Adeno-associated virus type 2 modulates the host DNA damage response induced by herpes simplex virus 1 during coinfection

Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone.     

The immune response to ocular herpes simplex virus type 1 infection

Herpes simplex virus type 1 (HSV-1) is a prevalent microbial pathogen infecting 60% to 90% of the adult world population. The co-evolution of the virus with humans is due, in part, to adaptations that the virus has evolved to aid it in escaping immune surveillance, including the establishment of a latent infection in its human host. A latent infection allows the virus to remain in the host without inducing tissue pathology or eliciting an immune response. During the acute infection or reactivation of latent virus, the immune response is significant, which can ultimately result in corneal blindness or fatal sporadic encephalitis. In fact, HSV-1 is one of the leading causes of infectious corneal blindness in the world as a result of chronic episodes of viral reactivation leading to stromal keratitis and scarring. Significant inroads have been made in identifying key immune mediators that control ocular HSV-1 infection and potentially viral reactivation. Likewise, viral mechanisms associated with immune evasion have also been identified and will be discussed. Lastly, novel therapeutic strategies that are currently under development show promise and will be included in this review. Most investigators have taken full advantage of the murine host as a viable working in vivo model of HSV-1 due to the sensitivity and susceptibility to viral infection, ease of manipulation, and a multitude of developed probes to study changes at the cellular and molecular levels. Therefore, comments in this review will primarily be restricted to those observations pertaining to the mouse model and the assumption (however great) that similar events occur in the human condition.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

Hand-to-hand transmission of herpes simplex virus type 1

Droplets of tissue culture fluid containing herpes simplex virus type 1 were placed on the palm of the hand. Each 0.01 ml droplet was taken from a stock virus suspension with a titre of 10(7.5) TCID50/0.1 ml. At 0, 15, 30, 60 and 120 min a droplet was firmly touched with the middle finger of the right hand, after which, attempts were made to recover virus from the finger. At 0 min, when the virus-containing droplet was in a liquid state, there was a 100% rate of virus recovery. By 15 min the droplets had dried out, and after touching dried out droplets there was a 40% virus recovery rate, even though experimental procedures demonstrated that infectious virus was present in the dried out droplets at all test times. If the finger was moistened with tap water or saliva, there was a 100% recovery rate of virus after touching dried out droplets, as well as after touching droplets in a liquid state.     

Virion component of herpes simplex virus type 1 KOS interferes with early shutoff of host protein synthesis induced by herpes simplex virus type 2 186.

Herpes simplex virus (HSV) strains HSV type 1 (HSV-1) KOS and HSV-2 186 are representative of delayed and early shutoff strains, respectively, with regard to their ability to inhibit protein synthesis in Friend erythroleukemia cells. When these cells were simultaneously infected with HSV-1 KOS and HSV-2 186, HSV-1 KOS interfered with the rapid suppression of globin synthesis induced by HSV-2 186. The observed interference was competitive and not due to exclusion of HSV-2 by HSV-1 at the level of adsorption. Furthermore, UV-irradiated HSV-1 KOS was also effective at interfering with the early shutoff function of HSV-2 186, indicating that a virion component is responsible for the observed interference.     

Temperature-sensitive host range mutants of herpes simplex virus type 2.

Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblasts cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties.     

Functional expression of a cloned herpes simplex virus type 1 DNA polymerase gene.

A vector which expresses the herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene was constructed by ligating two separately cloned HSV DNA restriction fragments into an intermediate plasmid and then mobilizing the intact polymerase gene-encoding sequence into a pSV2 derivative. The expression vector (pD7) contains a functional simian virus 40 replication origin and early enhancer-promoter upstream from the HSV DNA polymerase-encoding sequence. COS-1 cells transfected with pD7 contained an RNA species, shown by Northern blot analysis to hybridize specifically with an HSV DNA pol probe and to be the same size (4.3 kilobases) as the pol mRNA found in HSV-1-infected COS-1 cells. A genetic complementation test was used to establish that pD7 expresses a functional pol gene product. COS-1 cells transfected with pD7 were able to partially complement the growth defect of an HSV-1 (KOS) temperature-sensitive mutant, tsC7, in the DNA polymerase gene at the nonpermissive temperature.     

On the mutation rate of herpes simplex virus type 1

All seven DNA-based microbes for which carefully established mutation rates and mutational spectra were previously available displayed a genomic mutation rate in the neighborhood of 0.003 per chromosome replication. The pathogenic mammalian DNA virus herpes simplex type 1 has an estimated genomic mutation rate compatible with that value.     

Functional analysis of the herpes simplex virus UL42 protein.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

Manipulation of herpes simplex virus type 1 by dielectrophoresis

The frequency-dependent dielectrophoretic behaviour of an enveloped mammalian virus, herpes simplex virus type 1 is described. It is demonstrated that over the range 10 kHz–20 MHz, these viral particles, when suspended in an aqueous medium of conductivity 5 mS m^?1, can be manipulated by both positive and negative dielectrophoresis using microfabricated electrode arrays. The observed transition from positive to negative dielectrophoresis at frequencies around 4.5 MHz is in qualitative agreement with a simple model of the virus as a conducting particle surrounded by an insulating membrane.     

PKR-dependent autophagic degradation of herpes simplex virus type 1

The lysosomal pathway of autophagy is the major catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Recent studies have also shown that autophagy (xenophagy) may be used to degrade bacterial pathogens that invade intracellularly. However, it is not yet known whether xenophagy is a mechanism for degrading viruses. Previously, we showed that autophagy induction requires the antiviral eIF2alpha kinase signaling pathway (including PKR and eIF2alpha) and that this function of eIF2alpha kinase signaling is antagonized by the herpes simplex virus (HSV-1) neurovirulence gene product, ICP34.5. Here, we show quantitative morphologic evidence of PKR-dependent xenophagic degradation of herpes simplex virions and biochemical evidence of PKR and eIF2alpha-dependent degradation of HSV-1 proteins, both of which are blocked by ICP34.5. Together, these findings indicate that xenophagy degrades HSV-1 and that this cellular function is antagonized by the HSV-1 neurovirulence gene product, ICP34.5. Thus, autophagy-related pathways are involved in degrading not only cellular constituents and intracellular bacteria, but also viruses.     

Functional and molecular analyses of the avirulent wild-type herpes simplex virus type 1 strain KOS.

It has been documented that KOS, a laboratory strain of herpes simplex virus type 1, is several orders of magnitude less neurovirulent than most other wild-type strains. Studies initiated to determine the functional nature of the block to neuroinvasiveness and to establish the genes involved have determined that, after footpad inoculation of mice, strain 17 syn+ induced neuropathologic signs (paralysis) at titers of 10(2) and yielded a PFU/50% lethal dose ratio of 10(4). In contrast, KOS was not lethal and did not induce paralysis at inoculation doses of 10(8) PFU. This reduced neurovirulence of KOS could not be explained by the lack of thymidine kinase activity, its inability to replicate in mouse cells maintained in culture at 38.5 degrees C, or its inefficient replication in nonneural tissues in vivo. Kinetic experiments tracing the virus through the nervous system after footpad inoculation showed that KOS was blocked at the level of the spinal ganglia. A cosmid library of strain 17 syn+ was utilized in recombination and in vivo selection experiments with strain KOS to establish the genomic region involved in 17 syn+ neuroinvasiveness. A cosmid clone containing the HindIII A fragment (0.25 to 0.53 map units) of strain 17 syn+ in mixed transfections with full-length KOS DNA yielded recombinants with enhanced neuroinvasiveness.     

Human cytomegalovirus TRS1 and IRS1 gene products block the double-stranded-RNA-activated host protein shutoff response induced by herpes simplex virus type 1 infection

Human cytomegalovirus (HCMV) attachment and entry stimulates the expression of cellular interferon-inducible genes, many of which target important cellular functions necessary for viral replication. Double-stranded RNA-dependent host protein kinase (PKR) is an interferon-inducible gene product that limits viral replication by inhibiting protein translation in the infected cell. It was anticipated that HCMV encodes gene products that facilitate the evasion of this PKR-mediated antiviral response. Using a deltagamma1 34.5 herpes simplex virus type 1 (HSV-1) recombinant that triggers PKR-mediated protein synthesis shutoff, experiments identified an HCMV gene product expressed in the initial hours of infection that allows continued protein synthesis in the infected cell. Recombinant HSV-1 viruses expressing either the HCMV TRS1 or IRS1 protein demonstrate that either of these HCMV gene products allows the deltagamma1 34.5 recombinant viruses to evade PKR-mediated protein shutoff and maintain late viral protein synthesis.