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  • 1
    Online Resource
    Online Resource
    A hydrophobic domain within the small capsid protein of Kaposi’s sarcoma-associated herpesvirus is required for assembly
    Microbiology Society ; 2014
    In:  Journal of General Virology Vol. 95, No. 8 ( 2014-08-01), p. 1755-1769
    Details
    In: Journal of General Virology, Microbiology Society, Vol. 95, No. 8 ( 2014-08-01), p. 1755-1769
    Abstract: Kaposi’s sarcoma-associated herpesvirus (KSHV) capsids can be produced in insect cells using recombinant baculoviruses for protein expression. All six capsid proteins are required for this process to occur and, unlike for alphaherpesviruses, the small capsid protein (SCP) ORF65 is essential for this process. This protein decorates the capsid shell by virtue of its interaction with the capsomeres. In this study, we have explored the SCP interaction with the major capsid protein (MCP) using GFP fusions. The assembly site within the nucleus of infected cells was visualized by light microscopy using fluorescence produced by the SCP–GFP polypeptide, and the relocalization of the SCP to these sites was evident only when the MCP and the scaffold protein were also present – indicative of an interaction between these proteins that ensures delivery of the SCP to assembly sites. Biochemical assays demonstrated a physical interaction between the SCP and MCP, and also between this complex and the scaffold protein. Self-assembly of capsids with the SCP–GFP polypeptide was evident. Potentially, this result can be used to engineer fluorescent KSHV particles. A similar SCP–His 6 polypeptide was used to purify capsids from infected cell lysates using immobilized affinity chromatography and to directly label this protein in capsids using chemically derivatized gold particles. Additional studies with SCP–GFP polypeptide truncation mutants identified a domain residing between aa 50 and 60 of ORF65 that was required for the relocalization of SCP–GFP to nuclear assembly sites. Substitution of residues in this region and specifically at residue 54 with a polar amino acid (lysine) disrupted or abolished this localization as well as capsid assembly, whereas substitution with non-polar residues did not affect the interaction. Thus, this study identified a small conserved hydrophobic domain that is important for the SCP–MCP interaction.
    Type of Medium: Online Resource
    ISSN: 0022-1317 , 1465-2099
    URL: Article
    DOI: 10.1099/vir.0.064303-0
    RVK:
    XA 53770
    RVK:
    WA 15000
    Language: English
    Publisher: Microbiology Society
    Publication Date: 2014
    detail.hit.zdb_id: 2007065-2
    SSG: 12
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  • 2
    Online Resource
    Online Resource
    A Domain in the Herpes Simplex Virus 1 Triplex Protein VP23 Is Essential for Closure of Capsid Shells into Icosahedral Structures
    American Society for Microbiology ; 2011
    In:  Journal of Virology Vol. 85, No. 23 ( 2011-12), p. 12698-12707
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 85, No. 23 ( 2011-12), p. 12698-12707
    Abstract: VP23 is a key component of the triplex structure. The triplex, which is unique to herpesviruses, is a complex of three proteins, two molecules of VP23 which interact with a single molecule of VP19C. This structure is important for shell accretion and stability of the protein coat. Previous studies utilized a random transposition mutagenesis approach to identify functional domains of the triplex proteins. In this study, we expand on those findings to determine the key amino acids of VP23 that are required for triplex formation. Using alanine-scanning mutagenesis, we have made mutations in 79 of 318 residues of the VP23 polypeptide. These mutations were screened for function both in the yeast two-hybrid assay for interaction with VP19C and in a genetic complementation assay for the ability to support the replication of a VP23 null mutant virus. These assays identified a number of amino acids that, when altered, abolish VP23 function. Abrogation of virus assembly by a single-amino-acid change bodes well for future development of small-molecule inhibitors of this process. In addition, a number of mutations which localized to a C-terminal region of VP23 (amino acids 205 to 241) were still able to interact with VP19C but were lethal for virus replication when introduced into the herpes simplex virus 1 (HSV-1) KOS genome. The phenotype of many of these mutant viruses was the accumulation of large open capsid shells. This is the first demonstration of capsid shell accumulation in the presence of a lethal VP23 mutation. These data thus identify a new domain of VP23 that is required for or regulates capsid shell closure during virus assembly.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.05791-11
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2011
    detail.hit.zdb_id: 1495529-5
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  • 3
    Online Resource
    Online Resource
    Expression of the HSV-1 capsid protein VP19C in Escherichia coli: A single amino acid change overcomes an expression block of the full-length polypeptide
    Elsevier BV ; 2011
    In:  Protein Expression and Purification Vol. 77, No. 1 ( 2011-5), p. 80-85
    Details
    In: Protein Expression and Purification, Elsevier BV, Vol. 77, No. 1 ( 2011-5), p. 80-85
    Type of Medium: Online Resource
    ISSN: 1046-5928
    URL: Article
    DOI: 10.1016/j.pep.2010.12.013
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2011
    detail.hit.zdb_id: 1471688-4
    SSG: 12
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  • 4
    Online Resource
    Online Resource
    Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes
    American Society for Microbiology ; 2013
    In:  Journal of Virology Vol. 87, No. 7 ( 2013-04), p. 3915-3929
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 87, No. 7 ( 2013-04), p. 3915-3929
    Abstract: All herpesviruses encode a complex of two proteins, referred to as the nuclear egress complex (NEC), which together facilitate the exit of assembled capsids from the nucleus. Previously, we showed that the Kaposi's sarcoma-associated herpesvirus (KSHV) NEC specified by the ORF67 and ORF69 genes when expressed in insect cells using baculoviruses for protein expression forms a complex at the nuclear membrane and remodels these membranes to generate nuclear membrane-derived vesicles. In this study, we have analyzed the functional domains of the KSHV NEC proteins and their interactions. Site-directed mutagenesis of gammaherpesvirus conserved residues revealed functional domains of these two proteins, which in many cases abolish the formation of the NEC and remodeling of nuclear membranes. Small in-frame deletions within ORF67 in all cases result in loss of the ability of the mutant protein to induce cellular membrane proliferation as well as to interact with ORF69. Truncation of the C terminus of ORF67 that resides in the perinuclear space does not impair the functions of ORF67; however, deletion of the transmembrane domain of ORF67 produces a protein that cannot induce membrane proliferation but can still interact with ORF69 in the nucleus and can be tethered to the nuclear membrane by virtue of its interaction with the wild-type-membrane-anchored ORF67. In-frame deletions in ORF69 have varied effects on NEC formation, but all abolish remodeling of nuclear membranes into circular structures. One mutant interacts with ORF67 as well as the wild-type protein but cannot function in membrane curvature and fission events that generate circular vesicles. These studies genetically confirm that ORF67 is required for cellular membrane proliferation and that ORF69 is the factor required to remodel these duplicated membranes into circular-virion-size vesicles. Furthermore, we also investigated the NEC encoded by Epstein-Barr virus (EBV). The EBV complex comprised of BFRF1 and BFLF2 was visualized at the nuclear membrane using autofluorescent protein fusions. BFRF1 is a potent inducer of membrane proliferation; however, BFLF2 cannot remodel these membranes into circular structures. What was evident is the superior remodeling activity of ORF69, which could convert the host membrane proliferations induced by BFRF1 into circular structures.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.03418-12
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2013
    detail.hit.zdb_id: 1495529-5
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  • 5
    Online Resource
    Online Resource
    Plaquing of Herpes Simplex Viruses
    MyJove Corporation ; 2021
    In:  Journal of Visualized Experiments , No. 177 ( 2021-11-05)
    Details
    In: Journal of Visualized Experiments, MyJove Corporation, , No. 177 ( 2021-11-05)
    Type of Medium: Online Resource
    ISSN: 1940-087X
    URL: Article
    DOI: 10.3791/62962
    Language: English
    Publisher: MyJove Corporation
    Publication Date: 2021
    detail.hit.zdb_id: 2259946-0
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  • 6
    Online Resource
    Online Resource
    Plaquing of Herpes Simplex Viruses
    MyJove Corporation ; 2021
    In:  Journal of Visualized Experiments , No. 177 ( 2021-11-05)
    Details
    In: Journal of Visualized Experiments, MyJove Corporation, , No. 177 ( 2021-11-05)
    Type of Medium: Online Resource
    ISSN: 1940-087X
    URL: Article
    DOI: 10.3791/62962-v
    Language: English
    Publisher: MyJove Corporation
    Publication Date: 2021
    detail.hit.zdb_id: 2259946-0
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  • 7
    Online Resource
    Online Resource
    The Assembly Domain of the Small Capsid Protein of Kaposi's Sarcoma-Associated Herpesvirus
    American Society for Microbiology ; 2012
    In:  Journal of Virology Vol. 86, No. 21 ( 2012-11), p. 11926-11930
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 86, No. 21 ( 2012-11), p. 11926-11930
    Abstract: Self-assembly of Kaposi's sarcoma-associated herpesvirus capsids occurs when six proteins are coexpressed in insect cells using recombinant baculoviruses; however, if the small capsid protein (SCP) is omitted from the coinfection, assembly does not occur. Herein we delineate and identify precisely the assembly domain and the residues of SCP required for assembly. Hence, six residues, R14, D18, V25, R46, G66, and R70 in the assembly domain, when changed to alanine, completely abolish or reduce capsid assembly.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.01430-12
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 1495529-5
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  • 8
    Online Resource
    Online Resource
    Nelfinavir Inhibits Maturation and Export of Herpes Simplex Virus 1
    American Society for Microbiology ; 2014
    In:  Journal of Virology Vol. 88, No. 10 ( 2014-05-15), p. 5455-5461
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 88, No. 10 ( 2014-05-15), p. 5455-5461
    Abstract: Nelfinavir (NFV) is an HIV-1 protease inhibitor with demonstrated antiviral activity against herpes simplex virus 1 (HSV-1) and several other herpesviruses. However, the stages of HSV-1 replication inhibited by NFV have not been explored. In this study, we investigated the effects of NFV on capsid assembly and envelopment. We confirmed the inhibitory effects of NFV on HSV-1 replication by plaque assay and found that treatment with NFV did not affect capsid assembly, activity of the HSV-1 maturational protease, or formation of DNA-containing capsids in the nucleus. Confocal and electron microscopy studies showed that these capsids were transported to the cytoplasm but failed to complete secondary envelopment and subsequent exit from the cell. Consistent with the microscopy results, a light-scattering band corresponding to enveloped virions was not evident following sucrose gradient rate-velocity separation of lysates from drug-treated cells. Evidence of a possibly related effect of NFV on viral glycoprotein maturation was also discovered. NFV also inhibited the replication of an HSV-1 thymidine kinase mutant resistant to nucleoside analogues such as acyclovir. Given that NFV is neither a nucleoside mimic nor a known inhibitor of nucleic acid synthesis, this was expected and suggests its potential as a coinhibitor or alternate antiviral therapeutic agent in cases of resistance. IMPORTANCE Nelfinavir (NFV) is a clinically important antiviral drug that inhibits production of infectious HIV. It was reported to inhibit herpesviruses in cell culture. Herpes simplex virus 1 (HSV-1) infections are common and often associated with several diseases. The studies we describe here confirm and extend earlier findings by investigating how NFV interferes with HSV-1 replication. We show that early steps in virus formation (e.g., assembly of DNA-containing capsids in the nucleus and their movement into the cytoplasm) appear to be unaffected by NFV, whereas later steps (e.g., final envelopment in the cytoplasm and release of infectious virus from the cell) are severely restricted by the drug. Our findings provide the first insight into how NFV inhibits HSV-1 replication and suggest that this drug may have applications for studying the herpesvirus envelopment process. Additionally, NFV may have therapeutic value alone or in combination with other antivirals in treating herpesvirus infections.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.03790-13
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
    detail.hit.zdb_id: 1495529-5
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  • 9
    Online Resource
    Online Resource
    A Null Mutation in the UL36 Gene of Herpes Simplex Virus Type 1 Results in Accumulation of Unenveloped DNA-Filled Capsids in the Cytoplasm of Infected Cells
    American Society for Microbiology ; 2000
    In:  Journal of Virology Vol. 74, No. 24 ( 2000-12-15), p. 11608-11618
    Details
    In: Journal of Virology, American Society for Microbiology, Vol. 74, No. 24 ( 2000-12-15), p. 11608-11618
    Abstract: The UL36 open reading frame (ORF) encodes the largest herpes simplex virus type 1 (HSV-1) protein, a 270-kDa polypeptide designated VP1/2, which is also a component of the virion tegument. A null mutation was generated in the UL36 gene to elucidate its role in the virus life cycle. Since the UL36 gene specifies an essential function, complementing cell lines transformed for sequences encoding the UL36 ORF were made. A mutant virus, designated KΔUL36, that encodes a null mutation in the UL36 gene was isolated and propagated in these cell lines. When noncomplementing cells infected with KΔUL36 were analyzed, both terminal genomic DNA fragments and DNA-containing capsids (C capsids) were detected; therefore, UL36 is not required for cleavage or packaging of DNA. Sedimentation analysis of lysates from mutant-infected cells revealed the presence of particles that have the physical characteristics of C capsids. In agreement with this, polypeptide profiles of the mutant particles revealed an absence of the major envelope and tegument components. Ultrastructural analysis revealed the presence of numerous unenveloped DNA containing capsids in the cytoplasm of KΔUL36-infected cells. The UL36 mutant particles were tagged with the VP26-green fluorescent protein marker, and their movement was monitored in living cells. In KΔUL36-infected cells, extensive particulate fluorescence corresponding to the capsid particles was observed throughout the cytosol. Accumulation of fluorescence at the plasma membrane which indicated maturation and egress of virions was observed in wild-type-infected cells but was absent in KΔUL36-infected cells. In the absence of UL36 function, DNA-filled capsids are produced; these capsids enter the cytosol after traversing the nuclear envelope and do not mature into enveloped virus. The maturation and egress of the UL36 mutant particles are abrogated, possibly due to a late function of this complex polypeptide, i.e., to target capsids to the correct maturation pathway.
    Type of Medium: Online Resource
    ISSN: 0022-538X , 1098-5514
    URL: Article
    DOI: 10.1128/JVI.74.24.11608-11618.2000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2000
    detail.hit.zdb_id: 1495529-5
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  • 10
    Online Resource
    Online Resource
    Rapid and efficient in vitro excision of BAC sequences from herpesvirus genomes using Cre-mediated recombination
    Elsevier BV ; 2018
    In:  Journal of Virological Methods Vol. 261 ( 2018-11), p. 67-70
    Details
    In: Journal of Virological Methods, Elsevier BV, Vol. 261 ( 2018-11), p. 67-70
    Type of Medium: Online Resource
    ISSN: 0166-0934
    URL: Article
    DOI: 10.1016/j.jviromet.2018.08.006
    RVK:
    WA 15000
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2018
    detail.hit.zdb_id: 2007929-1
    SSG: 12
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