Inhibition of the stress-activated kinase, p38, does not affect the virus transcriptional program of herpes simplex virus type 1

To investigate the impact of stress kinase p38 activation on HSV-1 transcription, we performed a global transcript profile analysis of viral mRNA using an oligonucleotide-based DNA microarray. RNA was isolated from Vero cells infected with the KOS strain of HSV-1 in the presence or absence of SB203580, a pyridinyl imidazole inhibitor of p38. Under conditions that eliminated ATF2 activation but had no effect on c-, and reduced virus yield by 85–90%, no effect on accumulation of viral IE, DE, or L transcripts was observed by array analysis or selected Northern blot analysis at 2, 4, and 6 h post infection. Results of array data from cells infected with the ICP27 mutant d27-1 in the presence or absence of SB203580 only reflected the known restricted transcription phenotype of the ICP27 mutant. This result is consistent with a role for p38 activation on virus replication lying downstream of the essential role of ICP27 in DE and perhaps late transcription regulation. No effect of SB203580 on transcription was detected after infection with the ICP0 mutant 7134, at 0.5 or 5.0 PFU/cell, though decreases in the rate of accumulation of all kinetic classes of mRNA could be detected, relative to virus. These results indicate that inhibiting p38 activity in Vero cells, while significantly reducing virus yield, demonstrated no obvious impact on the program of viral transcription

Herpes Simplex Virus ICP27 Activation of Stress Kinases JNK and p38

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0(−)/4(−)) and d103 (4(−)/22(−)/47(−)) mutants activated p38 and JNK, while the d106 (4(−)/22(−)/27(−)/47(−)) and d107 (4(−)/27(−)) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary

Herpes Simplex Virus Type 1 ICP27-Dependent Activation of NF-κB

The ability of herpes simplex virus type 1 (HSV-1) to activate NF-κB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-κB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IκBα and IκBβ. In this study, we identified loss of IκBα as a marker of NF-κB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IκBα loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IκBα loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-κB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-κB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IκBα and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-κB activation during HSV infection, highlighting the important role that NF-κB may play in infection

HCMV Protein LUNA Is Required for Viral Reactivation from Latently Infected Primary CD14⁺ Cells

<div><p>Human cytomegalovirus (HCMV) is a member of the <em>Herpesviridae</em> family that infects individuals throughout the world. Following an initial lytic stage, HCMV can persist in the individual for life in a non-active (or latent) form. During latency, the virus resides within cells of the myeloid lineage. The mechanisms controlling HCMV latency are not completely understood. A latency associated transcript, UL81-82ast, encoding the protein LUNA (Latency Unique Natural Antigen) was identified from latently infected donors <em>in vivo</em>. To address the role of the UL81-82ast protein product LUNA, in the context of the viral genome, we developed a recombinant HCMV bacterial artificial chromosome (BAC) that does not express LUNA. This construct, LUNA knockout FIX virus (FIX-ΔLUNA), was used to evaluate LUNA's role in HCMV latency. The FIX-ΔLUNA virus was able to lytically infect Human Fibroblast (HF) cells, showing that LUNA is not required to establish a lytic infection. Interestingly, we observed significantly higher viral copy numbers in HF cells infected with FIX-ΔLUNA when compared to FIX-WT virus. Furthermore, FIX-WT and FIX-ΔLUNA genomic DNA and transcription of UL81-82ast persisted over time in primary monocytes. In contrast, the levels of UL138 transcript expression in FIX-ΔLUNA infected HF and CD14⁺ cells was 100 and 1000 fold lower (respectively) when compared to the levels observed for FIX-WT infection. Moreover, FIX-ΔLUNA virus failed to reactivate from infected CD14⁺ cells following differentiation. This lack of viral reactivation was accompanied by a lack of lytic gene expression, increase in viral copy numbers, and lack of the production of infectious units following differentiation of the cells. Our study suggests that the LUNA protein is involved in regulating HCMV reactivation, and that in the absence of LUNA, HCMV may not be able to enter a proper latent state and therefore cannot be rescued from the established persistent infection in CD14⁺ cells.</p> </div

FIX-ΔLUNA viral infection of CD14⁺ cells fail to produce infectious virus after IL6 differentiation.

<p>A) Cell lysates plus supernatant from FIX-WT, FIX-ΔLUNA, or FIX-Rev infected cells were collected at 10 dpi and 20 dpi. IL6 was added at 10 dpi; at 5 (15dpIL6) and 10 (20dpIL6) days after differentiation, cell lysates and sups were also collected. The collected lysate/supernatant mix was serially diluted and used to infect HF cells. Two days post infection, cells were fixed with 4× paraformaldehyde, then stained with mouse anti-IE1/2 (1∶250) overnight. Cells were then incubated with Alexa Fluor 488 anti-mouse IgG for 1 hr, and incubated with DAPI for 30 min. IE positive cells were counted to determine the number of infectious units (IU). Only the time points post reactivation showed any significance when comparing FIX-WT or FIX-Rev to FIX-ΔLUNA (10dpIL6: p<3×10⁻⁵; 20dpIL6: p<0.0006). The ** indicates a <i>P</i>-value<0.001.</p

LUNA deletion mutants do not disrupt pp71.

<p>A) Sequence of the start codon region for UL8-82ast in FIX-WT and FIX-ΔLUNA viruses. The upper strand represents the coding frame for UL82 and its translation, the bottom strand represents the coding frame for UL81-82ast and its translation. Red-labeled nucleotides and amino acids indicate those that changed after mutagenesis. B) Hind III digest of FIX-BAC-WT (lane 1), FIX-BAC-(GalK-KAN)-A (lane 2), FIX-BAC-ΔLUNA (lane 3), FIX-BAC-(GalK-KAN)-B (lane 4) and FIX-BAC-Rev (lane 5). FIX-BAC-(GalK-KAN)-A contains the Galk-Kan insert before the mutation; FIX-BAC-(GalK-KAN)-B is the re-introduction of the insert in the process of creating the revertant. C) Southern blot of the digest in (B) with a GalK specific probe. The Galk-Kan insert is indicated by the arrow. D) HF cells were infected with FIX-WT, FIX-ΔLUNA or FIX-Rev respectively over a 20day time course. The presence of the pp71 protein was detected via western blotting. 20 µg of protein was added per well, samples were loaded in the following order for each time point: FIX-WT, FIX-ΔLUNA and FIX-Rev. Lane 1: Mock HF, lanes 2–4: HF 1 dpi, lanes 5–7: HF 3 dpi, lanes 8–10: HF 5 dpi, lanes 11–13: HF 10 dpi, lanes 14–16: HF 20 dpi. Blots were incubated with primary antibodies goat anti-pp71 (1∶500) and mouse anti-actin (1∶10,000) and secondary antibodies anti-goat or anti-mouse IgG HRP (1∶1000).</p

Primers for BAC mutagenesis and RT-PCR.

a<p>F: Forward Primer; R: Reverse Primer.</p

The LUNA protein is dispensable for growth in HF infected cells and to maintain latent infection in CD14⁺ cells.

<p>A) HF and CD14⁺ cells were infected at an MOI = 5, MOI = 1, or MOI = 0.5 with either FIX-WT, FIX-ΔLUNA or FIX-Rev. Supernatants from each infection were collected at the indicated time points and titered via standard plaque assay. Samples were tested in triplicate. Student <i>t</i> test analysis determined no significant difference among the viral growth of FIX-WT vs. FIX-Rev or FIX-ΔLUNA in either HF cells or in CD14⁺ cells (p>0.5 in both cell lines). B) HF and CD14⁺ cells were infected at an MOI = 3 with either FIX-WT, FIX-ΔLUNA, or FIX-Rev. Samples were fixed at 1 dpi and stained with rabbit anti-LUNA and Alexa Fluor 594 anti rabbit IgG (red) for LUNA detection, and mouse anti-IE primary antibodies along with Alexa Fluor 488 anti-mouse IgG (green). Cell nuclei were stained with DAPI. C) HF and CD14⁺ cells were infected at an MOI = 3 and protein was harvested at 1day post infection for western blot analysis. Proteins were detected using a rabbit anti-LUNA monoclonal antibody (1∶500), mouse anti-IE1 monoclonal antibody (1∶100) and mouse anti-tubulin as a loading control.</p