animal imaging is an emerging technique to measure protein expression in vivo using luminescence. Currently, it has only been possible to determine VZV protein expression in xenografts postmortem. Therefore, to measure immediate early (IE63) and late (glycoprotein E [gE]) protein expression in vivo viruses expressing IE63 or gE as luciferase fusion proteins were generated. Viable recombinant viruses pOka-63-luciferase and pOka-63/70-luciferase, which had luciferase genes fused to ORF63 and its duplicate ORF70, or pOka-gE-CBR were recovered that expressed IE63 or gE as fusion proteins and generated luminescent plaques. In contrast to pOka-63/70-luciferase viruses, the luciferase gene was rapidly lost in vitro when fused to a single copy of ORF63 or ORF68. IE63 expression was successfully measured in human skin and dorsal root ganglia xenografts infected with the genomically stable pOka-63/70-luciferase viruses. The progress of VZV infection in dorsal root ganglia xenografts was delayed in valacyclovir treated mice but followed a similar trend in untreated mice when the antiviral was withdrawn 28 days post-inoculation. Thus, IE63-luciferase fusion proteins were effective for investigating VZV infection and antiviral activity in human xenografts.
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http://dx.doi.org/10.1016/j.jviromet.2008.07.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2657092PMC
December 2008

Functions of Varicella-zoster virus ORF23 capsid protein in viral replication and the pathogenesis of skin infection.

Authors:
Vaishali Chaudhuri Marvin Sommer Jaya Rajamani Leigh Zerboni Ann M Arvin

J Virol 2008 Oct 6;82(20):10231-46. Epub 2008 Aug 6.

Stanford University School of Medicine, 300 Pasteur Dr., S-354, Stanford, CA 94305, USA.

The assembly of herpesvirus capsids is a complex process involving interactions of multiple proteins in the cytoplasm and in the nucleus. Based on comparative genome analyses, varicella-zoster virus (VZV) open reading frame 23 (ORF23) encodes a conserved capsid protein, referred to as VP26 (UL35) in other alphaherpesviruses. Mutagenesis using a VZV bacterial artificial chromosome system showed that ORF23 was dispensable for replication in vitro. However, the absence of ORF23 disrupted capsid assembly in a melanoma cell line. Expression of ORF23 as a red fluorescent protein (RFP) fusion protein appeared to have a dominant negative effect on replication that was rescued by ORF23 expression from a nonnative site in the VZV genome. In contrast to its VP26 homolog, ORF23 has an intrinsic nuclear localization capacity that was mapped to an SRSRVV motif at residues 229 to 234 in the extreme C terminus of ORF23. In addition, coexpression with ORF23 resulted in nuclear import of the major capsid protein, ORF40. VZV ORF33.5 also translocated ORF40, which may provide a redundant mechanism in vitro but appears insufficient to overcome the dominant negative effect of the monomeric RFP-ORF23 (mRFP23) fusion protein. ORF23 was required for VZV infection of human skin xenografts, indicating that ORF33.5 does not compensate for lack of ORF23 in vivo. These observations suggest a model of VZV capsid assembly in which nuclear transport of the major capsid protein and associated proteins requires ORF23 during VZV replication in the human host. If so, ORF23 expression could be a target for a novel antiviral drug against VZV.
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http://dx.doi.org/10.1128/JVI.01890-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566272PMC
October 2008

Functions of the ORF9-to-ORF12 gene cluster in varicella-zoster virus replication and in the pathogenesis of skin infection.

Authors:
Xibing Che Mike Reichelt Marvin H Sommer Jaya Rajamani Leigh Zerboni Ann M Arvin

J Virol 2008 Jun 9;82(12):5825-34. Epub 2008 Apr 9.

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

The gene cluster composed of varicella-zoster virus (VZV) open reading frame 9 (ORF9) to ORF12 encodes four putative tegument proteins and is highly conserved in most alphaherpesviruses. In these experiments, the genes within this cluster were deleted from the VZV parent Oka (POKA) individually or in combination, and the consequences for VZV replication were evaluated with cultured cells in vitro and with human skin xenografts in SCID mice in vivo. As has been reported for ORF10, ORF11 and ORF12 were dispensable for VZV replication in melanoma and human embryonic fibroblast cells. In contrast, deletion of ORF9 was incompatible with the recovery of infectious virus. ORF9 localized to the virion tegument and formed complexes with glycoprotein E, which is an essential protein, in VZV-infected cells. Recombinants lacking ORF10 and ORF11 (POKADelta10/11), ORF11 and ORF12 (POKADelta11/12), or ORF10, ORF11 and ORF12 (POKADelta10/11/12) were viable in cultured cells. Their growth kinetics did not differ from those of POKA, and nucleocapsid formation and virion assembly were not disrupted. In addition, these deletion mutants showed no differences compared to POKA in infectivity levels for primary human tonsil T cells. Deletion of ORF12 had no effect on skin infection, whereas replication of POKADelta11, POKADelta10/11, and POKADelta11/12 was severely reduced, and no virus was recovered from skin xenografts inoculated with POKADelta10/11/12. These results indicate that with the exception of ORF9, the individual genes within the ORF9-to-ORF12 gene cluster are dispensable and can be deleted simultaneously without any apparent effect on VZV replication in vitro but that the ORF10-to-ORF12 cluster is essential for VZV virulence in skin in vivo.
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http://dx.doi.org/10.1128/JVI.00303-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395146PMC
June 2008

Cellular and viral factors regulate the varicella-zoster virus gE promoter during viral replication.

Authors:
Barbara Berarducci Marvin Sommer Leigh Zerboni Jaya Rajamani Ann M Arvin

J Virol 2007 Oct 18;81(19):10258-67. Epub 2007 Jul 18.

Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Dr., Rm. G312, Stanford, CA 94305-5208, USA.

Varicella-zoster virus (VZV) glycoprotein E (gE) is essential for viral replication and is involved in cell-to-cell spread, secondary envelopment, and entry. We created a set of mutations in the gE promoter to investigate the role of viral and cellular transcriptional factors in regulation of the gE promoter. Deletion or point mutation of the two Sp1 sites in the gE promoter abolished Sp1 binding and IE62-mediated transactivation of the gE promoter in vitro. Incorporation of the deletion or the point mutations disrupting both of the Sp1 binding sites into the VZV genome was not compatible with viral replication. A point mutation altering the atypical Sp1 binding site was lethal, while altering the second site impaired VZV replication significantly, indicating functional differences between the two Sp1 binding sites. Deletions in the gE promoter that abolished putative binding sites for cellular transcriptional factors other than Sp1, identified by bioinformatics analysis, were inserted in the VZV genome. Replication of the viruses with mutations of the gE promoter did not differ from control recombinants in melanoma cells or primary human tonsil T cells in vitro. These deletions did not affect infection of human skin xenografts in SCIDhu mice. These results indicate that Sp1 is required for IE62-mediated transactivation of the gE promoter and that this transcriptional factor appears to be the only cellular factor essential for regulation of the gE promoter.
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http://dx.doi.org/10.1128/JVI.00553-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045477PMC
October 2007

Frequent occult infection with Cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis.

Authors:
Luciano Potena Cecile T J Holweg Marcy L Vana Leena Bashyam Jaya Rajamani A Louise McCormick John P Cooke Hannah A Valantine Edward S Mocarski

J Clin Microbiol 2007 Jun 4;45(6):1804-10. Epub 2007 Apr 4.

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

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.
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http://dx.doi.org/10.1128/JCM.01362-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933112PMC
June 2007

Origin and instability of GAA repeats: insights from Alu elements.

Authors:
Chitra Chauhan Debasis Dash Deepak Grover Jaya Rajamani Mitali Mukerji

J Biomol Struct Dyn 2002 Oct;20(2):253-63

Functional Genomics Unit, Centre for Biochemical Technology (CSIR), Delhi University Campus, Mall Road, Delhi-110007, India.

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7- 38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3' end of an "A" stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed.
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http://dx.doi.org/10.1080/07391102.2002.10506841DOI Listing
October 2002