Transcriptional control signals of a herpes simplex virus type 1 late (gamma 2) gene lie within bases -34 to +124 relative to the 5' terminus of the mRNA

1986 ◽  
Vol 6 (11) ◽  
pp. 3652-3666
Author(s):  
F L Homa ◽  
T M Otal ◽  
J C Glorioso ◽  
M Levine
Keyword(s):  
Herpes Simplex ◽  
Dna Sequences ◽  
Viral Dna ◽  
Regulated Expression ◽  
Base Pair Fragment ◽  
Simplex Virus ◽  
Pair Fragment ◽  
Gc Gene ◽  
Hsv 1

The cis-acting DNA sequences required for regulated expression of a herpes simplex virus type 1 (HSV-1) late (gamma 2) gene were studied by using viruses containing specific deletions in the 5' transcribed noncoding and upstream regions of the HSV-1 glycoprotein C (gC) gene, a model gamma 2 gene. Nine mutant viruses which had variable 5' and 3' deletions within bases -569 to +124 relative to the 5' terminus of the gC mRNA were isolated. The mutants were isolated by a simple in situ hybridization screening procedure not requiring any prior selective pressure for or against expression of the gC gene. Analysis of RNA extracted from cells infected with individual mutants showed that the DNA sequences required for regulated expression of this gamma 2 gene lay within bases -34 to +124. This 158-base-pair fragment was sufficient to confer accurate and quantitative expression of gC mRNA and to maintain the stringent requirement on viral DNA replication for expression of this gene. Moreover, it was found that sequences located between -34 and +14 contained signals essential for expression of gC. To determine whether the -34 to +124 sequences would function as a gamma 2 promoter when moved to another region of the HSV-1 genome, the 158-base-pair fragment was substituted for the normal thymidine kinase promoter-regulatory sequences in the thymidine-kinase gene locus. Transcription of this chimeric gene was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis. The only recognizable consensus sequence upstream of the transcription initiation site for this gene was the TATAAA sequence at -30.

scholarly journals Transcriptional control signals of a herpes simplex virus type 1 late (gamma 2) gene lie within bases -34 to +124 relative to the 5' terminus of the mRNA.

1986 ◽  
Vol 6 (11) ◽  
pp. 3652-3666 ◽  
Author(s):  
F L Homa ◽  
T M Otal ◽  
J C Glorioso ◽  
M Levine
Keyword(s):  
Herpes Simplex ◽  
Dna Sequences ◽  
Viral Dna ◽  
Regulated Expression ◽  
Base Pair Fragment ◽  
Simplex Virus ◽  
Pair Fragment ◽  
Gc Gene ◽  
Hsv 1

The cis-acting DNA sequences required for regulated expression of a herpes simplex virus type 1 (HSV-1) late (gamma 2) gene were studied by using viruses containing specific deletions in the 5' transcribed noncoding and upstream regions of the HSV-1 glycoprotein C (gC) gene, a model gamma 2 gene. Nine mutant viruses which had variable 5' and 3' deletions within bases -569 to +124 relative to the 5' terminus of the gC mRNA were isolated. The mutants were isolated by a simple in situ hybridization screening procedure not requiring any prior selective pressure for or against expression of the gC gene. Analysis of RNA extracted from cells infected with individual mutants showed that the DNA sequences required for regulated expression of this gamma 2 gene lay within bases -34 to +124. This 158-base-pair fragment was sufficient to confer accurate and quantitative expression of gC mRNA and to maintain the stringent requirement on viral DNA replication for expression of this gene. Moreover, it was found that sequences located between -34 and +14 contained signals essential for expression of gC. To determine whether the -34 to +124 sequences would function as a gamma 2 promoter when moved to another region of the HSV-1 genome, the 158-base-pair fragment was substituted for the normal thymidine kinase promoter-regulatory sequences in the thymidine-kinase gene locus. Transcription of this chimeric gene was regulated as a gamma 2 gene in that its expression in infected cells was dependent on viral DNA synthesis. The only recognizable consensus sequence upstream of the transcription initiation site for this gene was the TATAAA sequence at -30.

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 The Conserved Carboxyl-Terminal Half of Herpes Simplex Virus Type 1 Regulatory Protein ICP27 Is Dispensable for Viral Growth in the Presence of Compensatory Mutations

2000 ◽  
Vol 74 (16) ◽  
pp. 7362-7374 ◽  
Author(s):  
Scott M. Bunnell ◽  
Stephen A. Rice
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Vero Cells ◽  
Terminal Portion ◽  
Viral Dna ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early protein that regulates viral gene expression by poorly characterized mechanisms. Previous data suggest that its carboxyl (C)-terminal portion is absolutely required for productive viral infection. In this study, we isolated M16R, a second-site revertant of a viral ICP27 C-terminal mutant. M16R harbors an intragenic reversion, as demonstrated by the fact that its cloned ICP27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alteration in a homopolymeric run of C residues at codons 215 to 217. This results in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation assays confirmed that the truncated, frameshifted protein can partially substitute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshift mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed which possesses the frameshift in the context of an ICP27 gene with the C terminus deleted. We found that both M16exC and exCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required for the viability of M16R. Consistent with this interpretation, we isolated two viable derivatives ofexCd305 which grow productively in Vero cells despite being incapable of encoding the C-terminal portion of ICP27. Studies of viral DNA synthesis in mutant-infected cells indicated that the truncated, frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved widely in herpesviruses and previously believed to be absolutely essential, is dispensable for HSV-1 lytic replication in the presence of compensatory genomic mutations.

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 Determination of Minimum Herpes Simplex Virus Type 1 Components Necessary To Localize Transcriptionally Active DNA to ND10

2003 ◽  
Vol 77 (10) ◽  
pp. 5821-5828 ◽  
Author(s):  
Qiyi Tang ◽  
Luge Li ◽  
Alexander M. Ishov ◽  
Valerie Revol ◽  
Alberto L. Epstein ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Host Protein ◽  
Transcript Accumulation ◽  
Viral Dna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT DNA viruses such as herpes simplex virus type 1 (HSV-1) appear to start their replicative processes at specific nuclear domains known as ND10. In analyses to determine the minimum viral components needed for transcript accumulation at ND10, we find that a specific viral DNA sequence, OriS, and the viral immediate-early proteins ICP4 and ICP27 are sufficient for a reporter gene placed in cis to the OriS sequence to transcribe at ND10. A chromatin immunoprecipitation assay demonstrated expected critical intermediates in retaining the minimal genome at ND10 for the HSV-1 replication origin through direct or indirect binding to the host protein Daxx. Coimmunoprecipitation assays with antibodies to Daxx and ICP4, ICP27, and ICP8 showed that the respective proteins interact, possibly forming a complex. A potential complex between the origin, early viral DNA-binding protein ICP8 and Daxx did not result in transcription at ND10. Thus, the deposition of transcriptionally active HSV-1 genomes at ND10 is most likely a consequence of retention at ND10 through the interaction of viral genome-bound ICP4 and ICP27 with Daxx. Such a complex might be more likely immobilized at the outside of ND10 by the PML-interacting Daxx than at other nuclear sites.

scholarly journals Hematogenous Vertical Transmission of Herpes Simplex Virus Type 1 in Mice

2006 ◽  
Vol 80 (6) ◽  
pp. 2823-2831 ◽  
Author(s):  
Javier S. Burgos ◽  
Carlos Ramirez ◽  
Fernando Guzman-Sanchez ◽  
Juan M. Alfaro ◽  
Isabel Sastre ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Vertical Transmission ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Severe Disease ◽  
Viral Dna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that causes severe disease and death in newborn humans but, to date, it remains unclear how neonatal infection occurs. We show here that the vertical transmission of HSV-1 in mice is mainly hematogenous and involves the colonization of the neonate central nervous system (CNS). HSV-1 DNA was mainly detected in the blood and CNS of the offspring born to latently infected mothers; no significant differences were seen between the viral DNA concentrations in the blood of these mothers and their female progeny (either neonate or adult). The administration of acyclovir during gestation reduced or eliminated both the maternal and the neonatal viral DNA in the blood. Embryo transfer was performed to ensure (as far as possible) that only vertical hematogenous infection took place. Immunohistochemical analysis detected viral proteins in the encephalon of the offspring. Immunofluorescence studies provided immunoreactive evidence of HSV-1 proteins in the neurons of the hippocampus and showed that these viruses can molecularly reactivate after hyperthermia. Neonatal HSV-1 infection therefore appears to be mainly caused by hematogenous vertical transmission, and the viruses that colonize the offspring CNS are capable of molecular reactivation after a period of latency.

scholarly journals Mutational Analysis of the Herpes Simplex Virus Type 1 UL25 DNA Packaging Protein Reveals Regions That Are Important after the Viral DNA Has Been Packaged

2010 ◽  
Vol 84 (9) ◽  
pp. 4252-4263 ◽  
Author(s):  
Maureen O'Hara ◽  
Frazer J. Rixon ◽  
Nigel D. Stow ◽  
Jill Murray ◽  
Mary Murphy ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Deletion Mutant ◽  
Dna Packaging ◽  
The Other ◽  
Viral Dna ◽  
Mutant Proteins ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The herpes simplex virus type 1 (HSV-1) UL25 gene encodes a minor capsid protein, pUL25, that is essential for packaging the full-length viral genome. Six regions which contain disordered residues have been identified in the high-resolution structure of pUL25. To investigate the significance of these flexible regions, a panel of plasmids was generated encoding mutant proteins, with each member lacking the disordered residues in one of the six regions. In addition, UL25 constructs were produced, which specified proteins that contained missense mutations individually affecting two of the four regions on the surface of pUL25 predicted from evolutionary trace analysis to be important in protein-protein interactions. The impacts of these mutations on viral DNA packaging, virus assembly, and growth were examined. Of the nine mutant proteins analyzed, five failed to complement the growth of a UL25 deletion mutant in Vero cells. These noncomplementing proteins fell into three classes. Proteins in one class did not alter the DNA packaging phenotype of an HSV-1 UL25 deletion mutant, whereas proteins from the other two classes allowed the UL25 null mutant to package full-length viral DNA. Subsequent analysis of the latter classes of mutant proteins demonstrated that one class enabled the null virus to release enveloped virus particles from U2OS cells, whereas the other class prevented egress of mature HSV-1 capsids from the nucleus. These findings reveal a new role for pUL25 in virion assembly, consistent with its flexible structure and location on the capsid.

scholarly journals Herpes Simplex Virus Type 1 DNA Polymerase Requires the Mammalian Chaperone Hsp90 for Proper Localization to the Nucleus

2005 ◽  
Vol 79 (16) ◽  
pp. 10740-10749 ◽  
Author(s):  
April D. Burch ◽  
Sandra K. Weller
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Hsp90 Chaperone ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Many viruses and bacteriophage utilize chaperone systems for DNA replication and viral morphogenesis. We have previously shown that in the herpes simplex virus type 1 (HSV-1)-infected cell nucleus, foci enriched in the Hsp70/Hsp40 chaperone machinery are formed adjacent to viral replication compartments (A. D. Burch and S. K. Weller, J. Virol. 78:7175-7185, 2004). These foci have now been named virus-induced chaperone-enriched (VICE) foci. Since the Hsp90 chaperone machinery is known to engage the Hsp70/Hsp40 system in eukaryotes, the subcellular localization of Hsp90 in HSV-1-infected cells was analyzed. Hsp90 is found within viral replication compartments as well as in the Hsp70/Hsp40-enriched foci. Geldanamycin, an inhibitor of Hsp90, results in decreased HSV-1 yields and blocks viral DNA synthesis. Furthermore, we have found that the viral DNA polymerase is mislocalized to the cytoplasm in both infected and transfected cells in the presence of geldanamycin. Additionally, in the presence of an Hsp90 inhibitor, proteasome-dependent degradation of the viral polymerase was detected by Western blot analysis. These data identify the HSV-1 polymerase as a putative client protein of the Hsp90 chaperone system. Perturbations in this association appear to result in degradation, aberrant folding, and/or intracellular localization of the viral polymerase.

scholarly journals The UL15 protein of herpes simplex virus type 1 is necessary for the localization of the UL28 and UL33 proteins to viral DNA replication centres

2008 ◽  
Vol 89 (7) ◽  
pp. 1709-1715 ◽  
Author(s):  
Martin R. Higgs ◽  
Valerie G. Preston ◽  
Nigel D. Stow
Keyword(s):  
Herpes Simplex Virus ◽  
Dna Replication ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Simplex Virus ◽  
Hsv 1

The UL15, UL28 and UL33 proteins of herpes simplex virus type 1 (HSV-1) are thought to comprise a terminase complex responsible for cleavage and packaging of the viral genome into pre-assembled capsids. Immunofluorescence studies confirmed that shortly after infection with wild-type HSV-1 these three proteins localize to viral DNA replication compartments within the nucleus, identified by the presence of the single-stranded DNA-binding protein, ICP8. In cells infected with either UL28- or UL33-null mutants, the other two terminase proteins also co-localized with ICP8. In contrast, neither UL28 nor UL33 was detectable in replication compartments following infection with a UL15-null mutant, although Western blot analysis showed they were present in normal amounts in the infected cells. Provision of UL15 in a complementing cell line restored the ability of all three proteins to localize to replication compartments. These data indicate that UL15 plays a key role in localizing the terminase complex to DNA replication compartments, and that it can interact independently with UL28 and UL33.

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