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STAT1 is activated by IFNs and other cell signals. Following activation, STAT1 is translocated to the nuclei and activates transcription of IFN-stimulated genes. Although the activation of STAT1 by IFNs is classically associated with antiviral defense and tumor-suppressive functions, emerging data indicate that expression of the STAT1 pathway confers cellular resistance to DNA-damaging agents and mediates aggressive tumor growth. Recent advances in the development of Janus-activated kinase/Stat inhibitors and peptide inhibitors specific for individual Stat proteins may provide new insights into the controversial functions of this pathway. Clin Cancer Res; 18(11); 3015–21. ©2012 AACR.

We are public health scientists who have closely followed the emergence of 2019 novel coronavirus disease (COVID-19) and are deeply concerned about its impact on global health and wellbeing. We have watched as the scientists, public health professionals, and medical professionals of China, in particular, have worked diligently and effectively to rapidly identify the pathogen behind this outbreak, put in place significant measures to reduce its impact, and share their results transparently with the global health community. This effort has been remarkable. We sign this statement in solidarity with all scientists and health professionals in China who continue to save lives and protect global health during the challenge of the COVID-19 outbreak. We are all in this together, with our Chinese counterparts in the forefront, against this new viral threat.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals. IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

Systemic administration of checkpoint inhibitors alone and especially concurrent with intratumoral administration of oncolytic herpesviruses (oHSV) has a major impact on cancer therapy marred by rare failures of healthy organs. Furthermore, tumors vary with respect to susceptibility to oncolytic effects of oHSV. Here we report the construction and properties of 3 families of oncolytic herpes simplex viruses expressing no immunomodulatory genes (T1 series), murine IL-12 (T2 series) or murine or human IL-12 and anti PD-1 antibody (T3 series). We report that insertion of the gene encoding PD-1 Ab significantly augmented the oncolytic activity of oHSV bereft of immunostimulatory genes (T1 series) or expressing IL-12 alone (T2 series). The T3 oHSV expressed IL-12, PD-1 Ab were restricted to the tumor bed whereas the induced IFN-γ accumulated to high levels both in tumor bed and in blood. Furthermore, the T3 oHSV was superior to systemic administration of IL-12 and antibody to PD-1. This report also shows that the oncolytic activity of T3 oHSV in a relatively resistant tumor was enhanced by concurrent intratumoral administrationof genetically engineered exosomes carrying miRNA targeting CTLA-4 checkpoint.

The stress response genes encoding GADD45γ, and to a lesser extent GADD45β, are activated early in infection with herpes simplex virus 1 (HSV-1). Cells that had been depleted of GADD45γ by transfection of short hairpin RNA (shRNA) or in which the gene had been knocked out (ΔGADD45γ) yielded significantly less virus than untreated infected cells. Consistent with lower virus yields, the ΔGADD45γ cells (either uninfected or infected with HSV-1) exhibited significantly higher levels of transcripts of a cluster of innate immunity genes, including those encoding IFI16, IFIT1, MDA5, and RIG-I. Members of this cluster of genes were reported by this laboratory to be activated concurrently with significantly reduced virus yields in cells depleted of LGP2 or HDAC4. We conclude that innate immunity to HSV-1 is normally repressed in unstressed cells and repression appears to be determined by two mechanisms. The first, illustrated here, is through activation by HSV-1 infection of the gene encoding GADD45γ. The second mechanism requires constitutively active expression of LGP2 and HDAC4. IMPORTANCE Previous studies from our laboratory reported that knockout of some innate immunity genes was associated with increases in the expression of overlapping networks of genes and significant loss of the ability to support the replication of HSV-1; knockout of other genes was associated with decreases in the expression of overlapping networks of genes and had no effect on virus replication. In this report, we document that depletion of GADD45γ reduced virus yields concurrently with significant upregulation of the expression of a cluster of innate immunity genes comprising IFI16, IFIT1, MDA5, and RIG-I. This report differs from the preceding study in an important respect; i.e., the preceding study found no evidence to support the hypothesis that HSV-1 maintained adequate levels of LGP2 or HDAC4 to block upregulation of the cluster of innate immunity genes. We show that HSV-1 causes upregulation of the GADD45γ gene to prevent the upregulation of innate immunity genes.

I was shaped by World War II, years of near starvation as a war refugee, postwar chaos, life in several countries, and relative affluence in later life. The truth is that as I was growing up I wanted to be a writer. My aspirations came to an end when, in order to speed up my graduation from college, I took courses in microbiology. It was my second love at first sight—that of my wife preceded it. I view science as an opportunity to discover the designs in the mosaics of life. What initiates my search of discovery is an observation that makes no sense unless there exists a novel design. Once the design is revealed there is little interest in filling all the gaps. I was fortunate to understand that what lasts are not the scientific reports but rather the generations of scientists whose education I may have influenced.

On entry into the body herpes simplex viruses (HSV) replicate in a series of steps that involves derepression of viral DNA activated by VP16, a virion protein, and sequential transcription of viral genes in a cascade fashion. HSV also enters into neurons in which viral DNA maintained as heterochromatin and with few exceptions viral gene expression is silenced. A third face of the interaction of HSV with its host cells takes place at the moment when the silenced viral genome in neurons is abruptly derepressed. The available data do no reveal evidence that HSV encodes different regulatory programs for each facet of its interaction with its host cells. Rather the data point to significant gaps in our knowledge of the mechanisms by which each facet is initiated and the roles of the infected cells at each facet of the interaction of viral gene products with the host cell.

Analyses of the levels of mRNAs encoding IFIT1, IFI16, RIG-1, MDA5, CXCL10, LGP2, PUM1, LSD1, STING, and IFNβ in cell lines from which the gene encoding LGP2, LSD1, PML, HDAC4, IFI16, PUM1, STING, MDA5, IRF3, or HDAC 1 had been knocked out, as well as the ability of these cell lines to support the replication of HSV-1, revealed the following: (i) Cell lines lacking the gene encoding LGP2, PML, or HDAC4 (cluster 1) exhibited increased levels of expression of partially overlapping gene networks. Concurrently, these cell lines produced from 5 fold to 12 fold lower yields of HSV-1 than the parental cells. (ii) Cell lines lacking the genes encoding STING, LSD1, MDA5, IRF3, or HDAC 1 (cluster 2) exhibited decreased levels of mRNAs of partially overlapping gene networks. Concurrently, these cell lines produced virus yields that did not differ from those produced by the parental cell line. The genes up-regulated in cell lines forming cluster 1, overlapped in part with genes down-regulated in cluster 2. The key conclusions are that gene knockouts and subsequent selection for growth causes changes in expression of multiple genes, and hence the phenotype of the cell lines cannot be ascribed to a single gene; the patterns of gene expression may be shared by multiple knockouts; and the enhanced immunity to viral replication by cluster 1 knockout cell lines but not by cluster 2 cell lines suggests that in parental cells, the expression of innate resistance to infection is specifically repressed.

miRNAs are potent tools that in principle can be used to control the replication of infectious agents. The objectives of the studies reported here were to design miRNAs that can block the replication of herpes simplex virus 1 and which could be delivered to infected cells via exosomes. We report the following: (1) We designed three miRNAs targeting the mRNA encoding ICP4, an essential viral regulatory protein. Of the three miRNAs, one miRNA401 effectively blocked ICP4 accumulation and viral replication on transfection into susceptible cells. (2) To facilitate packaging of the miRNA into exosomes, we incorporated into the sequence of miRNA401 an exosome-packaging motif. miRNA401 was shown to be packaged into exosomes and successfully delivered by exosomes to susceptible cells, where it remained stable for at least 72 hr. Finally, the results show that miRNA401 delivered to cells via exosomes effectively reduced virus yields in a miRNA401 dose-dependent fashion. The protocol described in this report can be applied to study viral gene functions without actually deleting or mutagenizing the gene.

Our Phase I trials of experimental virotherapy for recurrent glioblastoma (GBM) have shown that inoculation with a conditionally replication-competent early generation oncolytic herpes simplex virus (oHSV), G207, is safe. However, while 17 of 37 subjects experienced objective clinical responses, the highly attenuated oHSV did not uniformly improve survival. We sought to identify predictors that would identify mechanisms contributing to survival and improve future trial design, by studying accrued samples. We analyzed pre-treatment biopsy and post-G207-treatment tumor samples (collected D2-5 post injection) banked from the patients enrolled in the phase IB G207 trial. The key findings from these patients suggest that productive G207 infection and G207-induced changes in gene expression were predictive of oHSV therapeutic success. RNAseq-based transcriptome analysis of these samples revealed that both the intrinsic IFN mediated antiviral response and adaptive immune functional response in patients correlated significantly with improved survival following G207 inoculation. Further, GBM tissue stained using multiplex fluorescent immunohistochemistry supported differences in the tumor microenvironments that were identified from RNAseq data analysis. Our data indicate that both viral gene expression and the resulting intrinsic anti-viral and recruited adaptive response were critical for survival after G207 inoculation and predict survival with this early generation oHSV in patients with recurrent malignant glioma.

The herpes simplex virus host shutoff RNase (VHS-RNase) is the major early block of host responses to infection. VHS-RNase is introduced into cells during infection and selectively degrades stable mRNAs made before infection and the normally short-lived AU-rich stress response mRNAs induced by sensors of innate immunity. Through its interactions with pU L 47, another tegument protein, it spares from degradation viral mRNAs. Analyses of embedded motifs revealed that VHS-RNase contains a nuclear export signal (NES) but not a nuclear localization signal. To reconcile the potential nuclear localization with earlier studies showing that VHS-RNase degrades mRNAs in polyribosomes, we constructed a mutant in which NES was ablated. Comparison of the mutant and wild-type VHS-RNases revealed the following. (i) On infection, VHS-RNase is transported to the nucleus, but only the wild-type protein shuttles between the nucleus and cytoplasm. (ii) Both VHS-RNases localized in the cytoplasm following transfection. On cotransfection with pU L 47, a fraction of VHS-RNase was translocated to the nucleus, suggesting that pU L 47 may enable nuclear localization of VHS-RNase. (iii) In infected cells, VHS-RNase lacking NES degraded the short-lived AU-rich mRNAs but not the stable mRNAs. In transfected cells, both wild-type and NES mutant VHS-RNases effectively degraded cellular mRNAs. Our results suggest that the stable mRNAs are degraded in the cytoplasm, whereas the AU-rich mRNAs may be degraded in both cellular compartments. The selective sparing of viral mRNAs may take place during the nuclear phase in the course of interaction of pU L 47, VHS-RNase, and nascent viral mRNAs.

On entry into the nucleus, herpes simplex virus 1 (HSV-1) DNA localizes to nuclear bodies known as ND10. Gene repression imposed by ND10 is released by a viral protein, ICP0, via degradation of the ND10 constituents promyelocytic leukemia protein (PML) and Sp100 and the subsequent dispersal of ND10 bodies. In order to understand the dynamic interaction between ICP0 and ND10, we carried out deletion mapping to identify the domains of ICP0 responsible for its association with ND10. Here, we report the following. (i) An ND10 entry signal (ND10-ES), located between residues 245 and 474, is required for ICP0 to penetrate and fuse with ND10. ICP0 lacking ND10-ES adheres to the surface of ND10 but fails to enter. (ii) In the absence of ND10-ES, the E3 ubiquitin ligase of ICP0 facilitates the transient adhesion of the truncated ICP0 to the ND10 surface, whereas the presence of ND10-ES in ICP0 renders ND10 fusion regardless of the E3 ligase activity. (iii) The C terminus of ICP0 is required for retention of ICP0 in ND10 but plays no role in the recruitment process. (iv) The adverse effects of an inactive RING domain on viral replication are partially reversed by deleting either ND10-ES or the C-terminal retention domain, suggesting that additional ICP0 functions require the release of ICP0 from ND10. Based on these results, we conclude that association of ICP0 and ND10 is a dynamic process, in which three sequential steps—adhesion, fusion, and retention—are adopted to stabilize the interaction. A faithful execution of these steps defines the ultimate productivity of the virus.

Herpes simplex virus (HSV) evolved an elegant strategy that enables the virus to impact a large fraction of the human population. The virus replicates at the portal of entry (mouth, genitals) and concurrently it is transported retrograde to sensory neurons. In sensory neurons it establishes a silent (latent) infection. A variety of stimuli can reactivate the virus. The reactivated virus is transmitted anterograde to a site at the portal of entry for transmission by physical contact between infected and uninfected tissues to other individuals. The central issue is how a virus that vigorously replicates and successfully blocks the innate immune defenses of the host at the portal of entry into the body remains silent in sensory neurons. The presentation focuses on three key issues: (a) current assessment of the impact of HSV on human health, (b) the mechanisms by which the virus overcomes a key host defense mechanism at the portal of entry into the body and yet is silenced in latently infected neurons, and (c) the mechanisms by which the virus reactivates.

A herpes simplex virus tegument protein brought into the cell during infection and designated the virion host shutoff protein (VHS) is an endoribonuclease that degrades mRNA. The prevailing view for many years has been that the VHS-RNase does not discriminate between cellular and viral RNAs and that the viruses prevail because the accumulation of viral transcripts outpaces their degradation. Here we report the following. (i) The degradation of viral mRNA made during infection of Vero or HEp-2 cells proceeds at a much-reduced rate compared to that of cellular mRNA. In effect, viral mRNAs are largely stable, whereas cellular mRNAs are rapidly degraded or, in the case of AU-rich mRNA, cleaved and rendered dysfunctional. (ii) In contrast to viral mRNAs made after infection, viral mRNAs expressed by plasmids transfected into cells prior to infection are degraded after infection at a rate comparable to that of cellular mRNAs. Moreover, the mRNA encoded by the transfected plasmid is hyperadenylated in the infected cell. Hyperadenylation but not degradation of mRNAs is blocked by actinomycin D. The results indicate that VHS-mRNA discriminates between viral and cellular mRNA but only in the context of infection and that discrimination is not based on the sequence of the mRNA but most likely on one or more viral factors expressed in the infected cell.

After entry into the nucleus, herpes simplex virus (HSV) DNA is coated with repressive proteins and becomes the site of assembly of nuclear domain 10 (ND10) bodies. These small (0.1-1 μM) nuclear structures contain both constant [e.g., promyelocytic leukemia protein (PML), Sp100, death-domain associated protein (Daxx), and so forth] and variable proteins, depending on the function of the cells or the stress to which they are exposed. The amounts of PML and the number of ND10 structures increase in cells exposed to IFN-β. On initiation of HSV-1 gene expression, ICP0, a viral E3 ligase, degrades both PML and Sp100. The earlier report that IFN-β is significantly more effective in blocking viral replication in murine PML(+/+) cells than in sibling PML(-/-) cells, reproduced here with human cells, suggests that PML acts as an effector of antiviral effects of IFN-β. To define more precisely the function of PML in HSV-1 replication, we constructed a PML(-/-) human cell line. We report that in PML(-/-) cells, Sp100 degradation is delayed, possibly because colocalization and merger of ICP0 with nuclear bodies containing Sp100 and Daxx is ineffective, and that HSV-1 replicates equally well in parental HEp-2 and PML(-/-) cells infected at 5 pfu wild-type virus per cell, but poorly in PML(-/-) cells exposed to 0.1 pfu per cell. Finally, ICP0 accumulation is reduced in PML(-/-) infected at low, but not high, multiplicities of infection. In essence, the very mechanism that serves to degrade an antiviral IFN-β effector is exploited by HSV-1 to establish an efficient replication domain in the nucleus.

Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.

Herpes simplex viruses replicate at the portal of entry into the body and are transported retrograde to sensory neurons in which they can establish a silent, latent infection characterized by the expression of a noncoding latency-associated transcript and a set of microRNAs. At the portal of entry into the body and in cell culture a viral protein, VP16, recruits cellular proteins that initiate a sequential derepression of several kinetic classes of viral genes. Earlier studies have shown that upon reactivation of latent virus in ganglionic organ cultures all genes are derepressed at once, thus obviating the need for VP16 to initiate sequential derepression of viral genes. One hypothesis that could explain the data is that the massive reactivation of all classes of viral genes is the consequence of activation of an apoptotic pathway. Here we show that two proapoptotic drugs, dexamethasone and 2[[3-(2,3-dichlorophenoxy)propyl]amino]-ethanol, each accelerates viral gene expression in ganglionic organ cultures. We also show that in cultured cells apoptosis induced by dexamethasone accelerates viral gene expression and accumulation of infectious virus. The results are surprising in light of the relatively large number of viral proteins that independently block apoptosis induced by viral gene products or exogenous agents. The results suggest that the virus may rely on apoptosis to exit from latency but that apoptosis may be detrimental for virus replication or spread at the portal of entry into the body.

Studies published elsewhere have shown that the herpes simplex virus regulatory protein ICP0 interacts with BMAL1, a partner and regulator of circadian histone acetyltransferase CLOCK, that both proteins localize at ND10 bodies and are stabilized by viral proteins, that enzymatically active CLOCK partially complements ΔICP0 mutants, and that silencing of CLOCK suppresses the expression of viral genes. Here we report that CLOCK is a component of the transcriptional complex that includes TFIID, ICP4, ICP27, and ICP22. The results suggest that the CLOCK histone acetyltransferase is a component of the viral transcriptional machinery throughout the replicative cycle of the virus and that ICP27 and ICP22 initiate their involvement in viral gene expression as components of viral transcriptome.

The U S 3 protein kinase of herpes simplex virus 1 plays a key role in blocking apoptosis induced by viral gene products or exogenous agents. The U S 3 protein kinase is similar to protein kinase A with respect to substrate range and specificity. We report that in the yeast two-hybrid system a domain of U S 3 essential for antiapoptotic activity reacted with programmed cell death protein 4 (PDCD4). We report that U S 3 interacts with PDCD4, that PDCD4 is posttranslationally modified in infected cells both in a U S 3-dependent and -independent fashion, and that depletion of PDCD4 by siRNA blocked apoptosis induced by a Δα4 mutant virus. In infected cells, PDCD4 accumulates in the nucleus, whereas U S 3 accumulates in the cytoplasm. Studies designed to elucidate the convergence of these proteins led to the discovery that U S 3 protein kinase cycles between the nucleus and cytoplasm and that U S 3 retains PDCD4 in infected cell nuclei.

At the portal of entry into the body, herpes simplex viruses (HSV) vigorously multiply and spread until curtailed by the adaptive immune response. At the same time, HSV invades nerve ending-abutting infected cells and is transported in a retrograde manner to the neuronal nucleus, where it establishes a latent (silent) infection. At intervals, as a consequence of physical or metabolic stress, the virus is activated and transported in an anterograde manner to the body surface. The progression of infection is regulated at four checkpoints. In cell culture or at the portal of entry into the body, HSV uses components of the HDAC1- or HDAC2/CoREST/LSD1/REST repressor complex to activate α genes (checkpoint 1) and then uses an α protein, ICP0, to suppress the same repressor complex from silencing post-α gene expression (checkpoint 2). In neurons destined to harbor latent virus (checkpoint 3), HSV hijacks the same repressor complex to silence itself as a first step in the establishment of the latent state. Suppression of histone deacetylases (HDACs) plays a key role in the reactivation from latency (checkpoint 4). HSV has evolved a strategy of using the same host repressor complex to meet its diverse lifestyle needs.

The virion host shutoff (VHS) RNase tegument protein released into cells by infecting virus has two effects. Preexisting stable mRNAs (e.g., GAPDH [glyceraldehyde-3-phosphate dehydrogenase]) are rapidly degraded. Stress response RNAs containing AU-rich elements (AREs) in the 3′ untranslated region (3′UTR) are deadenylated and cleaved, but the cleavage products persist for hours, in contrast to the short half-lives of ARE-containing mRNAs in uninfected cells. At late times, the VHS RNase is neutralized by the viral structural proteins VP16 and VP22. A recent study (J. A. Corcoran, W. L. Hsu, and J. R. Smiley, J. Virol. 80:9720-9729, 2006) reported that, at relatively late times after infection, ARE RNAs are rapidly degraded in cells infected with ΔICP27 mutant virus and concluded that ICP27 “stabilizes” ARE mRNAs. We report the following. (i) The rates of degradation of ARE mRNA at early times (3 h) after infection with the wild type or the ΔICP27 mutant virus are virtually identical, and hence ICP27 plays no role in this process. (ii) In noncomplementing cells, VHS RNase or VP22 is not synthesized. Therefore, the only VHS that is active is brought into cells by the ΔICP27 mutant. (ii) The VHS RNase brought into the cells by the ΔICP27 virus is reduced in potency relative to that of wild-type virus. Hence the rapid degradation of ARE mRNAs noted in ΔICP27 mutant-infected cells at late times is similar to that taking place in mock-infected or in ΔVHS RNase mutant-virus-infected cells and does not by itself support the hypothesis that ICP27 stabilizes ARE mRNAs. (iii) Concurrently, we present the first evidence that VHS RNase interacts with ICP27 most likely when bound to cap- and poly(A)-binding proteins, respectively.

In nonneuronal cells, herpes simplex virus 1 overcomes host defenses, replicates, and ultimately kills the infected cell. Among the host defenses suppressed by the virus is a repressor complex whose key components are histone deacetylase (HDAC) 1 or 2, RE-1 silencing transcription factor (REST), corepressor of REST (CoREST), and lysine-specific demethylase (LSD) 1. In neurons innervating cells at the portal of entry into the body, the virus establishes a “latent” infection in which viral DNA is silenced with the exception of a family of genes. The question posed here is whether the virus hijacks this repressor complex to silence itself in neurons during the latent state. To test this hypothesis, we inserted into the wild-type virus genome a wild-type REST [recombinant (R) 111], a dominant-negative REST (dnREST) lacking the N- and C-terminal repressor domains (R112), or an insertion control consisting of tandem repeats of stop codons (R113). The recombinant virus R112 carrying the dnREST replicated better and was more virulent than the wild-type parent or the other recombinant viruses when administered by the corneal or i.p. routes. Moreover, in contrast to other recombinants, corneal route inoculation by R112 recombinant virus resulted in higher DNA copy numbers, higher levels of infectious virus in eye, trigeminal ganglion, or brain, and virtually complete destruction of trigeminal ganglia in mice that may ultimately succumb to infection. These results support an earlier conclusion that the HDAC/CoREST/REST/LSD1 repressor complex is a significant component of the host innate immunity and are consistent with the hypothesis that HSV-1 hijacks the repressor to silence itself during latent infection.

ICP0, a promiscuous transactivator that enhances the expression of genes introduced by infection or transfection, functions in both nucleus and cytoplasm. The nuclear functions include degradation and dispersal of ND10 bodies and suppression of silencing of viral DNA. Subsequently, ICP0 shifts to the cytoplasm. Transfection of DNA prior to infection has no effect on the localization of ICP0 in cells that are efficient expressers of transgenes (e.g., Vero and HEK293) but results in delayed cytoplasmic localization of ICP0 in cells (e.g., HEp-2 and HEL) that are poor transgene expressers. Here, we examined by real-time PCR (qPCR) the accumulation of a transgene and of viral gI mRNAs in Vero or HEp-2 cells that were transfected and then infected with wild-type or ΔICP0 mutant viruses. The accumulation of transgene mRNA was unaffected by a ΔICP0 mutant, gradually increased in HEp-2 cells, but increased and then decreased in Vero cells infected with wild-type virus. In both cell lines, accumulation of gI mRNA increased with time and was less affected by the transfected DNA in Vero cells than in HEp-2 cells. The relative kinetics of mRNA accumulation reflected continued synthesis and degradation of the transgene and gI mRNAs. We conclude that the role of ICP0 is to render the DNA templates introduced by transfection or infection accessible by transcriptional factors, that the two cell lines differ with respect to the transcription-ready status of entering foreign DNA in the nucleus, and that ICP0 is not per se the recruiter of transcriptional factors to the accessible DNA templates.

Gamma interferon receptor α (IFN-γRα) 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-γRα is phosphorylated by the U S 3 kinase. The modification is abolished in cells infected with ΔU S 3, ΔU L 13, or Δ(U S 3/U L 13) mutant virus. Transcripts of the IFN-γ-dependent genes do not accumulate in cells transduced with the U S 3 protein kinase and treated with IFN-γ. In contrast, the accumulation of IFN-γ-dependent gene transcripts is suppressed in cells infected with the wild-type virus, in cells infected with the ΔU S 3 mutant virus, and to a lesser extent in the ΔU L 41 virus-infected cells. The accumulation of IFN-γ-dependent gene transcripts in ΔU 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-γ-dependent genes is blocked independently by the degradation of IFN-γ-dependent gene transcripts—a function of the virion host shutoff RNase—and by posttranslational modification of the IFN-γRα protein.

Earlier studies have shown that ICP22 and the U L 13 protein kinase but not the U S 3 kinase are required for optimal expression of a subset of late (γ 2 ) genes exemplified by U L 38, U L 41, and U S 11. In primate cells, ICP22 mediates the disappearance of inactive isoforms of cdc2 and degradation of cyclins A and B1. Active cdc2 acquires a new partner, the viral DNA synthesis processivity factor U L 42. The cdc2-U L 42 complex recruits and phosphorylates topoisomerase IIα for efficient expression of the γ 2 genes listed above. In uninfected cells, the cdc25C phosphatase activates cdc2 by removing two inhibitory phosphates. The accompanying report shows that in the absence of cdc25C, the rate of degradation of cyclin B1 is similar to that occurring in infected wild-type mouse embryo fibroblast cells but the levels of cdc2 increase, and the accumulation of a subset of late proteins and virus yields are reduced. This report links ICP22 with cdc25C. We show that in infected cells, ICP22 and U S 3 protein kinase mediate the phosphorylation of cdc25C at its C-terminal domain. In in vitro assays with purified components, both U L 13 and U S 3 viral kinases phosphorylate cdc25C and ICP22. cdc25C also interacts with cdc2. However, in infected cells, the ability of cdc25C to activate cdc2 by dephosphorylation of the inactive cdc2 protein is reduced. Coupled with the phosphorylation of cdc25C by the U S 3 kinase, the results raise the possibility that herpes simplex virus 1 diverts cdc25C to perform functions other than those performed in uninfected cells.

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 UL41 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 ΔUL41-infected cells gE is retained in thetrans-Golgi network and is not widely dispersed in cellular membranes.

Terminases comprise essential components of molecular motors required to package viral DNA into capsids in a variety of DNA virus systems. Previous studies indicated that the herpes simplex virus type 1 U L 15 protein (pU L 15) interacts with the pU L 28 moiety of a pU L 28-pU L 33 complex to form the likely viral terminase. In the current study, a novel temperature-sensitive mutant virus was shown to contain a mutation in U L 33 codon 61 predicted to change threonine to proline. At the nonpermissive temperature, this virus, designated ts8-22, replicated viral DNA and produced capsids that became enveloped at the inner nuclear membrane but failed to form plaques or to cleave or package viral DNA. Incubation at the nonpermissive temperature also precluded coimmunoprecipitation of U L 33 protein with its normal interaction partners encoded by U L 28 and U L 15 in ts8-22-infected cells and with pU L 28 in transient-expression assays. Moreover, a temperature-sensitive mutation in U L 15 precluded coimmunoprecipitation of pU L 15 with the U L 28 and U L 33 proteins at the nonpermissive temperature. We conclude that interactions between putative terminase components are tightly linked to successful viral DNA cleavage and packaging.

We report on the properties and function of two herpes simplex virus-1 (HSV-1) microRNAs (miRNAs) designated "miR-H28" and "miR-H29." Both miRNAs accumulate late in productive infection at a time when, for the most part, viral DNA and proteins have been made. Ectopic expression of miRNA mimics in human cells before infection reduced the accumulation of viral mRNAs and proteins, reduced plaque sizes, and at vey low multiplicities of infection reduced viral yields. The specificity of the miRNA mimics was tested in two ways. First, ectopic expression of mimics carrying mutations in the seed sequence was ineffective. Second, in similar tests two viral miRNAs made early in productive infection also had no effect. Both miR-H28 and miR-H29 are exported from infected cells in exosomes. A noteworthy finding is that both miR-H28 and miR-H29 were absent from murine ganglia harboring latent virus but accumulated in ganglia in which the virus was induced to reactivate. The significance of these findings rests on the principle that the transmission of HSV from person to person is by physical contact between the infected tissues of the donor and those of uninfected recipient. Diminished size of primary or recurrent lesions could be predicted to enhance person-to-person transmission. Reduction in the amount of reactivating latent virus would reduce the risk of retrograde transport to the CNS but would not interfere with anterograde transport to a site at or near the site of initial infection.

A preeminent phenotype of the infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is that it acts as a promiscuous transactivator. In most cell lines exposed to ΔICP0 mutant virus at low ratios of virus per cell infection, α genes are expressed but the transition to β and γ gene expression does not ensue, but can be enhanced by inhibitors of histone deacetylases (HDACs). Earlier studies have shown that ICP0 interacts with CoREST and displaces HDAC1 from the CoREST–REST–HDAC1/2 complex. HDAC1 and CoREST are then independently translocated to the cytoplasm. Here, we test the hypothesis that ICP0 blocks the silencing of HSV DNA by displacing HDAC1 from the CoREST–REST complex. Specifically, first, mapping studies led us to construct a truncated CoREST (CoREST 146–482 ) that in transfected cells displaced HDAC1 from the CoREST–REST complex. Second, we constructed two viruses. In BACs encoding the entire HSV-1, we replaced the gene encoding ICP0 with AmpR to yield a ΔICP0 mutant R8501. We also replaced ICP0 with CoREST 146–482 to yield recombinant R8502. The yield of R8502 mutant virus in Vero, HEp-2, and human embryonic lung cells exposed to 0.1 pfu of virus per cell was 100-, 10-, and 10-fold higher, respectively, than those of R8501 mutant virus. In Vero cells, the yield of R8502 was identical with that of wild-type virus. We conclude that CoREST 146–482 functionally replaced ICP0 and that, by extension, ICP0 acts to block the silencing of viral DNA by displacing HDAC1/2 from the CoREST–REST complex.

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-ΔU 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-ΔU 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.

Δγ134.5 mutant herpes simplex type 1 viruses are under active clinical investigation as oncolytic therapy for cancer. Mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) activity has been shown to suppress protein kinase R and thereby confer oncolytic susceptibility to some human tumors by R3616, a virus deleted for both copies of γ134.5. We report that systemic delivery of R3616 can selectively target and destroy human xenograft tumors that overexpress MEK activity compared with tumors that express lower MEK activity. These results suggest systemic delivery of R3616 may be effective in the treatment of some human tumors. [Cancer Res 2007;67(17):8301–6]

The virion host shutoff (vhs) protein encoded by the U L 41 gene of herpes simplex virus 1 is an endoribonuclease. The enzyme is introduced into the cell during unpackaging of the virion upon entry and selectively degrades mRNA for several hours. The RNase activity ceases after the onset of synthesis of late (γ) viral proteins. Here we report that vhs protein does not accumulate in cells transiently transfected with only a plasmid encoding the U L 41 gene. However, vhs does accumulate in cells cotransfected with plasmids expressing two other tegument proteins, VP16 and VP22. vhs does not directly interact with VP22 but, instead, binds VP22 only in the presence of VP16. In contrast to these findings, the amounts of vhs mRNA accumulating in the cells transfected solely with vhs are not significantly different from those detected in cells coexpressing vhs, VP16, and VP22. We conclude from these studies that the steady state of vhs mRNA, reflecting synthesis and turnover of mRNA, is not affected by the interaction of vhs protein with VP16 with VP22. A model is proposed in which the vhs protein may function to sequester mRNAs in compartments inaccessible to the cellular translational machinery and that VP16 and VP22 rescue the mRNAs by interacting with the vhs protein.

A key property of herpes simplex viruses (HSVs) is their ability to establish latent infection in sensory or autonomic ganglia and to reactivate on physical, hormonal, or emotional stress. In latently infected ganglia, HSVs express a long noncoding RNA, a latency-associated transcript (LAT), which plays a key role in maintaining latently infected neurons, but not viral proteins. To investigate the events leading to reactivation, we examined the use of ganglionic organ cultures that enable rapid reactivation in medium containing antibody to nerve growth factor (NGF) or delayed reactivation in medium containing NGF and epidermal growth factor (EGF). Here we report the discovery that activating transcription factor 3 (ATF3), a stress response protein, profoundly affects the interaction of HSV with its host. Specifically, (i) ATF3 is induced by stress, such as inhibition of protein synthesis or infection; (ii) in infected cells, ATF3 enhances the accumulation of LAT by acting on the response elements in the promoter of the LAT precursor RNA; (iii) ATF3 is induced nearly 100-fold in ganglionic organ cultures; and (iv) ATF3 plays a key role in the maintenance of the latent state, inasmuch as expression of ATF3 bereft of the C-terminal activation domain acts as a dominant negative factor, inducing HSV gene expression in ganglionic organ cultures harboring latent virus and incubated in medium containing NGF and EGF. Thus, ATF3 is a component of a cluster of cellular proteins that together with LAT maintain the integrity of the neurons harboring latent virus.

Viral oncolytic therapy has been pursued with renewed interest as the molecular basis of carcinogenesis and viral replication has been elucidated. Genetically engineered, attenuated viruses have been rationally constructed to achieve a therapeutic index in tumor cells compared with surrounding normal tissue. Many of these attenuated mutant viruses have entered clinical trials. Here we review the preclinical literature demonstrating the interaction of oncolytic viruses with ionizing radiation and provides a basis for future clinical trials.

Earlier, our laboratory reported that purified glutathione S -transferase-virion host shutoff (GST- vhs ) protein exhibited endoribonucleolytic activity in in vitro assays using as substrates in vitro-transcribed regions of IEX-1 mRNA. Here, we report that studies of the cleavage patterns of synthetic RNA oligonucleotides defined the activity of GST- vhs as being similar to that of RNase A. Thus, GST- vhs cleaved the RNA at the 3′ end of single-stranded cytidine and uridine residues. Since the GST-m vhs nuclease-defective mutant protein failed to cleave the synthetic RNAs, the results unambiguously attribute the activity to vhs .

SK-N-SH cells exposed to low ratios of ICP0-null (ΔICP0) mutants of herpes simplex virus per cell express the viral α proteins, but the progression to β and γ gene expression does not ensue. In these restrictive cells, post-α gene expression can be induced after exposure of the infected cells to sodium butyrate, an indication that VP16 brought into cells by the virus and the α gene products made after infection cannot block the silencing of viral post-α genes by histone deacetylases (HDACs). This observation is consistent with evidence reported earlier that ICP0 dissociates HDAC1/2 from the CoREST/REST complex. In permissive U2OS cells, replication is independent of the ratio of ΔICP0 mutant per cell. To determine whether other viral genes are involved in blocking HDACs, we used a surrogate system consisting of baculoviruses carrying viral or cellular genes driven by CMV immediate–early promoter. Expression of these genes requires blocking of histone deacetylation. We report that ( i ) cotransduced U S 3 or U S 3.5 protein kinase substitutes for sodium butyrate in enabling the expression of a reporter gene in restrictive cells and enhancing it in permissive cells; ( ii ) HDAC1 is phosphorylated concomitant with the expression of reporter genes; and ( iii ) the amounts and appearance of HDAC1 are altered in transduced cells expressing U S 3 protein kinase in the absence of other viral proteins. We conclude that the U S 3 protein kinase blocks histone deacetylation by a mechanism distinct from that of ICP0 and that debilitated histone deacetylation contributes to the permissiveness of U2OS cells for ΔICP0 mutants.

Previously, we reported that the U S 3 protein kinase blocks apoptosis, that it activates protein kinase A (PKA), that activation of PKA blocks apoptosis in cells infected with a U S 3 deletion mutant, and that an overlapping transcriptional unit encodes a truncated kinase designated U S 3.5. Here, we report the properties of the kinases based on comparisons of herpes simplex virus and baculoviruses expressing U S 3 or U S 3.5 kinase. Specifically, we report the following. (i) Both kinases mediate the phosphorylation of HDAC1, HDAC2, and the PKA regulatory IIα subunit in the absence of other viral proteins. (ii) Both enzymes mediate the phosphorylation of largely identical sets of proteins carrying the phosphorylation consensus site of PKA, but only U S 3 blocks apoptosis, suggesting that it is U S 3 and not PKA that is responsible for the phosphorylation of the proteins bearing the shared consensus phosphorylation site and the antiapoptotic activity. (iii) Both kinases cofractionate with mitochondria. Immune depletion of the U S 3 and U S 3.5 kinases from the cytoplasm removed the kinases from the supernatant fraction, but not from the mitochondrial fraction, and therefore, if the antiapoptotic activity of the U S 3 kinase is expressed in mitochondria, the localization signal and the antiapoptotic functions are located on different parts of the protein. (iv) The U S 3 protein kinase is required for the translocation of virus particles from the nucleus. Although the U L 31 protein is phosphorylated in cells infected with the mutant expressing U S 3.5 kinase, the release of virus particles from nuclei was impeded in some cells, suggesting that the U S 3 kinase affects the modification of the nuclear membrane more efficiently than the U S 3.5 kinase.

Current design of genetically engineered viruses for selective destruction of cancer cells is based on the observation that attenuated viruses replicate better in tumor cells than in normal cells. The ideal virus, however, is one that can infect only cancer cells by virtue of altered host range. Such a virus can be made more robust than the highly attenuated viruses used in clinical trials. Earlier, we reported the construction of a recombinant herpes simplex virus 1 (R5111) in which the capacity to bind heparan sulfate was disabled and which contained a chimeric IL-13-glycoprotein D that enabled the virus to infect cells expressing the IL-13alpha2 receptor (IL-13Ralpha2) commonly found on the surface of malignant glioblastomas or high-grade astrocytomas. In the earlier report, we showed that the recombinant R5111 was able to enter and infect cells via the interaction of the chimeric glycoprotein D with IL-13Ralpha2 but that the virus retained the capacity to bind and replicate in cells expressing the natural viral receptors HveA or nectin-1. Here, we report the construction of a recombinant virus (R5141) that can only enter and replicate in cells that express the IL-13Ralpha2. The recombinant R5141 does not depend on endocytosis to infect cells. It does not infect cells expressing HveA or nectin-1 receptors or cells expressing IL-13Ralpha2 that had been exposed to soluble IL-13 before infection. The studies described here show that the host range of herpes simplex viruses can be altered by genetic manipulation to specifically target cancer cells.

An earlier report showed that infected cell protein no. 0 (ICP0) of herpes simplex virus 1 (HSV-1) interacts with the SH3 domains of a recently discovered adaptor protein, CIN85. Here, we report the following. (i) ICP0 also interacts with other SH3 domain-containing proteins and, in particular, with nonneuronal members of the Src kinase family. (ii) HSV-1 infection enhanced the activating phosphorylation of Tyr416 of the members of the Src kinase family, modestly enhanced the kinase activity of Src, and posttranslationally modified at least one additional member of the Src kinase family by phosphorylation in a manner dependent on the viral gene products ICP0, unique short 3 (U S 3), and unique long 13 (U L 13). (iii) To define the roles of Src kinase family members, we examined the accumulation of viral proteins, DNA, and mRNA and virus yields from wild-type mouse embryo fibroblasts and sibling cells lacking Src, Fyn, and Yes (SYF − ); a mutant cell line, +Src, in which Src was restored to SYF − cells; and the mutant cell line (CSK − ) lacking the negative regulator Csk gene of the Src kinase family. Representative α, β, and γ 2 proteins accumulated in the largest amounts in SYF − cells and the smallest amounts in +Src compared to wild-type cells. The CSK − cells yielded smaller amounts of the γ 2 protein and at least 10-fold less virus than wild-type cells. We conclude that HSV-1 proteins regulate the activities of Src family kinases to achieve optimal viral yields in the course of viral replication.

The herpes simplex virus 1 ORF U L 41 encodes a protein (virion host shutoff or vhs ) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or β-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3′ domain disappears rapidly, a large 5′ domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: ( i ) a GST- vhs protein produced in Escherichia coli , solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST- vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. ( ii ) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3′ untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. ( iii ) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.

Earlier this laboratory constructed a herpes simplex virus 1 recombinant (R5111) that carries a IL13 ligand inserted into glycoprotein D and can enter cells via the IL13Ralpha2 receptor commonly expressed on the surface of malignant glioma cells. In this report, we describe the properties of two recombinant viruses carrying chiemric gD genes. In R5181 recombinant virus the chimeric gene consisted on the residues 20-155 of urokinase plaminogen activator (uPA) inserted between residues 24 and 25 of gD. In R5182 the insert consisted of a 23-residue sequence encoding the uPA binding domain for the urokinase plaminogen activator receptor (uPAR). These viruses were constructed for three reasons, to increase the number of viruses that specifically target receptors on the surface of malignant glioma cells, to determine whether viruses exhibiting novel ligands could enter cells via receptors anchored to the cell surface via glycosylphosphatidylinositol anchor as has been recently demonstrated for nectin1, and to determine whether receptors other than IL13Ralpha2 could be targeted by genetic engineering of the virus. We report that R5181 but not R5182 recombinant virus was able to enter cells expressing uPAR. The results indicate that HSV-1 recombinant viruses can be engineered to enter cells via a variety of unrelated nonviral receptors, including receptors that are anchored to the cells surface but without transmembrane domains.

Long-term survivors of glioblastoma multiforme, the most common form of primary intracranial malignancy in adults, are extremely rare. Experimental animal models that more closely resemble human disease are essential for the identification of effective novel therapies. We report here an extensive analysis of the 4C8 glioma model to assess its suitability for evaluating novel type 1 herpes simplex virus (HSV-1) therapies of malignant glioma. We first determined that expression of major histocompatibility complex I and II and of αvβ3 in the 4C8 model was comparable to that seen in human glioma cells. Next, using a panel of Δγ134.5 HSVs, we demonstrated that, in vitro, 4C8 cells were as sensitive as human glioma cells to both infection and lysis and that the 4C8 cells supported the production of foreign gene products. Replication competence of HSV was demonstrated in vitro. Finally, 4C8 intracranial gliomas were established in immunologically competent syngeneic B6D2F1 mice, treated by intratumoral injection of selected engineered HSVs, including the interleukin-12-expressing virus, M002. Survival data from these studies demonstrated that 4C8 cells in vivo are sensitive to both direct oncolysis and HSV-mediated interleukin-12 expression. Fluorescence-activated cell sorting analyses of immune-related infiltrating cells supported the concept that survival was prolonged in part because of antitumor actions of these cells. We conclude that the 4C8/B6D2F1 syngeneic glioma model is suitable for preclinical evaluation of HSV-based therapies and that M002 is a superior virus for the treatment of murine glioma in this model.

The infected cell protein (ICP)0 enables gene expression and the replication of herpes simplex virus (HSV)-1 in cells infected at low multiplicities and enhances the expression of genes introduced into cells by transfection or infection. We report that a short sequence of ICP0 is similar to a sequence in the amino terminus of CoREST, a corepressor that exists in complexes with the repressor REST and histone deacetylases (HDACs) 1 or 2 to repress cellular gene expression. In wild-type-virus-infected cells, HDAC1 dissociates from the CoREST/REST complex, CoREST and HDAC1 are phosphorylated by a process mediated by viral protein kinases, and CoREST and HDAC1 are partially translocated to the cytoplasm. In cells infected with a virus mutant (ΔICP4), in which ICP0 accumulates, but post-α gene expression is blocked, HDAC1 is dissociated from the CoREST/REST complex, but translocation to the cytoplasm does not occur. After infection with a mutant virus from which ICP0 is deleted, the complex remains intact, but, under conditions of productive infection, the complex is partially translocated to the cytoplasm. These results suggest that, at low multiplicities of infection, ICP0 blocks CoREST-mediated silencing of viral genes by dissociation of HDAC1, whereas subsequent modifications and translocation of the components of the complex are the functions of other viral gene products made later in infection.

Malignant glioma tumor cells in situ exhibit on their surfaces the interleukin 13 (IL-13) receptor designated IL13R2. To target herpes simplex virus 1 to this receptor, we constructed a recombinant virus (R5111) in which the known heparan sulfate binding sites in glycoproteins B and C were deleted and IL-13 was inserted into both glycoproteins C and D. We also transduced a baby hamster kidney cell line lacking the known viral receptors (J1-1) and Vero cells with a plasmid encoding IL13R2. The J1-1 derivative (J-13R) cell line is susceptible to and replicates the R5111 recombinant virus but not the wild-type parent virus. We report the following. (i) Expression of IL13R2 was rapidly lost from the surface of transduced cells grown in culture. The loss appeared to be related to ligands present in fetal bovine serum in the medium. None of the malignant glioma cell lines cultivated in vitro and tested to date exhibited the IL13R2 receptor. (ii) Soluble IL-13 but not IL-4 or IL-2 blocked the replication of R5111 recombinant virus in J-13R cells. (iii) The endocytosis inhibitor PD98059 blocked the replication in J1-1 cells of a mutant lacking glycoprotein D (gD / ) but not the replication of R5111 in the J-13R cells. We conclude that R5111 enters cells via its interaction with the IL13R2 receptor in a manner that cannot be differentiated from the interaction of wild-type virus with its receptors. The experiments described in this report stemmed from two considerations. In the last decade, several laboratories have attempted to develop herpes simplex virus 1(HSV-1) mutants suitable for cytoreductive therapy of human malignant gliomas. It is estimated that these incurable tumors claim 10,000 lives per year in the United States alone, and current therapeutic modalities seldom produce long-lasting remissions (14). Of the various mutants tested in the mouse model, two such mutants found their way to clinical trials. Both mutants lack the 134.5 genes shown earlier to be essential for viral replication in the central nervous system in experimental animal systems (15, 21). One mutant, G207, lacked in addition the UL39 gene encoding the large subunit of ribonucleotide reductase (15, 16). While both mutants exhibited a satisfactory safety profile within the range of concentrations injected into the tumor mass, neither mutant exhibited unambiguous therapeutic potential as tested. The safety of the 134.5 null mutant hinges on the fact that it is unable to block the shutoff of protein synthesis resulting from the activation of protein kinase R (3, 12). In consequence, viral replication is reduced and the mutant is unable to spread from cell to cell. One potential counterindication for its use is the emergence of second-site mutations that block the activation of protein kinase R and show enhanced capacity to replicate in the central nervous system (2, 11, 18). The introduction of a second deletion reduced the risk of second-site, complementary mutation that partially restored virulence. However, the double mutant is further debilitated and replicates only in dividing cells. In malignant gliomas only a fraction of the tumor cells actively di