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Reactivation in vivo and in vitro of herpes simplex virus from mouse dorsal root ganglia which contain different levels of latency-associated transcripts.

In the dorsal root ganglia (DRG) of mice latently infected with the herpes simplex virus type 1 mutant in1814, there are more neurons that contain latency-associated transcripts (LATs) than in DRG of mice infected with a dose of equal infectivity of either a revertant or a wild-type virus. We investigated whether higher levels of LAT+ neurons resulted in more extensive reactivation either in vivo following neurectomy of the sciatic nerve or in vitro after explantation into culture. Neurectomy appeared to induce expression of immediate early 1 mRNA (IE1mRNA) in neurons of mice latently infected with each of three viruses. However IE1mRNA was detected in no more than 0.25% of the neurons of DRG from animals 2 to 4 days after neurectomy, irrespective of the percentage of LAT+ neurons present. Of the 22 neurons shown to express IE1mRNA, none expressed LATs also. However the lack of expression of viral antigen and the absence of a reduced potential for reactivation on explanation suggested that neurectomy had not induced full reactivation involving lytic replication leading to the death of the latently infected neurons. When DRG were explanted into culture, the distribution of the frequency of reactivation was similar to the distribution of DRG that contained LAT+ neurons. The presence of a high proportion of LAT+ neurons was not directly associated with earlier detection of reactivation but such experiments cannot be regarded as quantitative. We therefore concluded that neurectomy did not result in a reduced reactivation potential as described by others and that the frequency of expression of IE1mRNA following neurectomy did not correlate with the number of LAT+ neurons present.

Animals↗

Cloning and expression of the Epstein-Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme.

The gene encoding the Epstein-Barr virus (EBV)-specific dUTPase was amplified from virus DNA by PCR. The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein. The protein from E. coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs. Preliminary experiments yielded a Km value of about 0.8 microM for dUTP. MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated B cells harbouring either type 1 or type 2 EBV. The protein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression. We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein. The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from normal, healthy carriers and from patients with various diseases. While the sera of EBV-negative individuals (0/3) or healthy carriers (0/33) did not contain detectable levels of antibodies, patients with mononucleosis (5/18), chronic EBV infection (2/7), EBV reactivation (7/20) and human immunodeficiency virus infection (5/24) showed elevated antibody titres against the enzyme. This indicated that the dUTPase is expressed during EBV replication and reactivation. The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication.

Acute Disease↗

Continuous production of minute virus of mice by an untransformed variant of Fisher rat fibroblast (FR3T3).

Many tumour cells are killed by the lytic replication of the autonomous parvoviruses H-1 and minute virus of mice (MVMp), whereas most untransformed cells (although they take up these viruses efficiently) are resistant, i.e. they do not produce infectious virus and are not lysed. Therefore, cells able to continuously produce large quantities of infectious virus have not yet been described. We have isolated such cells from the resistant cell line FR3T3 (Fisher rat fibroblast). These cells (called FR3T3C) produce infectious MVMp virions without being detectably lysed. Furthermore, a persistently infected population (R100FR3T3C) was generated by repetitive infection of FR3T3C cells with MVMp. Indeed, R100FR3T3C cells were successfully cultivated for two years and continuously produced infectious virus. Seventeen clones of R100FR3T3C cells isolated by limiting dilution produced infectious virions, indicating that in the R100FR3T3C cell population, virus production was not limited to a few cells. These cell lines may be useful for the production of MVMp and for the generation of a cell line for the packaging of recombinant viral genomes.

Animals↗

Sensitivity to the parvovirus minute virus of mice as a probe for azatyrosine-mediated phenotypic reversion of spontaneously transformed cells.

It has been shown that cells transformed by known oncogenes could be reverted to an untransformed phenotype by the antibiotic Azatyrosine (AzTyr). In order to evaluate the reverting effect of AzTyr on five spontaneously transformed FR3T3C cell clones, we performed three assays: soft agar clonability, tumorigenicity in nude mice and susceptibility to killing by the parvovirus minute virus of mice (MVMp). In contrast to untransformed cells, transformed or tumorigenic cells are permissive for the lytic replication of MVMp and are killed. Our results demonstrate that although the cell populations that emerged after AzTyr treatment of FR3T3C clones had different phenotypes (two were untransformed and two had an altered transformed phenotype), they all behaved like untransformed cells, as judged from their resistance to MVMp infection. Our results demonstrate that susceptibility to MVMp is a valuable way to monitor the reversion.

3T3 Cells↗

Analysis of the human herpesvirus-6 immediate-early 1 protein.

Herpesvirus immediate-early (IE) gene products play key roles in establishing productive infections, regulating reactivation from latency and evading immune recognition. Analyses of HHV-6 IE gene expression have revealed that the IE1 gene of the HHV-6A and HHV-6B variants exhibits a higher degree of sequence variation than other regions of the genome and no obvious similarity to its positional analogue in HCMV. We have analysed expression of the HHV-6 U1102 (HHV-6A) and Z29 (HHV-6B) IE1 gene products using transient expression vectors, stable cell lines and in the context of lytic virus infection. The IE1 transcripts from both variants demonstrate a similar pattern of splice usage within their translated regions. The HHV-6 IE1 proteins from both variants traffic to, and form a stable interaction with, PML-bodies (also known as ND10 or PODS). Remarkably, PML-bodies remained structurally intact and associated with the IE1 protein throughout lytic HHV-6 infection. Immunoprecipitation studies demonstrated that HHV-6 IE1 from both variants is covalently modified by conjugation to the small ubiquitin-like protein SUMO-1. Overexpression of SUMO-1 in cell lines resulted in substantially enhanced levels of IE1 expression; thus sumoylation may bestow stability to the protein. These results indicate that the HHV-6 IE1 protein interacts with PML-bodies yet, unlike other herpesviruses, HHV-6 appears to have no requirement or mechanism to induce PML-body dispersal during lytic replication.

Cells, Cultured↗

Human herpesvirus-8 (Kaposi's sarcoma-associated virus) ORF50 increases in vitro cell susceptibility to human immunodeficiency virus type 1 infection.

ORF50, an immediate-early gene of human herpesvirus-8 (HHV-8), encodes a transactivating protein necessary for virus reactivation and lytic replication. ORF50 was reported recently to synergize with human immunodeficiency virus type 1 (HIV-1) tat at a post-transcriptional level. To study the effects of these molecular interactions on HIV replication and biology, cellular clones stably transformed with ORF50 were obtained by transfection of cell lines of different origin. These clones were infected subsequently with HIV. Experiments showed that ORF50 enhances HIV replication in T and B cells (Jurkat and BC-3 cells) and induces susceptibility and transient permissiveness in non-susceptible glial (A172) cells. Upregulation of viral receptors and co-receptors did not account for increased sensitivity to HIV infection and therefore the action of ORF50 might be modulated by the intracellular environment. Interestingly, non-susceptible cells transformed with ORF50 showed transient production of HIV particles that could spread to adjacent cells by direct contact. These findings show that HHV-8 ORF50 has an enhancing effect on HIV replication in vitro and suggest that the two viruses might interact in co-infected patients.

B-Lymphocytes↗

Murine gammaherpesvirus 68 bcl-2 homologue contributes to latency establishment in vivo.

The gammaherpesviruses are characteristically latent in lymphocytes and exploit lymphocyte proliferation to establish a large, persistent pool of latent genomes. Murine gammaherpesvirus 68 (MHV-68) allows the in vivo analysis of viral genes that contribute to this and other aspects of host colonization. In this study, the MHV-68 bcl-2 homologue, M11, was disrupted either in its BH1 homology domain or upstream of its membrane-localizing C-terminal domain. Each M11 mutant showed normal lytic replication in vitro and in vivo, but had a reduction in peak splenic latency. Lower infectious-centre titres correlated with lower in vivo B-cell activation, lower viral genome loads and reduced viral tRNA expression. This was therefore a true latency deficit, rather than a deficit in ex vivo reactivation. Stable, long-term levels of splenic latency were normal. M11 function therefore contributed specifically to viral latency amplification in infected lymphoid tissue.

Animals↗

Murine gammaherpesvirus-68 ORF28 encodes a non-essential virion glycoprotein.

Murine gammaherpesvirus-68 (MHV-68) ORF28 is a gammaherpesvirus-specific gene of unknown function. Analysis of epitope-tagged ORF28 protein indicated that it was membrane-associated and incorporated into virions in N-glycosylated, O-glycosylated and unglycosylated forms. The extensive glycosylation of the small ORF28 extracellular domain--most forms of the protein appeared to be mainly carbohydrate by weight--suggested that a major function of ORF28 is to attach a variety of glycans to the virion surface. MHV-68 lacking ORF28 showed normal lytic replication in vitro and in vivo and normal latency establishment. MHV-68 ORF28 therefore encodes a small, membrane-bound and extensively glycosylated virion protein, whose function is entirely dispensable for normal, single-cycle host colonization.

Animals↗

The herpesvirus saimiri Rta gene autostimulates via binding to a non-consensus response element.

Herpesvirus saimiri ORF 50a protein expression is sufficient to reactivate the entire lytic-replication cycle. ORF 50a functions as a sequence-specific transactivator that is capable of activating delayed-early gene expression via direct binding to an ORF 50 response element (RE) within the respective promoter. Here, it is shown that ORF 50a is capable of transactivating its own promoter. Deletion analysis of the ORF 50a promoter showed that the ORF 50-responsive element is contained within an 80 bp fragment, situated 293-373 bp from the transcription initiation site. Gel-retardation analysis further mapped the RE to a 34 bp fragment that was able to confer ORF 50 responsiveness to an enhancerless SV40 minimal promoter. Sequence analysis showed that this RE has no direct similarity to previously identified ORF 50 REs. Therefore, it is concluded that ORF 50a is capable of stimulating its own promoter via a novel RE.

Cell Line↗

Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae.

The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappaB and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.

Animals↗

Murine gammaherpesvirus-68 glycoprotein H-glycoprotein L complex is a major target for neutralizing monoclonal antibodies.

Herpesviruses characteristically persist in immune hosts as latent genomes, but to transmit infection they must reactivate and replicate lytically. The interaction between newly formed virions and pre-existing antibody is therefore likely to be a crucial determinant of viral fitness. Murine gammaherpesvirus-68 (MHV-68) behaves as a natural pathogen of conventional, inbred mice and consequently allows such interactions to be analysed experimentally in a relatively realistic setting. Here, monoclonal antibodies (mAbs) were derived from MHV-68-infected mice and all those recognizing infected-cell surfaces were tested for their capacity to neutralize MHV-68 virions. All of the neutralizing mAbs identified were specific for the viral glycoprotein H (gH)-gL heterodimer and required both gH and gL to reproduce their cognate epitopes. Based on antibody interference, there appeared to be two major neutralization epitopes on gH-gL. Analysis of a representative mAb indicated that it blocked infection at a post-binding step--either virion endocytosis or membrane fusion.

Animals↗

The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-Jkappa (CSL), the target of the Notch signaling pathway.

The RTA protein of the Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is responsible for the switch from latency to lytic replication, a reaction essential for viral spread and KS pathogenesis. RTA is a sequence-specific transcriptional activator, but the diversity of its target sites suggests it may act via interaction with host DNA-binding proteins as well. Here we show that KSHV RTA interacts with the RBP-Jkappa protein, the primary target of the Notch signaling pathway. This interaction targets RTA to RBP-Jkappa recognition sites on DNA and results in the replacement of RBP-Jkappa's intrinsic repressive action with activation mediated by the C-terminal domain of RTA. Mutation of such sites in target promoters strongly impairs RTA responsiveness. Similarly, such target genes are induced poorly or not at all by RTA in fibroblasts derived from RBP-Jkappa(-/-) mice, a defect that can be reversed by expression of RBP-Jkappa. In vitro, RTA binds to two adjacent regions of RBP-Jkappa, one of which is identical to the central repression domain that binds the Notch effector fragment. These results indicate that KSHV has evolved a ligand-independent mechanism for constitutive activation of the Notch pathway as a part of its strategy for reactivation from latency.

Animals↗

Herpes simplex virus latency in the nervous system--a new model.

Permissive herpes simplex virus (HSV) infection in tissue culture results in host cell destruction. Latent HSV infection in vivo occurs in neurons of peripheral sensory ganglia (PSG) and it therefore can not take place in neurons in which the virus has completed a lytic replication cycle similar to that present in vitro. Our hypothesis, based on experimental data and observations in humans, suggests that establishment of latent infection and reactivation of HSV-1 does not involve neuronal cell loss. Latency is established in neurons in which the virus does not replicate and is determined, in part, by the tissue levels of a herpes transactivating protein (Vmw65) that is a component of the viral tegument. We also suggest that reactivation of latent infection does not involve destruction of neurons and is due to replication of virus at the peripheral mucocutaneous tissues to where virus or viral DNA have been transported from the nervous tissue. Alternatively, reactivation is initiated in the PSG using a replication cycle which does not involve irreversible damage to neurons. This model explains the lack of damage to neurons which continue to serve as permanent reservoirs of latent virus for the entire life of the host.

Animals↗

Recent progress in herpes simplex virus immunobiology and vaccine research.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause prevalent, chronic infections that have serious outcomes in some individuals. Neonatal herpes may occur when the infant traverses the cervix during maternal genital herpes. Genital herpes is a major risk factor for human immunodeficiency virus type 1 transmission. Considerable efforts have been made to design and test vaccines for HSV, focusing on genital infection with HSV-2. Several protein subunit vaccines based on HSV-2 envelope glycoproteins have reached advanced-phase clinical trials. These antigens were chosen because they are the targets of neutralizing-antibody responses and because they elicit cellular immunity. Encouraging results have been reported in studies of treatment of HSV-seronegative women with a vaccine consisting of truncated glycoprotein D of HSV-2 and a novel adjuvant. Because most sexual HSV transmission occurs during asymptomatic shedding, it is important to evaluate the impact of vaccination on HSV-2 infection, clinically apparent genital herpes, and HSV shedding among vaccine recipients who acquire infection. There are several other attractive formats, including subunit vaccines that target cellular immune responses, live attenuated virus strains, and mutant strains that undergo incomplete lytic replication. HSV vaccines have also been evaluated for the immunotherapy of established HSV infection.

Forecasting↗

Interference of plasmid pCM194 with lysogeny of bacteriophage SP02 in Bacillus subtilis.

Three observations indicated that the 2-megadalton chloramphenicol resistance plasmid pCM194 interferes with SP02 lysogeny of Bacillus subtilis. SP02 plaques formed on B. subtilis(pCM194) appeared almost clear, whereas plaques produced on plasmid-free or pUB110-containing cells contained large turbid centers. The number of phages spontaneously liberated by B. subtilis(SP02) was increased 10-fold or more when pCM194 was also present in the lysogens. Lastly, growth of B. subtilis(SP02, pCM194) for approximately 20 to 25 generations resulted in essentially complete loss of the prophage. This interference was not observed with pUB110 or pE194, and the pCM194 interference was not directed against B. subtilis temperate phage phi 105, which is unrelated to SP02. Lytic replication of SP02 appeared to be unaffected by pCM194. pCM194 interference with SP02 lysogeny was demonstrable in recombination-proficient strains and a recE mutant of B. subtilis. SP02 prophage which were noninducible due to the phage ind mutation were resistant to pCM194 interference. pCM194 interference was lost when the entire pCM194 molecule was joined at its unique HpaII site or at one of the two MboI sites to pUB110 or pUB110 derivatives. pBR322 joined to pCM194 at the same MboI site or at the HindIII site produced chimeras that retained the ability to interfere with SP02 lysogeny. A three-part plasmid constructed by joining pBR322 to pCM194 (at HindIII sites) and to pE194 (at PstI sites) was compatible with the SP02 prophage and showed a temperature-sensitive replication phenotype characteristic of the pE194 replicon. One explanation for the interference involves competition for a host component between an SP02 genome attempting to establish lysogeny and plasmids whose replication is directed by the pCM194 replicon.

Bacillus subtilis↗

Tissue-specific splicing of the herpes simplex virus type 1 latency-associated transcript (LAT) intron in LAT transgenic mice.

To study the regulation of herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) expression and processing in the absence of other cis and trans viral functions, a transgenic mouse containing the region encompassing the LAT promoter (LAP1) and the LAT 5' exon through the 2.0-kb intron was created. LAT expression was detectable by reverse transcriptase PCR (RT-PCR) in a number of tissues, including the dorsal root ganglia (DRG), trigeminal ganglia (TG), brain, skin, liver, and kidney. However, when the accumulation of the 2.0-kb LAT intron was analyzed at the cellular level by in situ hybridization, little or no detectable accumulation was observed in the brain, spinal cord, kidney, or foot, although the 2.0-kb LAT intron was detected at high levels (over 90% of neurons) in the DRG and TG. Northern blot analysis detected the stable 2.0-kb LAT intron only in the sensory ganglia. When relative amounts of the spliced and unspliced LAT within the brain, liver, kidney, spinal cord, TG, and DRG were analyzed by real-time RT-PCR, splicing of the 2.0-kb LAT intron was significantly more efficient in the sensory ganglia than in other tissues. Finally, infection of both transgenic mice and nontransgenic littermates with HSV-1 revealed no differences in lytic replication, establishment of latency, or reactivation, suggesting that expression of the LAT transgene in trans has no significant effect on those functions. Taken together, these data indicate that the regulation of expression and processing of LAT RNA within the mouse is highly cell-type specific and occurs in the absence of other viral cis- and trans-acting factors.

Acute Disease↗

Detection of herpes simplex virus type 1 transcripts during latent infection in mice.

A latent infection can be established in the trigeminal ganglia of mice after corneal inoculation of herpes simplex virus type 1 (HSV-1). With a virion DNA probe, three transcripts (2.0, 1.5, and 1.45 kilobases [kb]) were detected by Northern blot (RNA blot) analysis of RNAs isolated from the ganglia of latently infected mice. All three transcripts hybridized to a nick-translated HSV-1 DNA probe from BamHI restriction fragment B (strain F). These RNAs were mapped with subfragments of BamHI-B and with strand-specific probes. They are at least partially colinear with each other, map to a 3.0-kb PstI-MluI subfragment of BamHI-B, and are transcribed from left to right. The latent HSV-1 RNAs partially overlap the 3' end of ICP0 mRNA but are transcribed in the opposite direction. The latent RNAs were not as extensively poly(A)+ as actin mRNA. The HSV-1 transcripts detected in latently infected trigeminal ganglia did not correspond with any that have been previously identified in permissively infected cells in tissue culture. However, the 2.0-kb HSV-1 RNA present during latency was detectable at reduced levels in the trigeminal ganglia of acutely infected mice and in infected tissue culture cells. The data indicate that the pattern of viral gene expression during HSV-1 latency in the trigeminal ganglia of mice does not represent restriction of the genes actively transcribed during the lytic replication cycle in tissue culture.

Animals↗

Latent herpes simplex virus type 1 transcription in human trigeminal ganglia.

We studied latent herpes simplex virus type 1 gene expression in human trigeminal ganglia. Two transcripts were mapped to a 3.0-kilobase region within the long repeat region and appeared to be located in neuronal nuclei. These viral RNAs were not abundant during lytic replication and may represent an alternative pattern of herpes simplex virus type 1 gene expression involved in the pathogenesis of latent infection.

Adult↗