Tyrosine 116 of the Herpes Simplex Virus Type 1 IEα22 Protein Is an Ocular Virulence Determinant and Potential Phosphorylation Site | IOVS

Tyrosine 116 of the Herpes Simplex Virus Type 1 IEα22 Protein Is an Ocular Virulence Determinant and Potential Phosphorylation Site | IOVS

The herpes simplex virus 1 (HSV-1) virion DNA contains nicks and gaps, and in this study a novel assay for estimating the size and number of gaps in virion DNA was developed. Previous studies have investigated the roles of Us8A in HSV-1 infection using null mutations in Us8A and gE; therefore, the role of Us8A remains to be elucidated. Research on the design and application of gene transfer vehicles and genetically altered cells to the treatment of a variety of diseases has greatly expanded the dimension and scope of this field such that almost any genetic alteration of cells and tissues to effect a therapeutic outcome is now considered to employ the principles of gene therapy. Here, we present data to support that hypothesis. This RNA sequence, designated 12/14, is present in the coterminal HSV-1 mRNAs UL12, UL13, and UL14. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase.

In addition, UL11 homologs from pseudorabies and Marek’s disease herpesviruses were also found to be capable of binding to the 40-kDa protein from HSV-1-infected cells, suggesting that the interaction is conserved among alphaherpesviruses. CLL cells transduced with hf-HSVCD40L were also more effective at stimulating T-cell proliferation than those transduced with HSVCD40L stocks and were successful in stimulating autologous CTL activity. Transcription factor Sp1 is a 95- to 105-kDa protein that binds to GC-rich recognition elements (GC-boxes) through C-terminal zinc finger motifs (30). EBV DNA replication acts in trans, likely initiating a cascade of events which culminates at specific late promoter sequences. A ≈8-kb primary LAT is spliced, yielding a stable ≈2-kb LAT intron (2). To determine whether ICP27 protein is associated with the translation machinery in HSV-1-infected cells, sucrose gradient analysis was used to separate polyribosomes from monoribosomes and uncomplexed ribosomal subunits. This report presents studies of the interaction between Us3 protein kinase and HSV-1 envelope glycoprotein B (gB).

Hence, gB, gC, gD, and the gH/gL complex mediate attachment and fusion of the virus at the cell surface (70). Although ICP34.5 is characterized as a γ1 late gene (8), the ICP34.5 protein is detectable as early as 2 h postinfection in infected cell cultures (9). Electron Microsc Soc Am Proc. Although recent reports indicate that viral proteins may function to promote recombination (49, 50, 57), it is likely that recombination during HSV-1 infection also may involve cellular recombination pathways (79). Immunoblot analysis of α22 protein isoforms in CJ394- and OD4-infected Vero cells. (E) Nocodazole-arrested HEp-2 cell lysates were treated with alkaline phosphatase in the presence or absence of phosphatase inhibitors and then reacted with GST fusion protein encoding the full-length UL42 protein. To begin to define the functions of the RISC-associated HSV-1 miRNAs, we have generated mutant viruses individually lacking three of the most highly expressed HSV-1 miRNAs, i.e., miR-H2, miR-H3, and miR-H4.

During wild-type infection, if viral DNA replication is blocked by the addition of the viral polymerase inhibitor phosphonoacetic acid (PAA), stage IIIb prereplicative sites containing all seven essential viral proteins form. Large open arrows: cluster of isoforms at 85 kDa. YK333(EGFP) carries an expression cassette consisting of the Egr-1 promoter, the EGFP open reading frame (ORF), and bidirectional polyadenylation signals of the HSV-1 UL21 and UL22 genes in the intergenic region between the UL3 and UL4 genes (31) and shows growth properties identical to those of wild-type HSV-1(F) (32). This protein has been shown to be extensively modified posttranslationally and appears to be phosphorylated, at least in part, by the virus-encoded kinase UL13 (17). The ability of ICP0 to target specific cellular proteins for ubiquitination and proteasome-dependent degradation has given rise to the hypothesis that ICP0 counteracts the cellular repression mechanism(s) that either initiates or maintains viral genomes in a state of transcriptional quiescence. 293T cells were maintained in DMEM containing 10% FCS. Extracts from equal numbers of cells were loaded in the comparable lanes 1 to 4 in the CJ394 and lanes 5 to 8 in the OD4 samples.
Tyrosine 116 of the Herpes Simplex Virus Type 1 IEα22 Protein Is an Ocular Virulence Determinant and Potential Phosphorylation Site | IOVS

Large open arrows: cluster of isoforms at 85 kDa. DNA repair proteins play both positive and negative roles during HSV infection, and HSV manipulates components of these pathways, activating some and disabling others (18–20, 24–32). Tryptic phosphopeptide mapping of α22 proteins. Infected cells were labeled with 32[P]-orthophosphate from 1 hour 50 minutes to 5 hours after virus was added and harvested, and the proteins were electrophoresed in a 12% denaturing polyacrylamide gel. In this report, we argue that as a regulator of fusion, gH/gL might not have to be in the same membrane as gB in order to regulate its activity, i.e., gH/gL on one cell might promote fusion of gB expressed by another cell, as long as gD and a gD receptor are also present. The objective of this study was to discover whether US11 had the potential to bind any other transcripts from HSV-infected cells that would be relevant to its role in the virus lytic cycle. A fragment containing the CRE-Luc2P-SV40 poly(A) cassette was excised from pGL4.29 plasmid (Promega, Genbank: DQ904461.1) using BamHI /SpeI, cloned into BamHI/ SpeI sites of a modified pBluescript vector containing two XmaI sites (pBS-Xma2) and then sub-cloned into the Ai9-tdTomato vector [15] using XmaI sites.

Here we show that ICP22 expression causes loss of phosphorylation of Ser2 and Tyr1 of the pol II CTD heptapeptide repeat without markedly affecting phosphorylation of Ser5 and Ser7. The product was cloned into pEGFP-N2 with SstI-EcoRI. After the incubations, the contents of the 2 tubes were combined over a period of 30 seconds and incubated for an additional 20 minutes at 22°C. Human glioma cell lines M059J and MJ-M6 (34) were maintained in DMEM-Ham F-12 nutrient mixture (Sigma) supplemented with 10% FCS and puromycin (0.5 μg/ml). , compare BcLF1 core to BMRF1 core). S1). Quantitative reverse transcription-PCR showed that the levels of reporter mRNA remained unchanged at 12 h postinjection in oocytes expressing either MS2 coat protein or MS2-ICP27 (Fig.

coli GS1783 containing pYEbac102 (55) (a kind gift from Gregory A. Experiments to evaluate the mechanism of gM targeting indicated it was not passively recycled from the TGN but rather actively and specifically targeted to the nucleus. Further, we showed the C-terminal domain of ICP27 is required for the efficient and specific inhibition of ICP34.5 splicing. Conway J F, Duda R L, Cheng N, Hendrix R W, Steven A C. We propose that HSV-1 genomes target and disrupt ND10 in order to gain access to certain ND10-associated proteins, including those involved in recombination and repair. Infected cells were lysed and incubated with agarose coupled anti-phosphotyrosine antibody. After extensive washing, the proteins were electrophoresed and blotted with the α22-specific RGST22 antibody.

Infected cells (MOI, 0.01 or 3) at each point were harvested, resuspended in 200 μl of a lysis buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.001% Triton X-100, 0.001% SDS) containing 50 mg/ml of proteinase K, and incubated at 50°C overnight. Mouse monoclonal antibodies against the 32-kDa subunit (71-9A) (16) and the 70-kDa subunit (2H10; also called 70c) (25) of RPA were supplied by Marc Wold (University of Iowa). Note that this doublet is visible in the OD4 lane, but migrates slightly faster, suggesting the loss of at least one phosphate group. In agreement with the growth properties of these viruses demonstrated in this and previous studies (42), YK491(ΔVP26/EGFP) and YK492(VP26T111A/EGFP) produced plaques similar in size but that were smaller than those observed with YK333(EGFP) and YK493(VP26Δ/TA-Repair/EGFP) (). Correct insertion of the 3×stop mutation was confirmed with SacII digests and Southern blot analyses (). Viral DNAs from plaque picks were isolated, and incorporation of each Phos mutation into the viral genome was confirmed by Southern blot analyses using restriction enzyme digests (see Fig. The recombinant viruses were verified by Southern blotting as described previously [12].

The second band in the CJ394 lane is actually a doublet migrating at approximately 75 kDa. Note that this doublet is visible in the OD4 lane, but migrates slightly faster, suggesting the loss of at least one phosphate group.

You may also like