Transcriptional activation by herpes simplex virus type 1 VP16 in vitro and its inhibition by oligopeptides.

VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression. We have reconstituted transcriptional activation by VP16 in an in vitro system that is dependent on DNA sequences from HSV immediate-early gene promoters and on protein-protein interactions between VP16 and Oct-1 that are required for VP16 activation in vivo. Activation increased synergistically with the number of TAATGARAT elements (the cis-acting element for VP16 activation in vivo) upstream of the core promoter, and mutations of this element that reduce Oct-1 or VP16 DNA binding reduced transactivation in vitro. A VP16 insertion mutant unable to interact with Oct-1 was inactive, but, surprisingly, a deletion mutant lacking the activation domain was approximately 65% as active as the full-length protein. The activation domains of Oct-1 were necessary for activation in reactions containing the VP16 deletion mutant, and they contributed significantly to activation by full-length VP16. Addition of a GA-rich element present in many HSV immediate-early gene enhancers synergistically stimulated VP16-activated transcription. Finally, oligopeptides that are derived from a region of VP16 thought to contact a cellular factor known as HCF (host cell factor) and that inhibit efficient VP16 binding to the TAATGARAT element also specifically inhibited VP16-activated, but not basal, transcription. Amino acid substitutions in one of these peptides identified three residues that are absolutely required for inhibition and presumably for interaction of VP16 with HCF.

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A Single Amino Acid Substitution in Herpes Simplex Virus Type 1 VP16 Inhibits Binding to the Virion Host Shutoff Protein and Is Incompatible with Virus Growth

Journal of Virology ◽

10.1128/jvi.77.5.2892-2902.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 2892-2902 ◽

Cited By ~ 24

Author(s):

J. Knez ◽

P. T. Bilan ◽

J. P. Capone

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immediate Early ◽

Virus Growth ◽

Amino Terminal ◽

Virion Host Shutoff ◽

Virion Host Shutoff Protein ◽

Simplex Virus

ABSTRACT In addition to its well-established role in the activation of herpes simplex virus immediate-early gene transcription, VP16 interacts with and downregulates the function of the virion host shutoff protein (vhs), thereby attenuating vhs-mediated destruction of viral mRNAs and translational arrest at late times of infection. We have carried out two-hybrid analysis in vivo and protein-protein interaction assays in vitro to identify determinants in VP16 necessary for interaction with vhs. The minimal amino-terminal subfragment of VP16 capable of binding to vhs encompassed residues 1 to 345. Alteration of a single leucine at position 344 to alanine (L344A) in the context of the amino-terminal fragment of VP16 containing residues 1 to 404 was sufficient to abolish interaction with vhs in vitro and in vivo. Leu344 could be replaced with hydrophobic amino acids (Ile, Phe, Met, or Val) but not by Asn, Lys, or Pro, indicating that hydrophobicity is an important property of binding to vhs. VP16 harboring a loss-of-function mutation at L344 was not compromised in its ability to interact with host cell factor (HCF-1) or to activate transcription of viral immediate-early genes in transient-transfection assays. Virus complementation assays using the VP16-null virus 8MA and the VP16/vhs double-mutant virus 8MAΔSma showed that VP16(L344A) was able to complement the growth of 8MAΔSma but not 8MA. Thus, a single point mutation in VP16 uncouples binding to vhs from other functions of VP16 required for virus growth and indicates that direct physical association between VP16 and vhs is necessary to sustain a productive infection.

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Sp1 binds to promoter sequences and activates herpes simplex virus ‘immediate-early’ gene transcription in vitro

Nature ◽

10.1038/317179a0 ◽

1985 ◽

Vol 317 (6033) ◽

pp. 179-182 ◽

Cited By ~ 137

Author(s):

Katherine A. Jones ◽

Robert Tjian

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Gene Transcription ◽

Immediate Early Gene ◽

Early Gene ◽

Immediate Early ◽

Promoter Sequences ◽

Transcription In Vitro ◽

Simplex Virus

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Evidence that the Immediate-Early Gene Product ICP4 Is Necessary for the Genome of the Herpes Simplex Virus Type 1 ICP4 Deletion Mutant Strain d120 To Circularize in Infected Cells

Journal of Virology ◽

10.1128/jvi.01869-06 ◽

2006 ◽

Vol 80 (23) ◽

pp. 11589-11597 ◽

Cited By ~ 14

Author(s):

Ying-Hsiu Su ◽

Xianchao Zhang ◽

Xiaohe Wang ◽

Nigel W. Fraser ◽

Timothy M. Block

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Mutant Strain ◽

Blot Analysis ◽

Deletion Mutant ◽

Vero Cells ◽

Early Gene ◽

Immediate Early ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Following infection, the physical state of linear herpes simplex virus (HSV) genomes may change into an “endless” or circular form. In this study, using Southern blot analysis of the HSV genome, we provide evidence that immediate-early protein ICP4 is involved in the process of converting the linear HSV-1 ICP4-deleted mutant strain d120 genome into its endless form. Under conditions where de novo viral DNA synthesis was inhibited, the genome of the ICP4 deletion mutant d120 failed to assume an endless conformation following infection of Vero cells (compared with the ability of wild-type strain KOS). This defect was reversed in the Vero-derived cell line E5, which produces the ICP4 protein, suggesting that ICP4 is necessary and sufficient to complement the d120 defect. When ICP4 protein was provided by the replication-defective DNA polymerase mutant HP66, the genomes of mutant d120 could assume an endless conformation in Vero cells. Western blot analysis using antibody specific to the ICP4 protein showed that although the d120 virions contained ICP4 protein, the majority of that ICP4 protein was in a 40-kDa truncated form, with only a small fraction present as a full-length 175-kDa protein. When expression of ICP4 protein from E5 cells was inhibited by cycloheximide, the d120 virion-associated ICP4 protein was unable to mediate endless formation after infection of E5 cells. Collectively, these data suggest that ICP4 protein has an important role in mediating the endless formation of the HSV-1 genome upon infection and that this function can be provided in trans.

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A mutant of herpes simplex virus type 1 immediate early polypeptide Vmw175 binds to the cap site of its own promoter in vitro but fails to autoregulate in vivo

Journal of General Virology ◽

10.1099/0022-1317-71-4-851 ◽

1990 ◽

Vol 71 (4) ◽

pp. 851-861 ◽

Cited By ~ 6

Author(s):

T. Paterson ◽

V. G. Preston ◽

R. D. Everett

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Immediate Early ◽

Simplex Virus

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Immediate-Early Expression of the Herpes Simplex Virus Type 1 ICP27 Transcript Is Not Critical for Efficient Replication In Vitro or In Vivo

Journal of Virology ◽

10.1128/jvi.78.19.10470-10478.2004 ◽

2004 ◽

Vol 78 (19) ◽

pp. 10470-10478 ◽

Cited By ~ 12

Author(s):

Aixu Sun ◽

G. V. Devi-Rao ◽

M. K. Rice ◽

L. W. Gary ◽

D. C. Bloom ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Immediate Early ◽

Wild Type ◽

Early Expression ◽

Simplex Virus

ABSTRACT We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.

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Construction of a US3 lacZ insertion mutant of herpes simplex virus type 2 and characterization of its phenotype in vitro and in vivo

Virology ◽

10.1016/0042-6822(92)91212-d ◽

1992 ◽

Vol 190 (1) ◽

pp. 256-268 ◽

Cited By ~ 59

Author(s):

Yukihiro Nishiyama ◽

Yoshinari Yamada ◽

Ryutaro Kurachi ◽

Tohru Daikoku

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Virus Type ◽

Herpes Simplex Virus Type ◽

Insertion Mutant ◽

Simplex Virus

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Structural Variability of the Herpes Simplex Virus 1 GenomeIn VitroandIn Vivo

Journal of Virology ◽

10.1128/jvi.00223-12 ◽

2012 ◽

Vol 86 (16) ◽

pp. 8592-8601 ◽

Cited By ~ 9

Author(s):

Charlotte Mahiet ◽

Ayla Ergani ◽

Nicolas Huot ◽

Nicolas Alende ◽

Ahmed Azough ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Considerable Proportion ◽

Inverted Repeats ◽

Herpes Simplex Virus 1 ◽

Cell Extracts ◽

Simplex Virus ◽

Hsv 1

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation.In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the onein vitrowas observed, along with a considerable proportion of noncanonical assortment.

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