David A. Leib

Regulation of starvation- and virus-induced autophagy by the eIF2α kinase signaling pathway

The eIF2α kinases are a family of evolutionarily conserved serine/threonine kinases that regulate stress-induced translational arrest. Here, we demonstrate that the yeast eIF2α kinase, GCN2, the target phosphorylation site of Gcn2p, Ser-51 of eIF2α, and the eIF2α-regulated transcriptional transactivator, GCN4, are essential for another fundamental stress response, starvation-induced autophagy. The mammalian IFN-inducible eIF2α kinase, PKR, rescues starvation-induced autophagy in GCN2-disrupted yeast, and pkr null and Ser-51 nonphosphorylatable mutant eIF2α murine embryonic fibroblasts are defective in autophagy triggered by herpes simplex virus infection. Furthermore, PKR and eIF2α Ser-51-dependent autophagy is antagonized by the herpes simplex virus neurovirulence protein, ICP34.5. Thus, autophagy is a novel evolutionarily conserved function of the eIF2α kinase pathway that is targeted by viral virulence gene products.

IFN-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and Sindbis viruses

Type I interferons (IFNs) play an essential role in the host response to viral infection through the induction of numerous IFN-stimulated genes (ISGs), including important antiviral molecules such as PKR, RNase L, Mx, and iNOS. Yet, additional antiviral ISGs likely exist. IFN-stimulated gene 15 (ISG15) is a ubiquitin homolog that is rapidly up-regulated after viral infection, and it conjugates to a wide array of host proteins. Although it has been hypothesized that ISG15 functions as an antiviral molecule, the initial evaluation of ISG15-deficient mice revealed no defects in their responses to vesicular stomatitis virus or lymphocytic choriomeningitis virus, leaving open the important question of whether ISG15 is an antiviral molecule in vivo. Here we demonstrate that ISG15 is critical for the host response to viral infection. ISG15−/− mice are more susceptible to influenza A/WSN/33 and influenza B/Lee/40 virus infections. ISG15−/− mice also exhibited increased susceptibility to both herpes simplex virus type 1 and murine gammaherpesvirus 68 infection and to Sindbis virus infection. The increased susceptibility of ISG15−/− mice to Sindbis virus infection was rescued by expressing wild-type ISG15, but not a mutant form of ISG15 that cannot form conjugates, from the Sindbis virus genome. The demonstration of ISG15 as a novel antiviral molecule with activity against both RNA and DNA viruses provides a target for the development of therapies against important human pathogens.

Noninvasive Bioluminescence Imaging of Herpes Simplex Virus Type 1 Infection and Therapy in Living Mice

ABSTRACT Mouse models of herpes simplex virus type 1 (HSV-1) infection provide significant insights into viral and host genes that regulate disease pathogenesis, but conventional methods to determine the full extent of viral spread and replication typically require the sacrifice of infected animals. To develop a noninvasive method for detecting HSV-1 in living mice, we used a strain KOS HSV-1 recombinant that expresses firefly (Photinus pyralis) and Renilla (Renilla reniformis) luciferase reporter proteins and monitored infection with a cooled charge-coupled device camera. Viral infection in mouse footpads, peritoneal cavity, brain, and eyes could be detected by bioluminescence imaging of firefly luciferase. The activity of Renilla luciferase could be imaged after direct administration of substrate to infected eyes but not following the systemic delivery of substrate. The magnitude of bioluminescence from firefly luciferase measured in vivo correlated directly with input titers of recombinant virus used for infection. Treatment of infected mice with valacyclovir, a potent inhibitor of HSV-1 replication, produced dose-dependent decreases in firefly luciferase activity that correlated with changes in viral titers. These data demonstrate that bioluminescence imaging can be used for noninvasive, real-time monitoring of HSV-1 infection and therapy in living mice.

Analysis of the Role of Autophagy in Replication of Herpes Simplex Virus in Cell Culture

ABSTRACT The herpes simplex virus type 1 (HSV-1) neurovirulence gene encoding ICP34.5 controls the autophagy pathway. HSV-1 strains lacking ICP34.5 are attenuated in growth and pathogenesis in animal models and in primary cultured cells. While this growth defect has been attributed to the inability of an ICP34.5-null virus to counteract the induction of translational arrest through the PKR antiviral pathway, the role of autophagy in the regulation of HSV-1 replication is unknown. Here we show that HSV-1 infection induces autophagy in primary murine embryonic fibroblasts and that autophagosome formation is increased to a greater extent following infection with an ICP34.5-deficient virus. Elimination of the autophagic pathway did not significantly alter the replication of wild-type HSV-1 or ICP34.5 mutants. The phosphorylation state of eIF2α and viral protein accumulation were unchanged in HSV-1-infected cells unable to undergo autophagy. These data show that while ICP34.5 regulates autophagy, it is the prevention of translational arrest by ICP34.5 rather than its control of autophagy that is the pivotal determinant of efficient HSV-1 replication in primary cell culture.

In vivo characterization of site-directed mutations in the promoter of the herpes simplex virus type 1 latency-associated transcripts.

Transient expression assays in PC12 cells showed that the cAMP response element (CRE) and the TATA box of the herpes simplex virus type 1 latency-associated transcripts (LATs) promoter are essential for basal expression. Recombinant viruses were generated containing site-specific mutations in these motifs. The abilities of these recombinants to replicate, express LATs and reactivate from latency were compared with wild-type and marker-rescued viruses in a murine ocular model. The acute replication of these TATA and CRE mutant viruses was at a level equivalent to their respective marker-rescued viruses. The reactivation of virus was unaffected by mutation in the TATA box as compared with wild-type or marker-rescued viruses. In situ hybridization of TATA box mutant virus-infected ganglia, however, showed threefold fewer LAT-positive neurons than wild-type virus-infected ganglia, with consistently weaker hybridization signals. Thus, this TATA box is required for normal expression of the LATs but not for efficient reactivation. The LATs CRE mutant reactivated with slightly but reproducibly reduced frequency and delayed kinetics relative to marker-rescued virus. By in situ hybridization, however, the percentage and intensity of LATs-positive neurons were found to be comparable for the CRE mutant- and wild-type virus-infected ganglia, suggesting that the CRE is dispensable for abundant LATs expression but that a reactivation function of the LATs may depend upon the presence of the CRE. Finally, using a modified assay for examining the timing of reactivation, we showed that the induction of viral reactivation by addition of exogenous cAMP can occur independently of the LATs.

Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats.

SummaryHerpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (<1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacyin vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or virus free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p=0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.

Protection from primary infection and establishment of latency by vaccination with a herpes simplex virus type 1 recombinant deficient in the virion host shutoff (vhs) function

A herpes simplex virus type 1 (HSV-1) recombinant virus deficient in the virion host shutoff (vhs) function was assessed for its ability to function as a live-attenuated vaccine. Protection of mice from wild-type challenge infection and the establishment and reactivation of HSV-1 latency was measured in a mouse ocular model. Challenge virus replication in corneas and trigeminal ganglia was significantly reduced for vaccinated mice. Consistent with these findings, the vaccinated groups showed no clinical signs during acute infection and high levels of virus-specific IgG and neutralizing antibodies were induced. The establishment of and reactivation from latency in trigeminal ganglia from the vaccinated group were also significantly reduced relative to controls. These data suggest that vhs deletion mutants may have significant utility as live-attenuated HSV-1 vaccines.

Functional Genomic Analysis of Herpes Simplex Virus Type 1 Counteraction of the Host Innate Response

ABSTRACT Herpes simplex virus type 1 (HSV-1) mutants lacking the ICP34.5 gene are severely attenuated in mouse models and have a significant growth defect in confluent mouse embryo fibroblasts. Previously, ICP34.5 was demonstrated to have a crucial role in evading the innate immune response to infection by mediating the dephosphorylation of eIF2α, a translation initiation factor phosphorylated by PKR during the antiviral response. To further understand the role of ICP34.5 in evasion of the antiviral response, we used transcriptional profiling to examine host cell gene expression in both wild-type and ICP34.5-null virus-infected mouse embryo fibroblasts over a time course of infection. Our study revealed that cells responded to infection within 3 h through PKR-dependent eIF2α phosphorylation and that the majority of up-regulated genes at 3 h postinfection were involved in the antiviral response. HSV-1 counters this response through early expression of ICP34.5 and dephosphorylation of eIF2α. By 12 h postinfection, the differences between the number and functional classification of genes differentially up- and down-regulated between wild-type and ICP34.5-null virus-infected cells were maximal. Specifically, in wild-type virus-infected cells, the majority of changed genes were involved in metabolic and biosynthetic processes, while in ICP34.5-null virus-infected cells, mostly antiviral genes were up-regulated. Further, ICP34.5-null virus-infected cells produced greater amounts of beta interferon than wild-type virus-infected cells. These results indicate that ICP34.5 expression and function at early times postinfection have a pivotal role in the ability of HSV-1 to gain control of the host cell and maintain an environment for successful viral replication.

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

ABSTRACT 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.

Postexposure vaccination with a virion host shutoff defective mutant reduces UV-B radiation-induced ocular herpes simplex virus shedding in mice

A herpes simplex virus type 1 (HSV-1) recombinant (UL41NHB) deficient in the virion host shutoff (vhs) function was tested as a therapeutic vaccine in an ultraviolet (UV) light-induced mouse ocular reactivation model. Mice were infected with HSV-1 via the cornea. Following the establishment of latency by HSV-1 the mice were subsequently vaccinated intraperitoneally with one dose of UL41NHB or with uninfected cell extract. Mice were subsequently UV-irradiated to induce viral reactivation and during the 7 days post-UV irradiation, numbers of mice shedding virus were reduced from 13/23 (57%) to 3/25 (12%), and numbers of virus-positive eye swabs were reduced from 40/161 (25%) to 6/175 (3%) by the vaccine (P < 0.001). These data suggest that deletion of vhs may be a useful strategy in the development of attenuated therapeutic HSV vaccines.