Critical analysis of an oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor for the treatment of malignant melanoma.

Oncolytic viruses that selectively lyse tumor cells with minimal damage to normal cells are a new area of therapeutic development in oncology. An attenuated herpesvirus encoding the granulocyte-macrophage colony stimulating factor (GM-CSF), known as talimogene laherparepvec (T-VEC), has been identified as an attractive oncolytic virus for cancer therapy based on preclinical tumor studies and results from early-phase clinical trials and a large randomized Phase III study in melanoma. In this review, we discuss the basic biology of T-VEC, describe the role of GM-CSF as an immune adjuvant, summarize the preclinical data, and report the outcomes of published clinical trials using T-VEC. The emerging data suggest that T-VEC is a safe and potentially effective antitumor therapy in malignant melanoma and represents the first oncolytic virus to demonstrate therapeutic activity against human cancer in a randomized, controlled Phase III study.

Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain.

Viral hijacking of cellular processes relies on the ability to mimic the structure or function of cellular proteins. Many viruses encode ubiquitin ligases to facilitate infection, although the mechanisms by which they select their substrates are often unknown. The Herpes Simplex Virus type-1-encoded E3 ubiquitin ligase, ICP0, promotes infection through degradation of cellular proteins, including the DNA damage response E3 ligases RNF8 and RNF168. Here we describe a mechanism by which this viral E3 hijacks a cellular phosphorylation-based targeting strategy to degrade RNF8. By mimicking a cellular phosphosite, ICP0 binds RNF8 via the RNF8 forkhead associated (FHA) domain. Phosphorylation of ICP0 T67 by CK1 recruits RNF8 for degradation and thereby promotes viral transcription, replication, and progeny production. We demonstrate that this mechanism may constitute a broader viral strategy to target other cellular factors, highlighting the importance of this region of the ICP0 protein in countering intrinsic antiviral defenses.

The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0.

Cellular restriction factors responding to herpesvirus infection include the ND10 components PML, Sp100 and hDaxx. During the initial stages of HSV-1 infection, novel sub-nuclear structures containing these ND10 proteins form in association with incoming viral genomes. We report that several cellular DNA damage response proteins also relocate to sites associated with incoming viral genomes where they contribute to the cellular front line defense. We show that recruitment of DNA repair proteins to these sites is independent of ND10 components, and instead is coordinated by the cellular ubiquitin ligases RNF8 and RNF168. The viral protein ICP0 targets RNF8 and RNF168 for degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting this repair protein recruitment. This study highlights important parallels between recognition of cellular DNA damage and recognition of viral genomes, and adds RNF8 and RNF168 to the list of factors contributing to the intrinsic antiviral defense against herpesvirus infection.

Changing the ubiquitin landscape during viral manipulation of the DNA damage response.

Viruses often induce signaling through the same cellular cascades that are activated by damage to the cellular genome. Signaling triggered by viral proteins or exogenous DNA delivered by viruses can be beneficial or detrimental to viral infection. Viruses have therefore evolved to dissect the cellular DNA damage response pathway during infection, often marking key cellular regulators with ubiquitin to induce their degradation or change their function. Signaling controlled by ubiquitin or ubiquitin-like proteins has recently emerged as key regulator of the cellular DNA damage response. Situated at the interface between DNA damage signaling and the ubiquitin system, viruses can reveal key convergence points in this important cellular pathway. In this review, we examine how viruses harness the diversity of the cellular ubiquitin system to modulate the DNA damage signaling pathway. We discuss the implications of viral infiltration of this pathway for both the transcriptional program of the virus and for the cellular response to DNA damage.

Keeping viruses in Chk: DNA damage signaling puts the brakes on transformation.

Oncogenic viruses infect many cells but rarely lead to tumorigenesis. In this issue of Cell Host & Microbe, Nikitin et al. describe how a protective DNA damage response acts to suppress transformation in the majority of cells latently infected with Epstein-Barr virus (EBV).

Genomes in conflict: maintaining genome integrity during virus infection.

The cellular surveillance network for sensing and repairing damaged DNA prevents an array of human diseases, and when compromised it can lead to genomic instability and cancer. The carefully maintained cellular response to DNA damage is challenged during viral infection, when foreign DNA is introduced into the cell. The battle between virus and host generates a genomic conflict. The host attempts to limit viral infection and protect its genome, while the virus deploys tactics to eliminate, evade, or exploit aspects of the cellular defense. Studying this conflict has revealed that the cellular DNA damage response machinery comprises part of the intrinsic cellular defense against viral infection. In this review we examine recent advances in this emerging field. We identify common themes used by viruses in their attempts to commandeer or circumvent the host cell's DNA repair machinery, and highlight potential outcomes of the conflict for both virus and host.

A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses.

The ICP0 protein of herpes simplex virus type 1 is an E3 ubiquitin ligase and transactivator required for the efficient switch between latent and lytic infection. As DNA damaging treatments are known to reactivate latent virus, we wished to explore whether ICP0 modulates the cellular response to DNA damage. We report that ICP0 prevents accumulation of repair factors at cellular damage sites, acting between recruitment of the mediator proteins Mdc1 and 53BP1. We identify RNF8 and RNF168, cellular histone ubiquitin ligases responsible for anchoring repair factors at sites of damage, as new targets for ICP0-mediated degradation. By targeting these ligases, ICP0 expression results in loss of ubiquitinated forms of H2A, mobilization of DNA repair proteins and enhanced viral fitness. Our study raises the possibility that the ICP0-mediated control of histone ubiquitination may link DNA repair, relief of transcriptional repression, and activation of latent viral genomes.

Chromatin at the intersection of viral infection and DNA damage.

During infection, viruses cause global disruption to nuclear architecture in their attempt to take over the cell. In turn, the host responds with various defenses, which include chromatin-mediated silencing of the viral genome and activation of DNA damage signaling pathways. Dynamic exchanges at chromatin, and specific post-translational modifications on histones have recently emerged as master controllers of DNA damage signaling and repair. Studying viral control of chromatin modifications is identifying histones as important players in the battle between host and virus for control of cell cycle and gene expression. These studies are revealing new complexities of the virus-host interaction, uncovering the potential of chromatin as an anti-viral defense mechanism, and also providing unique insights into the role of chromatin in DNA repair.

Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection.

The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.

Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication.

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection.

Using or abusing: viruses and the cellular DNA damage response.

During infection, viruses attempt to hijack the cell while the host responds with various defense systems. Traditional defenses include the interferon response and apoptosis, but recent work suggests that this antiviral arsenal also includes the cellular DNA damage response machinery. The observation of interactions between viruses and cellular DNA repair proteins has not only uncovered new complexities of the virus-host interaction but is also reinforcing the view that viruses can reveal key regulators of cellular pathways through the proteins they target.

A hybrid vector for ligand-directed tumor targeting and molecular imaging.

Merging tumor targeting and molecular-genetic imaging into an integrated platform is limited by lack of strategies to enable systemic yet ligand-directed delivery and imaging of specific transgenes. Many eukaryotic viruses serve for transgene delivery but require elimination of native tropism for mammalian cells; in contrast, prokaryotic viruses can be adapted to bind to mammalian receptors but are otherwise poor vehicles. Here we introduce a system containing cis-elements from adeno-associated virus (AAV) and single-stranded bacteriophage. Our AAV/phage (AAVP) prototype targets an integrin. We show that AAVP provides superior tumor transduction over phage and that incorporation of inverted terminal repeats is associated with improved fate of the delivered transgene. Moreover, we show that the temporal dynamics and spatial heterogeneity of gene expression mediated by targeted AAVP can be monitored by positron emission tomography. This new class of targeted hybrid viral particles will enable a wide range of applications in biology and medicine.

APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons.

APOBEC3 proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and transposition of endogenous retroelements. One family member, APOBEC3A (hA3A), is an orphan, without any known antiviral activity. We show that hA3A is catalytically active and that it, but none of the other family members, potently inhibits replication of the parvovirus adeno-associated virus (AAV). hA3A was also a potent inhibitor of the endogenous LTR retroelements, MusD, IAP, and the non-LTR retroelement, LINE-1. Its function was dependent on the conserved amino acids of the hA3A active site, consistent with a role for cytidine deamination, although mutations in retroelement sequences were not found. These findings demonstrate the potent activity of hA3A, an APOBEC3 family member with no previously identified function. They also highlight the functional differences between APOBEC3 proteins. The APOBEC3 family members have distinct functions and may have evolved to resist various classes of genetic elements.

DNA repair proteins affect the lifecycle of herpes simplex virus 1.

We report that herpes simplex virus 1 (HSV-1) infection can activate and exploit a cellular DNA damage response that aids viral replication in nonneuronal cells. Early in HSV-1 infection, several members of the cellular DNA damage-sensing machinery are activated and accumulate at sites of viral DNA replication. When this cellular response is abrogated, formation of HSV-1 replication centers is retarded, and viral production is compromised. In neurons, HSV-1 replication centers fail to mature, and the DNA damage response is not initiated. These data suggest that the failure of neurons to mount a DNA damage response to HSV-1 may contribute to the establishment of latency.

Interactions of viruses with the cellular DNA repair machinery.

Mammalian cells are equipped with complex machinery to monitor and repair damaged DNA. In addition to responding to breaks in cellular DNA, recent studies have revealed that the DNA repair machinery also recognizes viral genetic material. We review some examples that highlight the different strategies that viruses have developed to interact with the host DNA repair apparatus. While adenovirus (Ad) inactivates the host machinery to prevent signaling and concatemerization of the viral genome, other viruses may utilize DNA repair to their own advantage. Viral interactions with the repair machinery can also have detrimental consequences for the host cells and their ability to maintain the integrity of the host genome. Exploring the interactions between viruses and the host DNA repair machinery has revealed novel host responses to virus infections and has provided new tools to study the DNA damage response.

The Mre11 complex is required for ATM activation and the G2/M checkpoint.

The maintenance of genome integrity requires a rapid and specific response to many types of DNA damage. The conserved and related PI3-like protein kinases, ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR), orchestrate signal transduction pathways in response to genomic insults, such as DNA double-strand breaks (DSBs). It is unclear which proteins recognize DSBs and activate these pathways, but the Mre11/Rad50/NBS1 complex has been suggested to act as a damage sensor. Here we show that infection with an adenovirus lacking the E4 region also induces a cellular DNA damage response, with activation of ATM and ATR. Wild-type virus blocks this signaling through degradation of the Mre11 complex by the viral E1b55K/E4orf6 proteins. Using these viral proteins, we show that the Mre11 complex is required for both ATM activation and the ATM-dependent G(2)/M checkpoint in response to DSBs. These results demonstrate that the Mre11 complex can function as a damage sensor upstream of ATM/ATR signaling in mammalian cells.

Deletion of the virion host shutoff protein (vhs) from herpes simplex virus (HSV) relieves the viral block to dendritic cell activation: potential of vhs- HSV vectors for dendritic cell-mediated immunotherapy.

Herpes simplex virus (HSV) infects dendritic cells (DC) efficiently but with minimal replication. HSV, therefore, appears to have evolved the ability to enter DC even though they are nonpermissive for virus growth. This provides a potential utility for HSV in delivering genes to DC for vaccination purposes and also suggests that the life cycle of HSV usually includes the infection of DC. However, DC infected with HSV usually lose the ability to become activated following infection (M. Salio, M. Cella, M. Suter, and A. Lanzavecchia, Eur. J. Immunol. 29:3245-3253, 1999; M. Kruse, O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, and A. Steinkasserer, J. Virol. 74:7127-7136, 2000). We report that for DC to retain the ability to become activated following HSV infection, the virion host shutoff protein (vhs) must be deleted. vhs usually functions to destabilize mRNA in favor of the production of HSV proteins in permissive cells. We have found that it also plays a key role in the inactivation of DC and is therefore likely to be important for immune evasion by the virus. Here, vhs would be anticipated to prevent DC activation in the early stages of infection of an individual with HSV, reducing the induction of cellular immune responses and thus preventing virus clearance during repeated cycles of virus latency and reactivation. Based on this information, replication-incompetent HSV vectors with vhs deleted which allow activation of DC and the induction of specific T-cell responses to delivered antigens have been constructed. These responses are greater than if DC are loaded with antigen by incubation with recombinant protein.