The DNA replication origins of herpes simplex virus type 1 strain Angelotti

The nucleotide sequences of the origins of DNA replication (ori) of the S- and L-component (oriS, oriL) of the herpes simplex virus type 1 (HSV-1) standard genome were determined from HSV-1 strain Angelotti (ANG). In contrast to other HSV-1 strains, the ANG oriS sequence revealed an insertion of an TA-dinucleotide in an otherwise very conserved but imperfect palindromic sequence of 47 bp. The oriL sequence of the standard ANG genome was found to be identical to that of an ANG class II defective genome which exhibits a duplication of a 144 bp palindrome. A model is presented to explain the origination of the amplified ANG oriL sequences from the parental genome with a single copy of oriL via illegitimate recombination. Alignment of the ori sequences of HSV, adeno- and papovaviruses unveiled that the HSV ori region can be subdivided into two distinct sites of homology to the DNA initiation signals of papova- and adenoviruses, suggesting that the HSV origins of replication comprise elements for DNA replication by both, cellular and virus-encoded DNA polymerases.     

Role of protein-protein interactions during herpes simplex virus type 1 recombination-dependent replication

Recombination-dependent replication is an integral part of the process by which double-strand DNA breaks are repaired to maintain genome integrity. It also serves as a means to replicate genomic termini. We reported previously on the reconstitution of a recombination-dependent replication system using purified herpes simplex virus type 1 proteins (Nimonkar A. V., and Boehmer, P. E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10201-10206). In this system, homologous pairing by the viral single-strand DNA-binding protein (ICP8) is coupled to DNA synthesis by the viral DNA polymerase and helicase-primase in the presence of a DNA-relaxing enzyme. Here we show that DNA synthesis in this system is dependent on the viral polymerase processivity factor (UL42). Moreover, although DNA synthesis is strictly dependent on topoisomerase I, it is only stimulated by the viral helicase in a manner that requires the helicase-loading protein (UL8). Furthermore, we have examined the dependence of DNA synthesis in the viral system on species-specific protein-protein interactions. Optimal DNA synthesis was observed with the herpes simplex virus type 1 replication proteins, ICP8, DNA polymerase (UL30/UL42), and helicase-primase (UL5/UL52/UL8). Interestingly, substitution of each component with functional homologues from other systems for the most part did not drastically impede DNA synthesis. In contrast, recombination-dependent replication promoted by the bacteriophage T7 replisome was disrupted by substitution with the replication proteins from herpes simplex virus type 1. These results show that although DNA synthesis performed by the T7 replisome is dependent on cognate protein-protein interactions, such interactions are less important in the herpes simplex virus replisome.     

Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system

Productive infection by adeno-associated virus type 2 (AAV) requires coinfection with a helper virus, e.g., adenovirus or herpesviruses. In the case of adenovirus coinfection, the replication machinery of the host cell performs AAV DNA replication. In contrast, it has been proposed that the herpesvirus replication machinery might replicate AAV DNA. To investigate this question, we have attempted to reconstitute AAV DNA replication in vitro using purified herpes simplex virus type 1 (HSV-1) replication proteins. We show that the HSV-1 UL5, UL8, UL29, UL30, UL42, and UL52 gene products along with the AAV Rep68 protein are sufficient to initiate replication on duplex DNA containing the AAV origins of replication, resulting in products several hundred nucleotides in length. Initiation can occur also on templates containing only a Rep binding site and a terminal resolution site. We further demonstrate that initiation of DNA synthesis can take place with a subset of these factors: Rep68 and the UL29, UL30, and UL42 gene products. Since the HSV polymerase and its accessory factor (the products of the UL30 and UL42 genes) are unable to efficiently perform synthesis by strand displacement, it is likely that in addition to creating a hairpin primer, the AAV Rep protein also acts as a helicase for DNA synthesis. The single-strand DNA binding protein (the UL29 gene product) presumably prevents reannealing of complementary strands. These results suggest that AAV can use the HSV replication apparatus to replicate its DNA. In addition, they may provide a first step for the development of a fully reconstituted AAV replication assay.     

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.     

Macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system

After corneal infection, herpes simplex virus type 1 (HSV-1) invades sensory neurons with cell bodies in the trigeminal ganglion (TG), replicates briefly, and then establishes a latent infection in these neurons. HSV-1 replication in the TG can be detected as early as 2 days after corneal infection, reaches peak titers by 3-5 days after infection, and is undetectable by 7-10 days. During the period of HSV-1 replication, macrophages and gammadelta TCR+ T lymphocytes infiltrate the TG, and TNF-alpha, IFN-gamma, the inducible nitric oxide synthase (iNOS) enzyme, and IL-12 are expressed. TNF-alpha, IFN-gamma, and the iNOS product nitric oxide (NO) all inhibit HSV-1 replication in vitro. Macrophage and gammadelta TCR+ T cell depletion studies demonstrated that macrophages are the main source of TNF-alpha and iNOS, whereas gammadelta TCR+ T cells produce IFN-gamma. Macrophage depletion, aminoguanidine inhibition of iNOS, and neutralization of TNF-alpha or IFN-gamma all individually and synergistically increased HSV-1 titers in the TG after HSV-1 corneal infection. Moreover, individually depleting macrophages or neutralizing TNF-alpha or IFN-gamma markedly reduced the accumulation of both macrophages and gammadelta TCR+ T cells in the TG. Our findings establish that after primary HSV-1 infection, the bulk of virus replication in the sensory ganglia is controlled by macrophages and gammadelta TCR+ T lymphocytes through their production of antiviral molecules TNF-alpha, NO, and IFN-gamma. Our findings also strongly suggest that cross-regulation between these two cell types is necessary for their accumulation and function in the infected TG.     

Rolling circle DNA replication by extracts of herpes simplex virus type 1-infected human cells.

Whole-cell extracts of herpes simplex virus type 1-infected human cells (293 cells) can promote the rolling circle replication of circular duplex DNA molecules. The products of the reaction are longer than monomer unit length and are the result of semiconservative DNA replication by the following criteria: (i) resistance to DpnI and susceptibility to MboI restriction enzymes, (ii) shift in density on a CsCl gradient of the products synthesized in the presence of bromo-dUTP to a position on the gradient consistent with those of molecules composed mainly of one parental DNA strand and one newly synthesized DNA strand, and (iii) the appearance in the electron microscope of molecules consisting of duplex circles with multiunit linear appendages, a characteristic of a rolling circle mode of DNA replication. The reaction requires ATP and is dependent on herpes simplex virus type 1-encoded DNA polymerase.     

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.     

Inhibition of PACT-mediated activation of PKR by the herpes simplex virus type 1 Us11 protein

PACT, a protein activator of PKR, can cause inhibition of cellular protein synthesis and apoptosis. Here, we report that the Us11 protein of herpes simplex virus type 1 can block PKR activation by PACT both in vitro and in vivo. Although Us11 can bind to both PKR and PACT, mutational analyses revealed that the binding of Us11 to PKR, and not to PACT, was essential for its inhibitory action. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. The binding of Us11 to PKR did not block the binding of PKR to PACT but prevented its activation. Us11 is the first example of a viral protein that can inhibit the action of PACT on PKR.     

Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.

Herpes simplex virus type 1 (HSV-1) infection of the murine cornea induces the rapid infiltration of neutrophils. We investigated whether these cells could influence virus replication. BALB/c mice treated with monoclonal antibody (MAb) RB6-8C5 experienced a profound depletion of neutrophils in the bloodstream, spleen, and cornea. In these animals, virus titers in the eye were significantly higher than those in the immunoglobulin G-treated controls at 3 days postinfection. By day 9, virus was no longer detectable in the controls, whereas titers of 10(3) to 10(6) PFU were still present in the neutrophil-depleted hosts. Furthermore, virus spread more readily to the skin and brains of MAb RB6-8C5-treated animals, rendering them significantly more susceptible to HSV-1-induced blepharitis and encephalitis. Only 25% of the treated animals survived, whereas all of the controls lived. Although MAb RB6-8C5 treatment did not alter the CD4+ T-cell, B-cell, natural killer cell, or macrophage populations, the CD8+ T-cell population was partially reduced. Therefore, the experiments were repeated in severe combined immunodeficiency mice, which lack CD8+ T cells. Again virus growth was found to be significantly elevated in the eyes, trigeminal ganglia, and brains of the MAb RB6-8C5-treated hosts. These results strongly indicate that in both immunocompetent and immunodeficient mice, neutrophils play a significant role in helping to control the replication and spread of HSV-1 after corneal infection.     

Monoclonal antibodies suppress replication of herpes simplex virus type 1 in trigeminal ganglia.

The effect of monoclonal antibodies on the growth of herpes simplex virus type 1 in trigeminal ganglia was investigated. Four-week-old mice were infected on an abrased cornea with herpes simplex virus type 1. Forty-eight hours after infection, trigeminal ganglia ipsilateral with infected eyes were removed and placed in culture. Incubation of infected ganglia in the presence of a pool of nonneutralizing monoclonal antibodies specific for glycoproteins of gB and gE suppressed virus growth by greater than 90%. This was comparable to the amount of suppression observed when infected ganglia were incubated in hyperimmune serum. Individual monoclonal antibodies were less efficient, being able to inhibit virus growth by only two- to threefold. The mechanism of suppression was examined. Reduction in virus growth was observed under conditions in which all susceptible ganglion cells were infected in vitro before nonneutralizing monoclonal antibody was added. Similar results were obtained in tests with virus-infected neuroblastoma cells. Furthermore, suppression of infectious progeny was seen in the absence of complement and immunologically reactive cells. Thus, neither virus neutralization nor immunocytolysis could account for the effects of antibody on virus growth. Rather, the data suggest that antibody can bind to herpes simplex virus type 1-infected neuronal cells and suppress intracellular virus replication.     

Inhibition of herpes simplex virus type 1 replication in temperature-sensitive cell cycle mutants.

Herpes simplex virus type 1 DNA synthesis and infections progeny production were studied in five different conditional hamster (BHK-21) cell cycle mutants. At the nonpermissive temperature (39.5 degrees C), both events were strongly inhibited in four of these cell lines. The degree of inhibition was a reproducible characteristic of each cell mutant and in two cases was dependent upon the multiplicity of infection. Experiments involving shifts to the nonpermissive temperature at least 3 h postinfection at 33.5 degrees C suggested that the defects in viral replication were not due to faulty adsorption, penetration, or uncoating, whereas experiments involving shifts of infected cells from the nonpermissive temperature to 33.5 degrees C revealed the reversible nature of the inhibition.     

Human natural killer cells limit replication of herpes simplex virus type 1 in vitro

Studies were undertaken to determine whether natural killer (NK) cells could inhibit the replication of herpes simplex virus type 1 (HSV-1) in culture. In the absence of effector cells, HSV-1 was found to replicate in fibroblasts with up to a 100-fold increase in virus titer from 4 to 16 hr after incubation at 37 degrees C. Human peripheral blood mononuclear cells were found to limit virus replication in a dose-dependent manner, with the greatest inhibition being observed at the highest concentration evaluated: i.e., an effector:target ratio of 800:1. The antiviral effect was not observed when nonactivated or virus-activated mononuclear cells were added to the virus preparations at the end (instead of the beginning) of the assay period, indicating that the observed effect was not due to a nonspecific toxicity of soluble factors released from freeze-thawed effectors. Neither was inhibition of HSV-1 replication due to the generation of interferon (IFN) during the NK assay, because the addition of anti-IFN did not abrogate the antiviral effect. Thus, the inhibition of viral replication was most likely due to a cytotoxic effector rather than to release of soluble factors. The effector cells responsible for limiting HSV-1 replication were shown to be NK cells by a number of criteria. Mononuclear cells from both HSV-1 seropositive and seronegative donors limited virus replication; their activity could be boosted by pretreatment of effector cells with IFN; the effector cells which limited virus replication were found in Percoll gradient fractions enriched for large granular lymphocytes; and the effector cells shared the cell surface phenotype of NK cells--they were enriched in populations depleted of T cells by panning with Leu-4 and were depleted of activity by treatment with the anti-NK antibody Leu-11b plus complement. We conclude that human NK cells are capable of recognizing and lysing HSV-1-infected target cells before infectious virus progeny are generated. These results suggest that NK cells, acting early in the course of an infection, might serve to limit HSV-1 replication and therefore reduce the virus load in the host before the development of the adaptive immune response and clearance of the infection.     

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.     

Inhibition of herpes simplex virus type 1 replication in fibroblast cultures by human blood mononuclear cells.

An assay was developed to test the effect of human blood mononuclear cells (MNCs) on herpes simplex virus (HSV) replication. In this assay, human fibroblast monolayers were inoculated with HSV and then cultured with or without blood MNCs. Fewer HSV-infected cells were recovered from human fibroblasts cultured in the presence than in the absence of blood MNCs. This inhibition of viral replication by MNCs was independent of HLA matching between the MNCs and fibroblasts and persisted even when T cells were depleted by antibody and complement. However, depletion of Leu11+ MNCs either by panning or with antibody and complement reduced the ability of the cells to suppress HSV infection, whereas enrichment of Leu11+ cells by fluorescence-activated cell sorting increased the viral suppression. Depletion of OKM1+ MNCs also reduced the viral suppression. After coculturing of MNCs and HSV-infected fibroblasts for 3 days, alpha interferon (IFN) and gamma IFN were detected in the supernatants. Predepletion of Leu11+ MNCs reduced the amount of gamma IFN produced in these cultures. Incubation of the MNCs and HSV-infected fibroblasts with antibody specific for either alpha or gamma IFN resulted in reduced viral suppression. Preincubation of MNCs for 18 h with either interleukin 2 or alpha IFN or for 7 days with antigen increased the suppression of HSV infection. These results suggest that natural killer cells with the Leu11+ phenotype participate in the recognition of HSV-infected cells at a point sufficiently early to interfere with the spread of infection in vitro and may inhibit viral replication by natural killer cell cytotoxicity, by generation of interferon, and by lymphokine-activated killing.     

Synthesis of aldehydo-sugar derivatives of pyrazoloquinoline as inhibitors of herpes simplex virus type 1 replication

Synthesis of a novel series of structurally related pyrazoloquinoline nucleosides is described. All the newly synthesized compounds were examined for their in vitro antiviral activity against herpes simplex type-1 as shown by two different bioassays, namely; crystal violet staining or the MTS tetrazolium dye measurement. The acute toxicity (LD50) values of the biologically active compounds were determined.     

Identification of the gene encoding the 65-kilodalton DNA-binding protein of herpes simplex virus type 1.

Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.     

Topoisomerase II cleavage of herpes simplex virus type 1 DNA in vivo is replication dependent

The genome of herpes simplex virus type 1 contains a large number of recognition sites for eucaryotic DNA type II topoisomerase. Topoisomerase II sites were identified by means of the consensus sequence described previously (J.R. Spitzner and M.T. Muller, Nucleic Acids Res. 16:5553-5556, 1988) and then confirmed by sequencing DNA cleavages introduced by purified topoisomerase II. In vivo, host topoisomerase II also introduced double-stranded DNA breaks in the viral genome at sites predicted by the consensus sequence. Host topoisomerase II acted on all immediate-early genes as well as on genes from other temporal classes; however, cleavages were not detected until 4 to 5 h postinfection and were most intense at 10 h postinfection. Topoisomerase II cleavages were not detected when viral DNA replication was prevented with phosphonoacetic acid. These data indicate that, although progeny viral genomes are acted upon by host topoisomerase II, this enzyme either does not act on parental viral genomes before DNA replication or acts on them with such low efficiency that cleavages are beyond our limit of detection. The findings suggest that host topoisomerase II is involved in aspects of viral replication at late times in the infectious cycle.