Early Protein ICP0 Research Articles

Herpes simplex virus type I disrupts the ATR-dependent DNA-damage response during lytic infection

Like other DNA viruses, herpes simplex virus type 1 (HSV-1) interacts with components of the cellular response to DNA damage. For example, HSV-1 sequesters endogenous, uninduced, hyperphosphorylated RPA (replication protein A) away from viral replication compartments. RPA is a ssDNA-binding protein that signals genotoxic stress through the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway. The sequestration of endogenous hyperphosphorylated RPA away from replicating viral DNA suggests that HSV-1 prevents the normal ATR-signaling response. In this study we examine the spatial distribution of endogenous hyperphosphorylated RPA with respect to ATR, its recruitment factor, ATRIP, and the cellular dsDNA break marker, gammaH2AX, during HSV-1 infection. The accumulation of these repair factors at DNA lesions has previously been identified as an early event in signaling genotoxic stress. We show that HSV-1 infection disrupts the ATR pathway by a mechanism that prevents the recruitment of repair factors, spatially uncouples ATRIP from ATR and sequesters ATRIP and endogenous hyperphosphorylated RPA within virus-induced nuclear domains containing molecular chaperones and components of the ubiquitin proteasome. The HSV-1 immediate early protein ICP0 is sufficient to induce the redistribution of ATRIP. This is the first report that a virus can disrupt the usually tight colocalization of ATR and ATRIP.

Activation of c-Jun N-terminal kinase (JNK) pathway by HSV-1 immediate early protein ICP0

The immediate early protein ICP0 encoded by herpes simplex virus 1 (HSV-1) is believed to activate transcription and consequently productive infection. The precise mechanisms of ICP0-mediated transactivation are under intensive study. Here, we demonstrate that ICP0 can strongly activate AP-1 responsive genes specifically. This activation is inhibited by c-Jun (S73A), c-Jun (S63/73A), TAK1 (K63W), but not by p38 (AF), ERK1 (K71R), ERK2 (K52R) and TRAF6 (C85A/H87A). We further investigate the relevancy of ERK, JNK and p38 MAPK pathways using their respective inhibitors PD98059, SP600125 and SB202190. Only SP600125 significantly attenuates the AP-1 responsive gene activation by ICP0. Consistent with these, the JNK is remarkably activated in response to ICP0, and this JNK activation is shown to be significantly attenuated by TAK1 (K63W). It turns out that ICP0 interacts specifically with TAK1 and stimulates its kinase activity. These findings reveal a new molecular mechanism ICP0 explores to regulate gene expression.

Alphaherpesvirus proteins related to herpes simplex virus type 1 ICP0 induce the formation of colocalizing, conjugated ubiquitin.

Herpes simplex virus type 1 immediate early protein ICP0 influences virus infection by inducing the degradation of specific cellular proteins via a mechanism requiring its RING finger and the ubiquitin-proteasome pathway. Many RING finger proteins, by virtue of their RING finger domain, interact with E2 ubiquitin-conjugating enzymes and act as a component of an E3 ubiquitin ligase. We have recently shown that ICP0 induces the accumulation of colocalizing, conjugated ubiquitin, suggesting that ICP0 can act as or contribute to an E3 ubiquitin ligase. In this report we demonstrate that the ICP0-related RING finger proteins encoded by other alphaherpesviruses also induce colocalizing, conjugated ubiquitin, thereby suggesting that they act by similar biochemical mechanisms.

Immunization with a LEAPS™ heteroconjugate containing a CTL epitope and a peptide from beta-2-microglobulin elicits a protective and DTH response to herpes simplex virus type 1

A ligand epitope antigen presentation system (LEAPS™) heteroconjugate vaccine containing a CTL epitope (H1) from the HSV-1 immediate early protein ICP27 (322–332) and a peptide sequence (J) from β-2-microglobulin (35–50) elicited protection from intraperitoneal viral challenge and promoted DTH responses. The H1 peptide and other H1 containing heteroconjugates did not elicit protection or DTH responses. Antibody to the H1 peptide could not be detected by ELISA following vaccination with peptide, heteroconjugate or natural infection. The LEAPS™ heteroconjugate appears to prime a Th1-like response which is subsequently boosted by infection. These studies show that attachment of the J peptide can make a CTL epitope into a vaccine which is immunogenic and promotes a protective Th1 type of response.

Protective immunity against herpes simplex virus (HSV) type 1 following oral administration of recombinant Salmonella typhimurium vaccine strains expressing HSV antigens.

Salmonella typhimurium strains expressing foreign antigens of various pathogens are capable of eliciting antigen-specific humoral and cellular immune responses. Attenuated S. typhimurium strain chi4550 (delta(cya) delta(crp) delta(asd)) was used as an expression vector for herpes simplex virus (HSV) antigens. Genes encoding glycoprotein D (gD) and the immediate-early protein ICP27 of HSV-1 were cloned and expressed in plasmid pYA292 (asd+) and subsequently placed into chi4550. Following two oral immunizations, the protective efficacy of recombinant strains against zosteriform challenge with HSV-1 was measured in 3-4-week-old BALB/c mice. Levels of protection observed were 77% with the ICP27 construct but only 31% with the gD construct. Zosteriform protection correlates with a CD4+-mediated delayed-type hypersensitivity (DTH) reaction against HSV. Accordingly, significant DTH was observed only in mice immunized orally with the S. typhimurium ICP27 construct. ELISA analysis of antigen-specific humoral responses failed to detect serum antibody responses following oral administration although recombinant S. typhimurium were isolated from spleens of orally dosed mice up to day 30. Intravenous (i.v.) immunization with the gD-expressing construct did, however, induce detectable serum antibody responses. Some humoral IgA responses against gD in faecal samples were detected as early as 3 weeks post-oral immunization while those induced by the i.v. route were slightly lower. These data suggest that recombinant S. typhimurium HSV antigens are capable of inducing immunity against HSV, some aspects of which are protective against HSV challenge.

The nuclear location of PML, a cellular member of the C3HC4 zinc-binding domain protein family, is rearranged during herpes simplex virus infection by the C3HC4 viral protein ICP0

ND10 are nuclear domains of unknown function that become abundant in response to stress. Infection by herpes simplex virus type 1 (HSV-1) causes the apparent disappearance of these domains, an effect that requires the expression of the immediate early protein ICP0. Previously, we have shown that there are a number of cellular antigens in the ND10. In this report, we show that one of these proteins is PML, a member of the C3HC4 zinc-binding domain family which also includes ICP0. The C3HC4 domain of ICP0 is essential for the apparent release of PML from the ND10, although the interaction of ICP0 with ND10 is determined by a small region near its carboxy terminus. PML and other ND10 proteins are not lost after removal from ND10 but deposited at the nuclear envelope or nuclear envelope modifications during later parts of the replication cycle. ICP0 is required for the onset of low multiplicity infections, and has been implicated in the process of reactivation from HSV latency. Therefore, the interaction between ICP0 and the ND10 domains, specifically PML, may be important for the outcome of virus-cell interactions.

The herpes simplex virus immediate early protein ICP27 encodes a potential metal binding domain and binds zinc in vitro

The herpes simplex virus type 1 (HSV-1) immediate-early regulatory proteins ICP27 and ICP0 each encode putative zinc-finger metal-binding domains. We utilized the technique of metal chelate affinity chromatography to demonstrate that ICP27 and ICP0 were able to bind to zinc in vitro. This property was further exploited to purify ICP27 from extracts of HSV-1-infected cells. The purification procedure also revealed that ICP27 possessed single-stranded DNA-binding activity. Analysis of ICP27 truncated peptides produced by in vitro translation verified that the zinc-binding region of ICP27 resides in the carboxy terminal 105 amino acids spanning the putative metal binding motif. However, a specific configuration of cysteine and histidine residues in this region was not required for binding to occur as demonstrated by the ability of a frame-shift mutation to bind with an efficiency similar to wild type. The mutated peptide retained four histidine and cysteine residues but in a configuration different from the consensus proposed for zinc-finger motifs. Therefore, while the region spanning the metal binding domain of ICP27 is essential for both the activator and repressor functions, and ICP27 binds zinc in vitro, it is not clear whether zinc binding in vivo is necessary for function.

Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression.

The herpes simplex virus (HSV-1) immediate early protein ICP27 is a regulatory protein which is essential for virus replication. The phenotype of temperature-sensitive and deletion mutants in ICP27 includes overexpression of some immediate early and early gene products and greatly reduced levels of late gene products. To determine whether regulation by ICP27 occurs primarily at the transcriptional level, we have studied the expression of two immediate early products (ICP4 and ICP27) and two late gene products (glycoprotein B and glycoprotein C) at the level of transcription initiation, accumulation of steady state mRNA, and protein synthesis in an ICP27 temperature-sensitive mutant tsLG4, compared to wild-type HSV-1. At the nonpermissive temperature in tsLG4-infected cells, the two immediate early gene products ICP4 and ICP27 were overexpressed both at the mRNA and protein level although synthesis of these transcripts as measured by nuclear runoff assays was reduced relative to the wild-type HSV-1 infections. The transcription of late gene products glycoprotein B (gB) and glycoprotein C (gC) was lower in runoff assays from tsLG4 infections suggesting that the reduction in the level of late products occurred at the transcriptional level. However, temperature shift experiments in which tsLG4-infected cells were shifted to the nonpermissive temperature at various times after infection showed that the synthesis of late transcripts was not altered 2 hr after the shift whereas both the accumulation of leaky late and late mRNA and the incorporation of [35S]methionine into newly synthesized gB and gC was reduced by 2 hr after the shift to nonpermissive temperature. Therefore, while the synthesis of new transcripts continued, the accumulation of late mRNAs and their translation into protein was reduced when ICP27 was defective, whereas, the converse was found for immediate early products. That is, the synthesis of new transcripts was reduced yet mRNA and protein accumulated to high levels. These results suggest that ICP27 acts at least in part post-transcriptionally to regulate the expression of immediate early and late gene products.