The herpes simplex virus 1 (HSV-1) immediate-early protein, infected cell protein 22 (ICP22), is required for efficient replication in restrictive cells, for virus-induced chaperone-enriched (VICE) domain formation, and for normal expression of a subset of viral late proteins. The ability of ICP0 to act as a potent viral transactivator is mediated by its N-terminal zinc-binding RING finger domain. In this study, we succeeded in generating a UL7 null HSV-1 mutant virus, MT102, and characterized it. Moreover, as with adenovirus, enhanced green fluorescent protein-tagged fusion proteins of UBF inhibit viral DNA replication. TRL, terminal repeat long; UL, unique long region; IRL, internal repeat long; IRS, internal repeat short; TRS, terminal repeat short. Virion DNA stimulated DNA-PKcs activity in transfected cells, and DNA with 5′ flaps stimulated a higher level of DNA-PKcs activity than that observed in cells transfected with untreated virion DNA. Us regions contain alphaherpesvirus-specific genes.
It has been reported that phosphorylation of viral capsid proteins regulates both subcellular localization and functions including transcriptional regulation, viral uncoating, and nucleic acid chaperone activity (7,–13). Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. It encodes over 76 polypeptides (22, 35) that are expressed as three temporal classes of genes, immediate early, early, and late, and are regulated in a coordinated cascade manner. Funding: This study was supported by grants from the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK092590 to RB), National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01-AR059847 to RB) and the American Heart Association (15POST25090134 to DA). – Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. As with other herpesviruses, the most recent model for HSV-1 envelopment suggests that assembled nucleocapsids are shuttled out of the nucleus by budding and fusion events on the inner and outer nuclear membranes, respectively, and then travel through the cytoplasm until reaching trans-Golgi network (TGN)-derived vesicles (reviewed in reference 27). Ten-milliliter samples of blood were obtained from 8 patients with established diagnoses of CLL following an approved University of Rochester Human Subject protocol.
Cyclin A-CDK-mediated phosphorylation increases DNA-binding activity of Sp1 (17, 23). In contrast, amplification of the viral genome does not alter the level of reporter gene expression from an early viral promoter, BMRF1 CAT, in comparison with the vector control, pCAT Basic (Fig. (A) Schematic diagram of HSV-2 LAT region and HSV-2 miR-I. ; infected-cell extracts showed slightly reduced levels of polyribosomes (data not shown), presumably due to shutoff of host protein synthesis as a consequence of infection. Membrane fusion also requires HSV-1 gD binding to one of its receptors (e.g., herpesvirus entry mediator, nectin-1, and specific sites on heparin sulfate generated by certain 3-O-sulfotransferases [16, 31, 52, 59]) and, probably, gB binding to one of its receptors, including the newly discovered gB-associated cellular protein paired immunoglobulin (Ig)-like type 2 receptor (PILRα) (2, 51). The viral glycoproteins are thus important players in the biology of herpesviruses. This regulated ICP34.5 expression may play an important role during establishment of viral latency and spontaneous reactivation.
Electron Microsc Soc Am Proc. Infected cells at stage I possess intact ND10 and display no staining for UL29. 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). R325 lacks the coding region for the carboxyl-terminal region of ICP22. In summary, the data presented here more fully define the miRNA expression profile of HSV-1 and provide the first demonstration of a phenotypic effect of these miRNAs in culture. If polymerase is present, PML can be recruited to these sites (9). To enrich YK494(VP26T111D/EGFP), infectious viruses obtained from the cotransfection were passaged two times in human neuroblastoma SK-N-SH cells (33); YK494(VP26T111D/EGFP) grew more efficiently than YK492(VP26T111A/EGFP), as described below ( to ).
The infected SK-N-SH cells were harvested and subjected to freeze-thawing and sonication. None of the studies conducted to characterize ICP22 has separated its functions from those of US1.5. Equivalent mutations were introduced into the HSV genome and virus isolates, and their revertants were characterized in cell culture. Suzuki. The genotype of each recombinant virus was confirmed by sequencing (data not shown). (A) CJ394 α22 protein. For instance, the immediate early protein ICP0 is a major player in counteracting cellular antiviral mechanisms by degrading antiviral proteins, such as DNA-PKcs (18, 19).
pBS-VP26-kpn+, into which a silent mutation in the wobble base of VP26 glycine-110 within pBS-VP26 was introduced to create a KpnI restriction site, pBS-VP26T111A with the VP26T111A mutation, and pBS-VP26T111D-bam+ with a VP26T111D mutation that created a BamHI restriction site, were constructed as described previously (34) (). The engineered KpnI or BamHI restriction sites were useful for screening recombinant viruses YK493(VP26Δ/TA-repair/EGFP) or YK494(VP26T111D/EGFP), respectively, as described previously (38). Our data show that gD, gH/gL, and gB act in a series of steps whereby gD is first activated by binding its cell receptor. We demonstrate using the electrophoretic mobility shift assay (EMSA) that US11 binds this RNA fragment directly in a sequence-specific manner via its C-terminal domain. Chimeric male mice were bred with albino female C57BL/6J-Tyrc-2J/J (“albino Bl6”) (Jackson). CTCF was used as a loading control. To make the plasmid that encodes GST.UL11H.d51-96, the NotI site was inserted along with a stop codon which was placed after codon 50.
The packaging flask was incubated 3 additional days before the virus was harvested and stored at −80°C until purification. HSV-1 strain 17+ was used throughout the experiments. and , compare cM to Mc). We detected miR-I in both 293 and HeLa cells transfected with pPstI-HincII (which includes miR-I sequences and ≈3 kb of upstream sequences) (E), implying that sequences more immediately upstream of miR-I can contribute to its expression under some circumstances. To define the ICP27 regions that contribute to this novel function, we examined the ability of MS2 fused with various ICP27 mutants to stimulate translation in the tethered function assay. As a result of the two-step Red-mediated mutagenesis procedure, E. This phenotype requires the expression of at least one other viral protein, as transfection of gM alone does not result in gM localization to the nuclear compartment.
Vero, 293, HeLa, and U2OS cell lines were obtained from ATCC. New Orleans, La. Cell lines.African green monkey kidney fibroblasts (Vero cells) were purchased from the American Type Culture Collection (Manassas, Va.) and maintained as monolayers in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, Calif.) supplemented with 5% fetal bovine serum (Gemini Bio-Products, Woodland, Calif.), penicillin, streptomycin, and amphotericin B (Invitrogen). 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) (). The plaque sizes for cells infected with YK494(VP26T111D/EGFP) were restored to those observed in cells infected with YK333(EGFP) or YK493(VP26Δ/TA-Repair/EGFP) (). To examine the effect of VP26 Thr-111 phosphorylation on HSV-1 pathogenesis, 3-week-old female mice (Charles River Laboratories) were infected intracranially with 500 PFU of YK491(ΔVP26/EGFP), YK492(VP26T111A/EGFP), YK493(VP26Δ/TA-Repair/EGFP), or YK494(VP26T111D/EGFP), as described previously (40, 43), and monitored daily for 14 days. Rabbit polyclonal anti-NBS1 antibody (NB 100-143) was purchased from Novus Biologicals (Littleton, Colo.).
As shown in , survival was significantly higher in mice infected with YK491(ΔVP26/EGFP) or YK492(VP26T111A/EGFP) than in mice infected with YK493(VP26Δ/TA-Repair/EGFP) (). Notably, the survival of mice infected with YK492(VP26T111A/EGFP) was similar to that of mice infected with YK491(ΔVP26/EGFP). J. MR viruses were plaque purified at least three times. Relative quantification of UL6 and UL8 to 18S rRNA was performed in a Thermal Cycler Dice Real Time System (Takara) by real-time RT PCR. The amount of protein present in immunoblot bands was quantified using the ImageQuant LAS 4000 system with ImageQuant TL7.0 analysis software (GE Healthcare Life Sciences). As shown in and , the level of VP26 expression in cells infected with YK492(VP26T111A/EGFP) was significantly lower than that in cells infected with wild-type HSV-1(F) or YK493(VP26Δ/TA-Repair/EGFP).
Virions were thawed, and SDS and proteinase K were added to final concentrations of 1% and 100 μg/ml, respectively. Thus, the VP26 T111D mutation in part restored the VP26 expression downregulated by the VP26 T111A mutation. Effects of the mutations in VP26 on its expression in SK-N-SH cells. All images were taken at ×60 magnification. For the preparation of cell extracts, the monolayers were washed with phosphate-buffered saline (PBS), and the cells were lysed by suspension in 1 ml of cell extract buffer (50 mM HEPES, pH 7.5, 50 mM NaCl, 0.1% NP-40 containing 1 μl of protease inhibitor cocktail [Roche Molecular Biochemicals]). RNA was isolated from liver using Aurum total RNA fatty and fibrous tissue kit (Bio-Rad). Ser2 phosphorylation of the CTD of transcriptionallyengaged pol II is also drastically reduced following expression of full-length Myc-ICP22 and Myc-193-256 (Figure 3C, D).
After the gel slice was dried in a Speed-vac for 20 min, the 40-kDa protein was reduced for 15 min with 2 mM Tris(2-carboxyethyl)phosphine (TCEP) in 25 mM NH4HCO3 (pH 8.0). HSV amplicon vectors encoding β-galactosidase (LacZ), CD80 (B7.1), or CD154 (CD40L) were packaged using either a standard helper virus (designated HSVlac, HSVB7.1, and HSVCD40L) or a helper virus–free method (designated hf-HSVlac, hf-HSVB7.1, and hf-HSVCD40L). The eluted protein was dialyzed against dialysis buffer (20 mM Tris-HCl [pH 8], 100 mM NaCl, 20% glycerol, and 1 mM PMSF) and combined with anti-Flag M2 affinity gel (Sigma). ). miR-I efficiently and specifically silenced ICP34.5 expression [by 93% at 6 h postinfection (hpi) and by 68% at 12 hpi] but not that of thymidine kinase (B). SK-N-SH cells were infected with wild-type HSV-1(F), YK491(ΔVP26/EGFP), YK492(VP26T111A/EGFP), YK493(VP26Δ/TA-repair/EGFP), or YK494(VP26T111D/EGFP) at an MOI of 5. Cells with or without fixation were incubated with mouse monoclonal antibodies to gB or gD in wash buffer on ice for 30 min.
Second, the above-mentioned PCR products were digested with NheI or EcoRV. Vero cells grown on four-well chamber slides (Fisher Scientific, MA) were infected with or without HSV-2 strain 333 at a multiplicity of infection (MOI) of 10. Vol. Cells were incubated with primary antibodies diluted in 3% NGS for at least 30 min. Furthermore, the frequencies of cells showing diffuse nuclear staining of VP5 and VP26 were restored to 86 and 89%, respectively, in cells infected with YK494(VP26T111D/EGFP) (). These results suggest that localization of VP5 and VP26 is regulated by phosphorylation at VP26 Thr-111 in HSV-1-infected cells. Effects of mutations in VP26 on the subcellular localization of VP26 and VP5 in SK-N-SH cells.
Tallies of cell populations were also performed with an Olympus BX60 microscope equipped with a mercury burner, a universal reflected-light fluorescent vertical illuminator, and a UPlanFl 100× objective (Olympus America Inc., Melville, N.Y.). Although all capsid proteins of HSV-1 have previously been reported to be phosphorylated in infected cells, as described above, the biological significance of HSV-1 capsid phosphorylation, as well as that of other herpesvirus capsid proteins, remains to be fully elucidated (22,–25). In this study, we have shown that the T111A mutation in VP26, which abolishes the phosphorylation of VP26 Thr-111, reduced HSV-1 replication and cell-cell spread in SK-N-SH cells and reduced HSV-1 neurovirulence in mice following intracranial inoculation. The same concentration of IFN-β was added to PBS washes and media during the course of infection. As shown in Fig. Subcellular fractionation was performed as described previously . Notably, the T111A mutation in VP26 impaired HSV-1 replication in SK-N-SH cells and neurovirulence in mice at levels similar to those observed with the VP26 null mutation.
These results suggest that phosphorylation of VP26 at Thr-111 is critical for efficient regulation of HSV-1 replication and pathogenesis. Samples were harvested at 48 h following transfection and titrated on Vero cells. (i) We have shown that the VP26 null mutation misdirects VP5 to nuclear punctate structures, indicating that VP26 regulates VP5 localization in infected cells. Since VP5 was diffusely distributed throughout the nucleus in wild-type HSV-1-infected cells, VP26 appears to prevent the aggregation of VP5 into punctate structures. 1A and Table 1). Seventy-five microliters of the 50% slurry was incubated with 50 μg of protein on a rotating wheel for 5 min at room temperature. However, the role of VP26 in HSV-1 pathogenesis was not investigated in that study.
Both recombinant full-length ICP22 and recombinant residues 193256 interact with recombinant CDK9 (Figure 5B), indicating that the interaction between ICP22 and CDK9 is direct and that residues 193 to 256 are sufficient. ). PMA was used to provide an extrinsic Signal One to potentially compensate for the adverse effect elicited by the helper virus on CLL cells, thereby allowing transduced B7.1 to elicit a costimulatory signal to T cells. The immune complex was washed three times with TBS buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl), and eluted with SDS gel loading buffer. For example, late gene expression in both EBV and HSV (γ2) is dependent upon lytic DNA replication, but the requirement is in cis for HSV (18, 26, 38, 48) and in trans for EBV (36). Although highly expressed, miR-I was not previously predicted, although several other miRNA were predicted for HSV-2 (10). Based upon the requirement for VP26 in efficient HSV-1 replication and pathogenesis shown in this and previous studies (42), it is reasonable to hypothesize that proper expression of VP26, regulated by its phosphorylation at Thr-111, is important for HSV-1 replication and pathogenesis.
, MBP-gB-P2 was labeled with [γ-32P]ATP in kinase assays using GST-Us3 (Fig. The following antibodies were used: PAS980 polyclonal anti-gM (courtesy of Lynn Enquist), TGN46 (Serotec), Calnexin (Stressgen), G1/93 anti-ERGIC53 (from Hans-Peter Hauri), Golgin97 (Molecular Probes), p230 (BD Transduction Laboratories), monoclonal antibody (MAb) 414 anti-nuclear pore complex (NPC) proteins (Covance Research Products), ICP5 anti-VP5 (Cedarlane), ID3 anti-gD (from Gary Cohen and Rosalyn Eisenberg), LP11 anti-gH (from Helena Brown), MAb 15betaB2 anti-gB (from David C. The nucleotide sequence for the C-terminal 19 aa and the stop codon is identical between the two viral strains (strains 333 and HG52) for which ICP34.5 sequences are available in GenBank. 3. Primary antibodies in TBST were used at the following dilutions: monoclonal anti-RAD51, anti-DNA-PKcs cocktail, and anti-Ku70 at 1:200; anti-RAD51 and antitubulin at 1:1000; 3-83, 71-9A, 2H10, anti-Mre11, and anti-Rad52 at 1:5,000; and anti-NBS1 and anti-Ku86 at 1:10,000. Support for this hypothesis comes from a report that FP tagging of a PRV VP26 homolog impairs viral replication and neuroinvasiveness at levels similar to those observed with the null mutation of PRV VP26 (45). Furthermore, as described above, the T111A mutation in HSV-1 VP26 reduced HSV-1 replication and neurovirulence at levels similar to those caused by the null mutation of HSV-1 VP26.
It is well-documented that phosphorylation of a protein can cause conformational changes (46). ). VP26 may prevent the aggregation of these HSV-1 capsid proteins, as suggested above, thereby distributing the capsid proteins throughout the nucleus and facilitating efficient capsid assembly. It is likely that phosphorylation of VP26 Thr-111 is required in order for VP26 to adopt the correct conformation required to prevent the aggregation of the HSV-1 capsid proteins in the nucleus, thus promoting not only efficient capsid assembly but also nuclear egress and viral replication. Light units are displayed as percentages normalized to the pICP0 values, and statistical analyses were performed using a Mann-Whitney U test. When the Phos mutant forms of ICP0 were examined for PML staining in Vero cells (Fig. Even at an MOI of 3, the yield of MT102 (ΔUL7) at 24 h post-infection was about 10-fold less than that of wild-type YK304 (Figure ).
In agreement with this hypothesis, it has been reported that HSV-2 UL14 has the ability to regulate the subcellular localization of VP26 (47). Blocking the phosphorylation of VP26 Thr-111 may cause a subtle imbalance among the factors that determine the localization of VP26 in infected cells, resulting in an aberrant accumulation of VP26 and VP5 in punctate structures in the nucleus in only a fraction of infected cells. Transfection experiments thus permit us to explore the infectivity of virion DNA in the absence of tegument proteins that may act to promote or disable host factors responding to incoming viral genomes. Thr-111 is located in the C-terminal domain of VP26 in HSV-1 and, interestingly, it has also been reported that the VP26 homolog in Epstein-Barr virus is phosphorylated in the C-terminal domain (48). Therefore, there is a possibility that the function(s) of the EBV VP26 homolog, as well as those of other herpesviruses, is also regulated by phosphorylation of the C-terminal domain. Conditions for coculture, fixation, and staining are the same as for Fig. Deletion analysis of the US11 binding site on 12/14 RNA.
We are currently pursuing this possibility.