Virion Host Shutoff Research Articles

Construction and properties of a herpes simplex virus 2 dl5-29 vaccine candidate strain encoding an HSV-1 virion host shutoff protein

The replication-defective herpes simplex virus 2 (HSV-2) dl5-29 mutant virus strain with deletions in the U L5 and U L29 genes has been shown to protect mice and guinea pigs against challenge with wild-type (wt) HSV-2 and to protect against ocular disease caused by HSV-1 infection. The dl5-29 strain is currently being prepared for clinical trials as a herpes vaccine candidate. As a possible approach to improve the efficacy of dl5-29 as a genital herpes vaccine, we replaced the U L41 gene encoding the virion host shutoff function (vhs) with the U L41 gene from HSV-1. While the HSV-2 U L41 and HSV-1 U L41 gene products have analogous functions, vhs-1 is 40-fold less active than vhs-2. Previously, it was shown that disruption of the U L41 gene can increase the efficacy of dl5-29 as a vaccine against HSV-2. These properties led us to hypothesize that replacement of vhs-2 by vhs-1 would decrease cytopathic effects in infected host cells, allowing longer survival of antigen-presenting cells and induction of stronger immune responses. The new recombinant dl5-29-41.1 virus shows nearly the same immunogenicity and protection against HSV-2 challenge as the parental dl5-29 virus or a triply deleted mutant virus, dl5-29-41, in the murine model of infection, and grows to higher titers than the parental strain in complementing cells, which is important for GMP production. The results have implications for the design of future HSV-2 vaccine candidates and mechanisms of induction of protective immunity against genital herpes.

Role of the UL41 Protein of Pseudorabies Virus in Host Shutoff, Pathogenesis and Induction of TNF-.ALPHA. Expression

The vhs (virion host shutoff) is highly conserved in alphaherpesvirus, including pseudorabies virus (PRV). In an attempt to explore the function of vhs of PRV, we constructed and characterized a mutant virus (Δ41). In the absence of vhs activity, Δ41 mutant is highly attenuated in mice model and the lethality is correlated with the virus dissemination in neural tissues. As with herpes simplex virus type 1 (HSV-1), the prototype virus of alphaherpesvirus, the pronounced decrease in cellular protein synthesis triggered by wild type PRV was largely restored in cells infected with Δ41 virus. Furthermore, tumor necrosis factor-α (TNF-α) protein expression was elevated significantly in spleen of mice infected with vhs mutant virus. Since TNF-α has been indicated to be an important cytokine in the innate immune response against various infections, our results implicate vhs may contribute to the protection against PRV lethality via the action of TNF-α. Overall, we confirm the shutoff function of vhs protein in PRV, and demonstrate the role that vhs protein plays in virulence, and regulation of cytokine production.

The virion-packaged endoribonuclease of herpes simplex virus 1 cleaves mRNA in polyribosomes

The virion host shutoff protein product of the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease that selectively degrades mRNAs during the first hours after infection. Specifically, in contrast to the events in uninfected cells or cells infected with a mutant lacking the RNase, in wild-type virus-infected cells mRNA of housekeeping genes exemplified by GAPDH is degraded rapidly, whereas mRNAs containing AU elements are cleaved and the 5' cleavage product of these RNAs persists for many hours. We report that in wild-type virus-infected cells there was a rapid increase in the number and size of processing bodies (P-bodies). These P-bodies were also preset in cycloheximide (CHX)-treated cells but not in either treated or untreated uninfected cells or cells infected with the RNase minus mutant. Additional studies revealed that polyribosomes extracted from cytoplasm of wild-type virus-infected cells treated with CHX and displayed in sucrose gradients contained ribosome-loaded, truncated AU-rich mRNAs lacking the 3' UTR and poly(A) tails. The results suggest that the virion RNase is bound to polyribosomes by virtue of the reported association with translation machinery and cleaves the RNAs 5' to the AU elements. In contrast to the slow degradation of the of the residual 5' domain, the 3' UTR of the AU-rich mRNA and the GAPDH mRNA are rapidly degraded in wild-type virus-infected cells.

Increased eIF2α Phosphorylation Attenuates Replication of Herpes Simplex Virus 2 vhs Mutants in Mouse Embryonic Fibroblasts and Correlates with Reduced Accumulation of the PKR Antagonist ICP34.5

Herpes simplex virus 2 (HSV-2) strains containing mutations in the virion host shutoff (vhs) protein are attenuated for replication compared with wild-type virus in mouse embryonic fibroblasts (MEFs). However, HSV-2 vhs mutants replicate to near wild-type levels in the absence of the RNA-activated protein kinase (PKR). PKR is one of several kinases that phosphorylates the eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation, and we previously found that more of the phosphorylated form of eIF2alpha accumulates in MEFs infected with HSV-2 vhs mutants than with wild-type virus. Here, we show that this increase in phosphorylated eIF2alpha is primarily PKR dependent. Using MEFs expressing nonphosphorylatable eIF2alpha, we demonstrate that phosphorylated eIF2alpha is the primary cause of attenuated replication of HSV-2 vhs mutants and that attenuation correlates with decreased accumulation of viral proteins. Normally, HSV antagonizes eIF2alpha phosphorylation through the action of ICP34.5, which redirects protein phosphatase 1alpha (PP1alpha) to dephosphorylate eIF2alpha during infection. We show that ICP34.5 does not accumulate efficiently in MEFs infected with HSV-2 vhs mutant viruses, suggesting that the accumulation of phosphorylated eIF2alpha and the attenuated phenotype of HSV-2 vhs mutants in MEFs result from a deficiency in ICP34.5.

Identification of Replication-competent HSV-1 Cgal+ Strain Signaling Targets in Human Hepatoma Cells by Functional Organelle Proteomics

In the present work, we have attempted a comprehensive analysis of cytosolic and microsomal proteomes to elucidate the signaling pathways impaired in human hepatoma (Huh7) cells upon herpes simplex virus type 1 (HSV-1; Cgal(+)) infection. Using a combination of differential in-gel electrophoresis and nano liquid chromatography/tandem mass spectrometry, 18 spots corresponding to 16 unique deregulated cellular proteins were unambiguously identified, which were involved in the regulation of essential processes such as apoptosis, mRNA processing, cellular structure and integrity, signal transduction, and endoplasmic-reticulum-associated degradation pathway. Based on our proteomic data and additional functional studies target proteins were identified indicating a late activation of apoptotic pathways in Huh7 cells upon HSV-1 Cgal(+) infection. Additionally to changes on RuvB-like 2 and Bif-1, down-regulation of Erlin-2 suggests stimulation of Ca(2+)-dependent apoptosis. Moreover, activation of the mitochondrial apoptotic pathway results from a time-dependent multi-factorial impairment as inferred from the stepwise characterization of constitutive pro- and anti-apoptotic factors. Activation of serine-threonine protein phosphatase 2A (PP2A) was also found in Huh7 cells upon HSV-1 Cgal(+) infection. In addition, PP2A activation paralleled dephosphorylation and inactivation of downstream mitogen-activated protein (MAP) kinase pathway (MEK(1/2), ERK(1/2)) critical to cell survival and activation of proapoptotic Bad by dephosphorylation of Ser-112. Taken together, our results provide novel molecular information that contributes to define in detail the apoptotic mechanisms triggered by HSV-1 Cgal(+) in the host cell and lead to the implication of PP2A in the transduction of cell death signals and cell survival pathway arrest.

Construction of a bovine herpesvirus 5 with a deletion on the gene virion host shutoff

Construction of a bovine herpesvirus 5 with a deletion on the gene virion host shutoff

Type I IFN response to Papiine herpesvirus 2 ( Herpesvirus papio 2; HVP2) determines neuropathogenicity in mice

Isolates of baboon α-herpesvirus Papiine herpesvirus 2 (HVP2) exhibit one of two distinct phenotypes in mice: extremely neurovirulent or apathogenic. Previous studies implicated the type I interferon (IFN) response as being a major factor in controlling infection by apathogenic isolates. To further investigate the possibility that the host IFN-β response underlies the pathogenicity of the two HVP2 subtypes, the susceptibility of mice lacking the IFN-β receptor (IFNAR − / − ) to infection was examined. Apathogenic isolates of HVP2 (HVP2ap) replicated in IFNAR − / − primary mouse dermal fibroblast (PMDF) cultures as well as neurovirulent (HVP2nv) isolates. IFNAR − / − mice were also susceptible to lethal infection by HVP2ap isolates. Unlike Balb/c or parental 129 mice, LD 50 and ID 50 values for HVP2ap were the same in IFNAR − / − mice indicating that in these mice infection always progressed to death. HVP2ap replicated in the skin at the site of inoculation and invaded dorsal root ganglia as efficiently as HVP2nv in IFNAR − / − mice. Since the virion host shutoff (vhs) protein encoded by the UL41 gene of herpes simplex virus has been implicated in circumventing the host IFN-β response and the phenotype of UL41 deletion mutants of HSV is very similar to that of HVP2ap isolates, the UL41 gene was deleted from HVP2nv (Δ41) and replaced with the UL41 ORF from HVP2ap (Δ41C). Like the parental HVP2nv virus, the Δ41C recombinant replicated efficiently in Balb/c PMDFs and did not induce a strong IFN-β response. The neuropathogenicity of the Δ41C recombinant was also the same as the parental HVP2nv virus in Balb/c mice, indicating that the vhs protein does not underlie the different neuropathogenic phenotype of HVP2ap and HVP2nv. In contrast, the Δ41 deletion virus induced a strong IFN-β response but was still able to undergo multiple rounds of replication in PMDF cultures, albeit at a slower pace than the parental HVP2nv. This was reflected in vivo as the Δ41 mutant had an LD 50 equivalent to that of the parental HVP2nv virus although the time to death was longer. These results indicate that while the vhs protein is involved in preventing and/or suppressing an IFN-β response, it is not responsible for the ability of HVP2nv to overcome IFN-β induced resistance of uninfected cells and does not underlie the divergent pathogenicity of the two HVP2 subtypes in mice.

Host Responses to Wild-Type and Attenuated Herpes Simplex Virus Infection in the Absence of Stat1

Humans and mice lacking the interferon signaling molecule Stat1 are sensitive to a variety of pathogens due to their presumed inability to mount a strong innate immune response. The herpes simplex virus type 1 (HSV-1) virion host shutoff (vhs) protein is a multifunctional immunomodulator that counteracts the innate immune response and viruses lacking vhs are attenuated and effective live vaccines in animal models. To investigate the interplay of viruses with an immunocompromised host, we performed functional genomics analyses on control and Stat1(-/-) mouse corneas infected with wild-type or vhs-null viruses. In control mice, correlative with viral growth, both viruses induced a transient increase in immunomodulators, followed by viral clearance. In contrast, infection of the Stat1(-/-) mice induced a heightened and prolonged induction of inflammatory modulators for both viruses, manifesting as a significant immune cell infiltrate and ocular disease. Moreover, while wild-type virus infection of Stat1(-/-) was always lethal, vhs-null infection was rarely lethal. There was a significant increase in Stat3- and interleukin-6 (IL-6)-dependent transcription in Stat1(-/-) mice, implicating the Stat3 and IL-6 pathways in the observed ocular pathology. Further, infected Stat1(-/-) mice showed phosphorylated Stat3 in the corneal epithelium. Our data show a role for vhs in evading innate host responses and a role for Stat1 in limiting virus infection and for facilitating an appropriate nonpathological inflammatory response.

Therapeutic Efficacy of a Herpes Simplex Virus With Radiation or Temozolomide for Intracranial Glioblastoma After Convection-enhanced Delivery

The herpes simplex virus-1 (HSV-1)-infected cell protein 0 (ICP0) is an E3 ubiquitin ligase implicated in cell cycle arrest and DNA repair inhibition. Convection-enhanced delivery (CED) of either the replication-defective, ICP0-producing HSV-1 mutant, d106, or the recombinant d109, devoid of all viral genome expression, was performed to determine the in vivo efficacy of ICP0 in combination with ionizing radiation (IR) or systemic temozolomide (TMZ) in the treatment of glioblastoma multiforme (GBM). Intracranial U87-MG xenografts were established in athymic nude mice. Animal survival was determined after mice underwent intracranial CED of either the replication-defective d106 or d109 viruses, or Hanks' balanced salt solution (HBSS), before a single session of whole-brain irradiation or TMZ treatment. Median survival for animals that underwent treatment with HBSS alone, d109 alone, d106 alone, HBSS + IR, HBSS + TMZ, d109 + IR, d106 + IR, and d106 + TMZ was 28, 35, 41, 39, 44, 39, 68 (P < 0.01), and 66 days (P < 0.01), respectively. Intracerebral d106 CED resulted in a significant increase in athymic nude mouse survival when combined with IR or TMZ. d106 CED allows for distribution of HSV-1 in human GBM xenografts and persistent viral infection.

Herpesvirus tegument protein activates NF-κB signaling through the TRAF6 adaptor protein

Viruses remodel the host cell to optimize their replication both by delivery of virion proteins into the cell and by de novo expression of viral proteins. The HSV particle contains several proteins that function to prepare the host cell for viral replication, including the VP16 transcriptional activator protein and virion host shutoff protein. HSV infection activates NF-kappaB pathways through Toll-like receptor (TLR) 2 and non-TLR pathways, and NF-kappaB activity is required for efficient viral replication. In a screen of the HSV proteome, we observed that the HSV U(L)37 tegument protein activates NF-kappaB signaling in a TLR2-independent manner. Expression of U(L)37 in transfected cells leads to IkappaB degradation and activation of both reporter genes and the endogenous IL-8 gene. This activation requires TNF receptor-associated factor 6 (TRAF6), and U(L)37 contains a TRAF6-binding domain that is required for interaction with TRAF6 and activation of NF-kappaB. A mutant virus encoding U(L)37 with an altered TRAF6-binding site shows reduced NF-kappaB activation in the early phase of infection. Therefore, the HSV U(L)37 virion structural protein can activate NF-kappaB through TRAF6. Activation of NF-kappaB by a virion tegument protein that is delivered into the host cell cytoplasm during viral entry represents a mechanism for activation of this pathway by a virus.

Further improvements in the technology of HSV-1-based amplicon vectors

Background: Herpes simplex virus type 1 (HSV-1) amplicon vectors can be used to deliver transgenes to the nuclear environment of mammalian cells without cytotoxicity or pathogenic consequences for the inoculated organisms. Previous efforts describe a method of making helper virus-free amplicons (hf-HSV particles) or cells that contain those particles (e.g., packaging cell lines or patient's cells infected in vivo or ex vivo). Objective/methods: Described herein are several advances in hf-HSV particle technology that were designed to improve both production, by expressing viral proteins in trans (e.g., virion host shutoff protein and/or viral protein 16) during production, and also amplicon genome expression stability in the infected cells, by including a transposon-encoding system (e.g., the Tcl-like Sleeping Beauty transposon system). Results/conclusions: Improved titers and chromosomal integration of the transgene enhanced hf-HSV amplicon applications. Nevertheless, further studies will be needed to improve the safety of treatments using an integrating amplicon vector.

Deletion of the Marek’s disease virus UL41 gene (vhs) has no measurable effect on latency or pathogenesis

Marek's disease is an economically important disease of poultry and is caused by an alphaherpesvirus, Marek's disease virus (MDV). The predicted protein product of the MDV UL41 open reading frame has significant protein sequence identity with the virion host shutoff (vhs) protein gene in other alphaherpesviruses. To determine whether the MDV UL41 gene functions as a vhs protein, we utilized a transient co-transfection assay and demonstrated that the MDV UL41 protein was as active in degrading RNA as the vhs protein of herpes simplex virus type 1. To evaluate whether the MDV UL41 gene was involved in pathogenesis, we deleted the MDV UL41 gene. The UL41 deletion mutant replicated in cell culture as well as the parental MDV. The deletion mutant was inoculated into susceptible day-old chicks. The pattern and degree of tumor lesions and neurovirulence produced by the deletion mutant was same as the pattern of lesions induced by the parental virus. The only observable difference between the inoculation of MDV and the MDV deletion mutant was that the early in vivo cytolytic infection with the deletion mutant was of a longer duration than in the non-mutant MDV.

Sequence determination of a mildly virulent strain (CU-2) of Gallid herpesvirus type 2 using 454 pyrosequencing

The complete DNA sequence of the mildly virulent Gallid herpesvirus type 2 strain CU-2 was determined and consists of 176,922 bp with an overall gene organization typical of class E herpesviruses. Phylogenetically, this strain partitions in its own branch between the virulent strains RB-1B, Md11, and Md5, and the vaccine strain CVI988. Overall, the genome of CU-2 is more similar to that of CVI988, with identically sized unique short regions of 11,651 bp. As in CVI988, an insertion of 177 bp was identified in the overlapping genes encoding the Meq, RLORF6, and 23 kDa proteins within the repeat long region of the genome. A total of 15 single nucleotide polymorphisms (SNPs) common to both CU-2 and CVI988, and not occurring in virulent strains, were identified in the genes encoding UL29, UL45, UL50, UL52, LORF10, RLORF14a, RLORF12, Meq(RLORF7), 23kDa, ICP4, US3, and two hypothetical proteins MDV071.4 and MDV076.4. Each gene encoding UL29 and Meq contained two SNPs. Only one major open reading frame (ORF) encoding UL41, the virus host shutoff (VHS) ribonuclease, was disrupted in the CU-2 genome. An additional cytosine after the 25 codon is predicted to produce a truncated protein of 97 aa. Since GaHV-2 mutants lacking UL41 have been reported to retain their virulence, other factors are likely responsible for the low virulence of CU-2. It is largely suspected that SNPs in common with CVI988 along with the insertions in the Meq loci are responsible for its phenotype. Conversely, we identified 43 nonsynonymous mutations (within 23 genes) that may contribute to the virulence of CU-2. These SNPs are shared exclusively with all sequenced virulent strains (Md5, Md11, and RB-1B) and not present within the CVI988 genome. Although most occur in proteins of unknown function, a significant percentage is in proteins involved in virion assembly.

Enhanced Pathogenesis of an Attenuated Herpes Simplex Virus for Mice Lacking Stat1

Mice lacking the Stat1 interferon signaling gene were infected with herpes simplex virus type 1 (HSV-1) or an attenuated recombinant lacking virion host shutoff (Delta vhs). Delta vhs virus-infected Stat1(-/-) mice showed levels of replication equivalent to that of the wild-type virus-infected control mice but reduced relative to wild-type virus-infected Stat1(-/-) mice. Stat1 deficiency relieves the immunomodulatory deficiency of Delta vhs virus, but not its inherent growth defect. Also Vhs is dispensable for reactivation.

Herpes Simplex Virus Virion Host Shutoff Attenuates Establishment of the Antiviral State

Herpes simplex virus mutants lacking the vhs protein are severely attenuated in animal models of pathogenesis and exhibit reduced growth in primary cell culture. As a result of these properties, viruses with vhs deleted have been proposed as live-attenuated vaccines. Despite these findings and their implications for vaccines, the mechanisms by which vhs promotes infection in cell culture and in vivo are not understood. In this study we demonstrate that vhs-deficient viruses replicate to reduced levels in interferon (IFN)-primed cells and that this deficit has both IFN-dependent and IFN-independent components. Furthermore, vhs-defective viruses induce increased and physiologically active levels of IFN, increased amounts of IFN-stimulated transcripts, and more phosphorylated eIF2alpha. In addition, we demonstrate greater accumulation of viral RNAs following infection with a vhs-deficient virus. This leads to the hypothesis that attenuation of viruses lacking vhs may be attributed to increased levels of double-stranded RNA, a potent pathogen-associated molecular pattern. Together these data show that vhs likely functions to reduce innate immune responses and thereby acts as a critical determinant of viral pathogenesis.

Expression of Gamma Interferon-Dependent Genes Is Blocked Independently by Virion Host Shutoff RNase and by US3 Protein Kinase

Gamma interferon receptor alpha (IFN-gammaR alpha) is stable but posttranslationally modified in herpes simplex virus 1(F) [HSV-1(F)]-infected cells. Studies with antibody directed to the phosphorylation site indicate that IFN-gammaR alpha is phosphorylated by the U(S)3 kinase. The modification is abolished in cells infected with DeltaU(S)3, DeltaU(L)13, or Delta(U(S)3/U(L)13) mutant virus. Transcripts of the IFN-gamma-dependent genes do not accumulate in cells transduced with the U(S)3 protein kinase and treated with IFN-gamma. In contrast, the accumulation of IFN-gamma-dependent gene transcripts is suppressed in cells infected with the wild-type virus, in cells infected with the DeltaU(S)3 mutant virus, and to a lesser extent in the DeltaU(L)41 virus-infected cells. The accumulation of IFN-gamma-dependent gene transcripts in DeltaU(L)41-infected cells could be due at least in part to a significant delay and reduction in the accumulation of the U(S)3 protein. The results suggest that the expression of IFN-gamma-dependent genes is blocked independently by the degradation of IFN-gamma-dependent gene transcripts--a function of the virion host shutoff RNase--and by posttranslational modification of the IFN-gammaR alpha protein.

Selective Ablation of Virion Host Shutoff Protein RNase Activity Attenuates Herpes Simplex Virus 2 in Mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.

Translocation and Colocalization of ICP4 and ICP0 in Cells Infected with Herpes Simplex Virus 1 Mutants Lacking Glycoprotein E, Glycoprotein I, or the Virion Host Shutoff Product of the UL41 Gene

In wild-type herpes simplex virus 1-infected cells, the major regulatory protein ICP4 resides in the nucleus whereas ICP0 becomes dynamically associated with proteasomes and late in infection is translocated and dispersed in the cytoplasm. Inhibition of proteasomal function results in retention or transport of ICP0 to the nucleus. We report that in cells infected with mutants lacking glycoprotein E (gE), glycoprotein I (gI), or the product of the U(L)41 gene, both ICP4 and ICP0 are translocated to the cytoplasm and coaggregate in small dense structures that, in the presence of proteasomal inhibitor MG132, also contain proteasomal components. Gold particle-conjugated antibody to ICP0 reacted in thin sections with dense protein aggregates in the cytoplasm of mutant virus-infected cells. Similar aggregates were present in the nuclei but not in the cytoplasm of wild-type virus-infected cells. Exposure of cells early in infection to MG132 does not result in retention of ICP0 as in wild-type virus-infected cells. The results suggest that the retention of ICP4 and ICP0 in the nucleus is a dynamic process that involves the function of other viral proteins that may include the Fc receptor formed by the gE/gI complex and is not merely the consequence of expression of a nuclear localization signal. It is noteworthy that in DeltaU(L)41-infected cells gE is retained in the trans-Golgi network and is not widely dispersed in cellular membranes.

Infection of human dendritic cells with herpes simplex virus type 1 dramatically diminishes the mRNA levels of the prostaglandin E 2 receptors EP2 and EP4

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) of the immune system. Their migration to secondary lymphoid tissues is a crucial step for the priming of T cells and ultimately for the initiation of adaptive immune responses. Therefore, DCs are potential targets for immune evasion strategies of pathogens. The migration of DCs to the T cell areas of lymph nodes is guided by a gradient of chemokines, CCL19 and CCL21, which are constitutively expressed there. CCR7, the receptor for these chemokines, is expressed on activated DCs, enabling their homing to the lymph nodes. However, CCR7 expression alone is not sufficient for efficient migration. Prostaglandin E 2 (PGE 2) is a mandatory factor for CCR7-mediated migration of DCs and exerts its effects via prostaglandin E 2 receptor 2 (EP2) and prostaglandin E 2 receptor 4 (EP4). In this study, we investigated the effect of herpes simplex virus type 1 (HSV-1) infection of mature monocyte-derived dendritic cells (MODCs) on the EP2 and EP4 receptor expression. Affymetrix analyses and real-time polymerase chain reaction (PCR) demonstrated a dramatic down-regulation of the expression of those receptors. Additional real-time PCR and migration assays with a Δvhs mutant virus lacking the virion host shutoff (vhs) gene implicate a vhs independent mechanism. Therefore, our results suggest a novel immune evasion strategy for HSV-1.

Replication-Competent Herpes Simplex Virus 1 Isolates Selected from Cells Transfected with a Bacterial Artificial Chromosome DNA Lacking Only the U L 49 Gene Vary with Respect to the Defect in the U L 41 Gene Encoding Host Shutoff RNase

To generate a null U(L)49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U(L)49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-DeltaU(L)49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U(L)41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U(L)49R DNA (R-U(L)49) accumulated a full-length vhs protein, indicating that in the parental BAC-DeltaU(L)49 DNA, the U(L)41 gene was intact. We conclude that expression of the vhs protein in the absence of U(L)49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U(L)49, to be neutralized.