scholarly journals gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes.

1985 ◽  
Vol 5 (3) ◽  
pp. 518-528 ◽  
Author(s):  
S Silver ◽  
B Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Viral Dna ◽  
Bamhi Fragment ◽  
Viral Genomes ◽  
Chimeric Genes ◽  
Simplex Virus ◽  
Hsv 1

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.

gamma 2-Thymidine kinase chimeras are identically transcribed but regulated a gamma 2 genes in herpes simplex virus genomes and as beta genes in cell genomes

1985 ◽  
Vol 5 (3) ◽  
pp. 518-528
Author(s):  
S Silver ◽  
B Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Thymidine Kinase ◽  
Viral Dna ◽  
Bamhi Fragment ◽  
Viral Genomes ◽  
Chimeric Genes ◽  
Simplex Virus ◽  
Hsv 1

True gamma or gamma 2 genes, unlike alpha, beta, and gamma 1 (beta gamma) genes of herpes simplex virus 1 (HSV-1), stringently require viral DNA synthesis for their expression. We report that gamma 2 genes resident in cells were induced in trans by infection with HSV-1 but that the induction did not require amplification of either the resident gene or the infecting viral genome. Specifically, to test the hypothesis that expression of these genes is amplification dependent, we constructed two sets of gamma 2-thymidine kinase (TK) chimeric genes. The first (pRB3038) consisted of the promoter-regulatory region and a portion of 5'-transcribed noncoding region of the domain of a gamma 2 gene identified by Hall et al. (J. Virol. 43:594-607) in the HSV-1(F) BamHI fragment D' to the 5'-transcribed noncoding and coding regions of the TK gene. The second (pRB3048) contained, in addition, an origin of HSV-1 DNA replication. Cells transfected with either the first or second construct and selected for the TK+ phenotype were then tested for TK induction after superinfection with HSV-1(F) delta 305, containing a deletion in the coding sequences of the TK gene, and viruses containing, in addition, a ts lesion in the alpha 4 regulatory protein (ts502 delta 305) or in the beta 8 major DNA-binding protein (tsHA1 delta 305). The results were as follows: induction by infection with TK- virus of chimeric TK genes with or without an origin of DNA replication was dependent on functional alpha 4 protein but not on viral DNA synthesis; the resident chimeric gene in cells selected for G418 (neomycin) resistance was regulated in the same fashion; the chimeric gene recombined into the viral DNA was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis; the gamma 2-chimeric genes resident in the host and in viral genomes were transcribed from the donor BamHI fragment D' containing the promoter-regulatory domain of the gamma 2 gene. The significance of the differential regulation of gamma 2 genes in the environments of host and viral genomes by viral trans-acting factors is discussed.

scholarly journals Accumulation of Herpes Simplex Virus Type 1 Early and Leaky-Late Proteins Correlates with Apoptosis Prevention in Infected Human HEp-2 Cells

2001 ◽  
Vol 75 (2) ◽  
pp. 1013-1030 ◽  
Author(s):  
Martine Aubert ◽  
Stephen A. Rice ◽  
John A. Blaho
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Amino Terminal ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803–2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5,d1-2, M11, M15, M16, n504R,n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3,d3-4, d4-5, d5-6, andd6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (γ2) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (β) and leaky-late (γ1) proteins correlates with the prevention of apoptosis in infected HEp-2 cells.

scholarly journals Roles of conserved residues within the pre-NH2-terminal domain of herpes simplex virus 1 DNA polymerase in replication and latency in mice

2014 ◽  
Vol 95 (4) ◽  
pp. 940-947 ◽  
Author(s):  
Shariya L. Terrell ◽  
Jean M. Pesola ◽  
Donald M. Coen
Keyword(s):  
Cell Culture ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Infectious Virus ◽  
Viral Dna ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) is essential for viral DNA synthesis and production of infectious virus in cell culture. While mutations that affect 5′–3′ polymerase activity have been evaluated in animal models of HSV-1 infection, mutations that affect other functions of HSV-1 Pol have not. In a previous report, we utilized bacterial artificial chromosome technology to generate defined HSV-1 pol mutants with lesions in the previously uncharacterized pre-NH2-terminal domain. We found that the extreme N-terminal 42 residues (deletion mutant polΔN43) were dispensable for replication in cell culture, while residues 44–49 (alanine-substitution mutant polA6) were required for efficient viral DNA synthesis and production of infectious virus. In this study, we sought to address the importance of these conserved elements in viral replication in a mouse corneal infection model. Mutant virus polΔN43 exhibited no meaningful defect in acute or latent infection despite strong conservation of residues 1–42 with HSV-2 Pol. The polA6 mutation caused a modest defect in replication at the site of inoculation, and was severely impaired for ganglionic replication, even at high inocula that permitted efficient corneal replication. Additionally, the polA6 mutation resulted in reduced latency establishment and subsequent reactivation. Moreover, we found that the polA6 replication defect in cultured cells was exacerbated in resting cells as compared to dividing cells. These results reveal an important role for the conserved motif at residues 44–49 of HSV-1 Pol for ganglionic viral replication.

scholarly journals Delaying the Expression of Herpes Simplex Virus Type 1 Glycoprotein B (gB) to a True Late Gene Alters Neurovirulence and Inhibits the gB-CD8+ T-Cell Response in the Trigeminal Ganglion

2010 ◽  
Vol 84 (17) ◽  
pp. 8811-8820 ◽  
Author(s):  
Srividya Ramachandran ◽  
Katherine A. Davoli ◽  
Michael B. Yee ◽  
Robert L. Hendricks ◽  
Paul R. Kinchington
Keyword(s):  
T Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Viral Dna ◽  
Cd8 Cells ◽  
Latently Infected ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Following herpes simplex virus type 1 (HSV-1) ocular infection of C57BL/6 mice, activated CD8+ T cells specific for an immunodominant epitope on HSV-1 glycoprotein B (gB-CD8 cells) establish a stable memory population in HSV-1 latently infected trigeminal ganglia (TG), whereas non-HSV-specific CD8+ T cells are lost over time. The retention and activation of gB-CD8 cells appear to be influenced by persistent viral antigenic exposure within the latently infected TG. We hypothesized that the low-level expression of gB from its native promoter before viral DNA synthesis is critical for the retention and activation of gB-CD8 cells in the TG during HSV-1 latency and for their ability to block HSV-1 reactivation from latency. To test this, we created a recombinant HSV-1 in which gB is expressed only after viral DNA synthesis from the true late gC promoter (gCp-gB). Despite minor growth differences compared to its rescuant in infected corneas, gCp-gB was significantly growth impaired in the TG and produced a reduced latent genome load. The gCp-gB- and rescuant-infected mice mounted similar gB-CD8 effector responses, but the size and activation phenotypes of the memory gB-CD8 cells were diminished in gCp-gB latently infected TG, suggesting that the stimulation of gB-CD8 cells requires gB expression before viral DNA synthesis. Surprisingly, late gB expression did not compromise the capacity of gB-CD8 cells to inhibit HSV-1 reactivation from latency in ex vivo TG cultures, suggesting that gB-CD8 cells can block HSV-1 reactivation at a very late stage in the viral life cycle. These data have implications for designing better immunogens for vaccines to prevent HSV-1 reactivation.

scholarly journals THYMIDINE KINASE AS A CAUSATIVE FACTOR FOR TYPE 1 HERPES SIMPLEX VIRUS RESISTANCE AGAINST ACYCLOVIR

2020 ◽  
Vol 5 (2) ◽  
pp. 159
Author(s):  
Yannie Febby Martina Lefaan ◽  
Riani Setiadhi
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Thymidine Kinase ◽  
Virus Resistance ◽  
Causative Factor ◽  
Competitive Inhibitor ◽  
Viral Dna ◽  
Simplex Virus ◽  
Immunocompetent Patients ◽  
Hsv 1

ABSTRACTBackground: Herpes Simplex Virus (HSV) infection demonstrates a high prevalence in the world. Acyclovir, one of guanine synthetic analogues, is commonly used to treat infections caused by HSV. HSV resistance against acyclovir may occur, especially in immunocompromised and immunocompetent patients, as the consequence of viral mutations. Thymidine kinase (TK) is an HSV tegument protein which plays an important role in HSV-1 resistance against acyclovir. Purpose: The purpose of this article is to review the mechanisms of TK mutation that cause HSV-1 resistance against acyclovir. Review: Acyclovir involves three stages of viral thymidine kinase phosphorylation to form acyclovir triphosphate. It prevents HSV replication by acting as a competitive inhibitor of viral DNA polymerase and a chain terminator in viral DNA synthesis. Resistance is associated with viral TK mutation that is encoded by UL23 gene. Long-term use of acyclovir may promote thymidine kinase mutation in immunocompromised and immunocompetent patients via three mechanisms, namely absolute insufficiency in TK activity (TK-negative), depletion in TK synthesis, and inability in TK phosphorylation which consequently hinders the phosphorylation of acyclovir. Herpes TK gene contains a series of cytosine and guanosine, that are important for the function and the  mutation of HSV by producing incomplete or fewer enzymes as the result of nucleotide addition or elimination in homopolymer process. Conclusion: HSV-1 resistance against acyclovir is evolved from TK mutations, in the form of TK-negative, TK low-producing, and TK altered mutants, that are unable to phosphorylate TK and accordingly disrupt acyclovir phosphorylation to convert acyclovir triphosphate. Keyword : Acyclovir, Herpes simplex virus, Resistance, Thymidine kinase

scholarly journals Blocks to herpes simplex virus type 1 replication in a cell line, tsBN2, encoding a temperature-sensitive RCC1 protein

2007 ◽  
Vol 88 (2) ◽  
pp. 376-383 ◽  
Author(s):  
Blair L. Strang ◽  
Nigel D. Stow
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Herpes Simplex Virus Type ◽  
Temperature Sensitive ◽  
Viral Dna ◽  
A Cell ◽  
Simplex Virus ◽  
Hsv 1

Circularization of the herpes simplex virus type 1 (HSV-1) genome is thought to be an important early event during the lytic cycle. Previous studies from another laboratory using a cell line, tsBN2, that carries a temperature-sensitive mutation in the gene encoding the regulator of chromatin condensation 1 (RCC1) indicated that functional RCC1 was required for HSV-1 genome circularization and subsequent viral DNA synthesis. Here, HSV-1 infection of tsBN2 cells has been re-examined by utilizing both wild-type HSV-1 and a derivative that enables a direct demonstration of circularization. At the non-permissive temperature, when RCC1 was absent, both circularization and viral DNA synthesis were reduced, but not abolished. However, no infectious progeny virus was detected under these conditions. An impairment in the cleavage of concatemeric DNA and the failure to express at least one capsid protein indicated that HSV-1 replication is also blocked at a late stage in the absence of RCC1. This conclusion was supported by a temperature-upshift experiment, which demonstrated a role for RCC1 at times later than 6 h post-infection. Finally, a virus constitutively expressing β-galactosidase produced the protein in a reduced number of cells when RCC1 was inactivated, suggesting that genome delivery to the nucleus or the initial stages of gene expression may also be affected.

scholarly journals Nucleolin Is Required for Efficient Nuclear Egress of Herpes Simplex Virus Type 1 Nucleocapsids

2009 ◽  
Vol 84 (4) ◽  
pp. 2110-2121 ◽  
Author(s):  
Ken Sagou ◽  
Masashi Uema ◽  
Yasushi Kawaguchi
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Nuclear Egress ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpesvirus nucleocapsids assemble in the nucleus and must cross the nuclear membrane for final assembly and maturation to form infectious progeny virions in the cytoplasm. It has been proposed that nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane, and these enveloped nucleocapsids then fuse with the outer nuclear membrane to enter the cytoplasm. Little is known about the mechanism(s) for nuclear egress of herpesvirus nucleocapsids and, in particular, which, if any, cellular proteins are involved in the nuclear egress pathway. UL12 is an alkaline nuclease encoded by herpes simplex virus type 1 (HSV-1) and has been suggested to be involved in viral DNA maturation and nuclear egress of nucleocapsids. Using a live-cell imaging system to study cells infected by a recombinant HSV-1 expressing UL12 fused to a fluorescent protein, we observed the previously unreported nucleolar localization of UL12 in live infected cells and, using coimmunoprecipitation analyses, showed that UL12 formed a complex with nucleolin, a nucleolus marker, in infected cells. Knockdown of nucleolin in HSV-1-infected cells reduced capsid accumulation, as well as the amount of viral DNA resistant to staphylococcal nuclease in the cytoplasm, which represented encapsidated viral DNA, but had little effect on these viral components in the nucleus. These results indicated that nucleolin is a cellular factor required for efficient nuclear egress of HSV-1 nucleocapsids in infected cells.

scholarly journals Viral DNA synthesis is required for the efficient expression of specific herpes simplex virus type 1 mRNA species

Virology ◽  
1980 ◽  
Vol 101 (1) ◽  
pp. 10-24 ◽  
Author(s):  
Louis E. Holland ◽  
Kevin P. Anderson ◽  
Charles Shipman ◽  
Edward K. Wagner
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Synthesis ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Mrna Species ◽  
Efficient Expression ◽  
Simplex Virus

scholarly journals A Domain of Herpes Simplex Virus pUL33 Required To Release Monomeric Viral Genomes from Cleaved Concatemeric DNA

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Kui Yang ◽  
Xiaoqun Dang ◽  
Joel D. Baines
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Replication ◽  
Dna Cleavage ◽  
Artificial Chromosome ◽  
Viral Dna ◽  
Viral Genomes ◽  
C Terminus ◽  
Simplex Virus

ABSTRACT Monomeric herpesvirus DNA is cleaved from concatemers and inserted into preformed capsids through the actions of the viral terminase. The terminase of herpes simplex virus (HSV) is composed of three subunits encoded by UL15, UL28, and UL33. The UL33-encoded protein (pUL33) interacts with pUL28, but its precise role in the DNA cleavage and packaging reaction is unclear. To investigate the function of pUL33, we generated a panel of recombinant viruses with either deletions or substitutions in the most conserved regions of UL33 using a bacterial artificial chromosome system. Deletion of 11 amino acids (residues 50 to 60 or residues 110 to 120) precluded viral replication, whereas the truncation of the last 10 amino acids from the pUL33 C terminus did not affect viral replication or the interaction of pUL33 with pUL28. Mutations that replaced the lysine at codon 110 and the arginine at codon 111 with alanine codons failed to replicate, and the pUL33 mutant interacted with pUL28 less efficiently. Interestingly, genomic termini of the large (L) and small (S) components were detected readily in cells infected with these mutants, indicating that concatemeric DNA was cleaved efficiently. However, the release of monomeric genomes as assessed by pulsed-field gel electrophoresis was greatly diminished, and DNA-containing capsids were not observed. These results suggest that pUL33 is necessary for one of the two viral DNA cleavage events required to release individual genomes from concatemeric viral DNA. IMPORTANCE This paper shows a role for pUL33 in one of the two DNA cleavage events required to release monomeric genomes from concatemeric viral DNA. This is the first time that such a phenotype has been observed and is the first identification of a function of this protein relevant to DNA packaging other than its interaction with other terminase components.

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