Conformational Changes in the Herpes Simplex Virus ICP8 DNA-Binding Protein Coincident with Assembly in Viral Replication Structures

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Herpes simplex virus 1 (HSV-1) ICP8 is a single-stranded DNA-binding protein that is necessary for viral DNA replication and exhibits recombinase activity in vitro . Alignment of the HSV-1 ICP8 amino acid sequence with ICP8 homologs from other herpesviruses revealed conserved aspartic acid (D) and glutamic acid (E) residues. Amino acid residue D1087 was conserved in every ICP8 homolog analyzed, indicating that it is likely critical for ICP8 function. We took a genetic approach to investigate the functions of the conserved ICP8 D and E residues in HSV-1 replication. The E1086A D1087A mutant form of ICP8 failed to support the replication of an ICP8 mutant virus in a complementation assay. E1086A D1087A mutant ICP8 bound DNA, albeit with reduced affinity, demonstrating that the protein is not globally misfolded. This mutant form of ICP8 was also recognized by a conformation-specific antibody, further indicating that its overall structure was intact. A recombinant virus expressing E1086A D1087A mutant ICP8 was defective in viral replication, viral DNA synthesis, and late gene expression in Vero cells. A class of enzymes called DDE recombinases utilize conserved D and E residues to coordinate divalent metal cations in their active sites. We investigated whether the conserved D and E residues in ICP8 were also required for binding metal cations and found that the E1086A D1087A mutant form of ICP8 exhibited altered divalent metal binding in an in vitro iron-induced cleavage assay. These results identify a novel divalent metal cation-binding site in ICP8 that is required for ICP8 functions during viral replication.

Section: Discussioncontrasting

confidence: 53%

Section: Resultsmentioning

confidence: 99%

Identification of a Divalent Metal Cation Binding Site in Herpes Simplex Virus 1 (HSV-1) ICP8 Required for HSV Replication

Bryant

Yan

Dreyfus

et al. 2012

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“…These interactions are regulated by the degree of phosphorylation of different sites on the protein (1,34). In addition, the ICP4 protein has been suggested to be involved in the initial stage of formation of viral DNA replication complexes (2,30), which appear to occur adjacent to ND10 sites at prereplicative domains (10,11,17,27,32). This suggests an important role of the ICP4 protein in initiating viral DNA replication, possibly by mediating the maturation of prereplicative domains.…”

mentioning

confidence: 98%

Evidence that the Immediate-Early Gene Product ICP4 Is Necessary for the Genome of the Herpes Simplex Virus Type 1 ICP4 Deletion Mutant Strain d 120 To Circularize in Infected Cells

Zhang

Wang

et al. 2006

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.

“…To identify potential ICP8-associated cellular and viral proteins, we immunoprecipitated ICP8 and associated molecules from wt HSV-infected or mock-infected whole-cell HEp-2 lysates prepared at 6.5 h postinfection using the conformation-specific 39S monoclonal antibody, which predominantly recognizes ICP8 at prereplicative sites or within HSV replication compartments (89). At this time of infection, the bulk of ICP8 is in replication compartments (18,72), and the majority of ICP8 is associated with replication complexes or progeny viral DNA (48).…”

Section: Coprecipitation Of Cellular and Viral Proteins With Icp8mentioning

confidence: 99%

Proteomics of Herpes Simplex Virus Replication Compartments: Association of Cellular DNA Replication, Repair, Recombination, and Chromatin Remodeling Proteinswith ICP8

Taylor

Knipe

2004

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212

262

In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous endjoining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.Herpes simplex virus 1 (HSV-1) is a large, double-stranded DNA virus that replicates in the host cell nucleus. HSV encodes over 80 gene products that contribute to viral replication in either cultured cells or animal hosts (76). Due to the limited size of the HSV-1 genome, the virus cannot code for every function required for its propagation; thus, HSV-1 must rely upon factors supplied by the host cell for replication. For example, HSV exclusively uses the host cell RNA polymerase II for the transcription of viral genes (4, 16). The exact number and identity of the cellular factors required for HSV replication is unknown, but the identification of such factors is an active area of research as it may shed light on mechanisms of viral replication, the cellular process, or the factor itself. It is this concept that induced us to identify cellular proteins that associate with HSV-1 ICP8.The HSV-1 single-stranded DNA-binding protein, ICP8, is a 128-kDa multifunctional zinc metalloprotein (31, 37) encoded by the U L 29 gene (61). ICP8, in concert with the other HSV DNA replication proteins, including the helicase-primase complex (U L 5, U L 8, U L 52), the origin-binding protein (U L 9), and the polymerase holoenzyme (U L 30/U L 42), is required for viral DNA synthesis (11,12). While the seven HSV DNA replication proteins are known, it is currently unclear as to what host proteins are involved in viral DNA replication. In addition to its role in DNA synthesis, IC...