- Volume 78, Issue 12, 1997

Human immunodeficiency virus type 1 (HIV-1) replication is restricted partially in SK-N-MC and completely in SK-N-SH neuronal cells. To investigate the molecular mechanism of this differential restriction of HIV-1 replication, cells infected with HIV-1 were analysed for their steady-state levels of: total and unintegrated HIV-1 DNA by DNA PCR, different species of HIV-1 RNA by RT-PCR, and HIV-1 p24 protein production by an ELISA procedure. We found that the kinetics of the infection were slower and there was a lower level of accumulation of HIV- 1 macromolecules (total and unintegrated circular DNA, unspliced and spliced RNAs and viral proteins) in the SK-N-MC cells than in the permissive CEM cells. In SK-N-SH cells, HIV-1 DNA was only transiently detected during the first 24 h post-infection, and the unspliced RNA was detected up to 1 week post-infection. However, the HIV-1 spliced RNAs and the 2-LTR circular DNA were not detected at all during the course of infection. Both SK-N-MC and SK-N-SH cells showed higher levels of HIV-1 DNA, RNA and p24 protein when infected with an HIV-1 (amphotropic retrovirus) pseudotype, HIV-1B. However, the level of HIV-1 replication was still lower in SK-N-SH than in SK-N-MC cells. Moreover, although the kinetics of viral protein production were comparable in SK-N-MC cells infected with HIV-1B and CEM cells infected with HIV-1, the overall level of virus replication was still much lower in HIV-1B- infected SK-N-MC cells. These data suggest that the restriction of HIV-1 replication in neuronal cell lines takes place at both virus-entry and post-entry levels, and cellular factors may be involved in the differential restriction of HIV-1 replication in these cells.

Two experimental feline immunodeficiency virus (FIV) vaccines were tested, either alone or in combination, in four groups of cats (A-D). One vaccine (SL3261-FIV) was composed of live attenuated Salmonella typhimurium aroA (SL3261) strains expressing the capsid (Gag) and part of the envelope (Env) proteins of FIV. The other was composed of FIV Gag and Env proteins incorporated into immune- stimulating complexes (iscom-FIV). Cats of group A were immunized four times with SL3261-FIV. Cats of group B were immunized twice with SL3261-FIV and then twice with iscom-FIV. Cats of group C were immunized twice with SL3261 expressing the B subunit of cholera toxin (SL3261-CtxB) and then twice with iscom-FIV. Cats of group D, which served as negative controls, were immunized twice with SL3261-CtxB and then twice with iscom into which the Gag and Env proteins of simian immunodeficiency virus (SIV) had been incorporated (iscom- SIV). Two weeks after the last immunization, all cats were challenged with FIV. At this time, cats immunized with iscom-FIV (groups B and C) showed strong plasma antibody responses to Gag and Env, whilst these responses were weak or undetectable in the cats immunized four times with SL3261-FIV (group A). Seven weeks after FIV challenge, Env-specific antibody responses had increased considerably in cats of all groups except group A. The mean virus loads in the cats of this group proved to be lower than those of the other groups at all time points, indicating partial protection.

Two distinct human diseases have been described in association with human T cell lymphotropic virus type I (HTLV-I) infection: adult T cell leukaemia and tropical spastic paraparesis/HTLV-I-associated myelopathy. Although comprehensive understanding of specific mechanisms underlying pathogenesis of either disease has not yet been achieved, the viral regulatory protein Tax is believed to play a significant role. Previous studies demonstrated the potential of Tax to transform host cells. Here, it is shown that the Tax transactivator has in addition the potential to induce T cell death by apoptosis. Using an inducible system (Jurkat cell line JPX-9), significant apoptotic cell death upon Tax expression was observed. In an attempt to detect the cellular genes mediating this effect, it was found that induction of Tax was associated with marked up- regulation of the Fas ligand (FasL) gene. Tax- induced apoptosis was inhibited when the Fas/FasL pathway was interrupted by YVAD-cmk,the inhibitor of ICE-like proteases. Transient expression experiments provided additional support for the putative role of endogenous FasL in Tax-induced apoptosis. Upon cotransfection with Tax-expressing plasmid, the transcriptional activity of the FasL promoter was found to be significantly upregulated in Jurkat cells and several other cell lines, as measured by reporter gene expression. Furthermore, cotransfection using different Tax mutants demonstrated that both CREB and NF-k -B activation domains of Tax protein were required for the transactivation to take effect.

Human papillomavirus type 8 (HPV-8) is a strictly cutaneous oncogenic virus known to induce malignant skin lesions in epidermodysplasia verruciformis patients. Our study shows that sequences surrounding transcription start sites of the HPV-8 oncogene E6 (nt 175–179) and comprising the presumable CCAAC and TATA boxes of the E6 promoter P175 contain a cluster of four motifs similar to the DNA recognition site of the multifunctional cellular transcription factor yin-yang 1 (YY1). Using DNase I footprinting and gel retardation tests it could be demonstrated that three of these motifs indeed act as YY1 binding sites. To test their functional relevance for P175 activity, engineered YY1 binding site mutants were analysed in the context of a P175 test vector using transient expression assays with human keratinocytes. YY1 turned out to exert both positive and negative effects upon the activity of the HPV-8 E6 promoter; binding of YY1 to a site upstream of the promoter’s cap-position (BS1) activated transcription, whereas the downstream site (BS2) mediated repression. The second downstream YY1 binding site (BS3) seemed to play an auxiliary role, enhancing the negative effect of YY1 BS2. These observations define YY1 as an important cellular protein involved in the control of E6 oncogene expression of the skin-specific ‘high risk’ HPV-8 and emphasize the potential regulatory role of sequences located downstream of the transcription start site.

The phosphorylation state of vaccinia virus (VV) protein H5R synthesized in infected cells was investigated by two-dimensional gel electrophoresis. Most of the H5R protein was underphosphorylated (pI 5 9 to 6 8) and, on centrifugation of cell lysates, was associated with virosomes sedimenting with nuclei. However, about a quarter of the H5R protein synthesized was highly phosphorylated (pI 5 5), and this was the major form of the H5R protein present in cytoplasmic extracts. Immunofluorescence of VV- infected cells in the absence of DNA replication showed that underphosphorylated H5R protein, specifically recognized by antibody, was abundantly distributed throughout the cytoplasm but also present in punctate particles, whereas most of the B1R protein detected was in the punctate particles. Late gene expression was not required for the H5R protein to accumulate in virosomes-viral DNA synthesis was sufficient. The different phosphorylation states and cytological locations of the H5R protein suggest it has multiple roles in VV development.

One of the main limitations of the vaccination of neonates from vaccinated or infected mothers is the interference by inherited maternal antibodies, which are known to inhibit the immune response against both live and inactivated vaccines. The efficiency of bypassing this inhibition by the transfer of an immunogenic glycoprotein gene, the gD gene of pseudorabies virus (PRV), into neonates was explored. The experiments were conducted in 1- day-old piglets, which are immunocompetent at birth. The same transcription unit (gD of PRV under the control of the adenovirus major late promoter) was delivered intramuscularly at birth either in the form of naked DNA or cloned in the genome of a replication-defective adenovirus. A booster injection of a conventional live PRV vaccine strain was given at 10 weeks of age, the replication of which was greatly restricted by the residual amounts of colostral antibodies in control animals. Piglets were challenged at the age of 16 weeks with a virulent PRV strain. The replication-defective adenovirus was able to efficiently prime piglets born to immune dams against gD in such a way that inoculation with the Bartha strain protected them against a subsequent challenge with the same level of efficacy in piglets born to naive or immune dams. In contrast, piglets born to immune dams into which the gD gene was not transferred, or transferred as naked DNA at birth, were not protected. These results open the way for early immunization of neonates born to vaccinated or infected mothers.

Envelope glycoprotein D (gD) of pseudorabies virus (PRV) is essential for penetration but is not required for cell-to-cell spread. When animals are inoculated with a phenotypically complemented PRV gD mutant, the virus is able to spread locally by means of direct cell-to-cell transmission, but progeny virions released by infected cells are non-infectious because they lack gD. Therefore, the virus cannot be transmitted from inoculated animals to other animals. This property makes a PRV gD mutant an attractive candidate as a safe vaccine vector. To examine whether a self-restricted, non-transmissible PRV mutant can be used as a biologically safe vaccine vector, a gD/gE-negative PRV recombinant virus which expresses envelope glycoprotein E2 of classical swine fever virus was constructed. Vaccination of pigs showed that the recombinant virus was able to protect pigs against both Aujeszky’s disease and classical swine fever.

Immunocytochemistry on serial paraffin sections was used to monitor the production dynamics of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α) and viral antigens in the trigeminal ganglion (TG) and the central side of the dorsal root entry zone (DRE) of mice, following infection of the cornea with herpes simplex virus type 1. In normal TG, scattered satellite cells were TNF-α and in the DRE, TNF-α and/or low numbers of IL-6 cells were detected. On day 3 after infection, foci of TG neurons with viral antigens were surrounded by large numbers of TNF-α and/or IL-6 cells and low numbers of IFN-γ cells. IL-2 and/or IL-4 cells appeared later, when viral antigens had almost cleared. In the TG, the most striking changes occurred with TNF-α, with respect to its source (satellite cells, Schwann cells and infiltrating cells) and the extent and long duration of its production. TNF-α was the predominant cytokine throughout acute and latent infection and even by day 30, numbers of satellite cells expressing this cytokine were three times higher than those in normal ganglia. Moreover, in the DRE, TNF-α was the only cytokine detected during virus clearance and again, its production continued, along with that of IL-6, on days 20 to 30, in both infiltrating cells and astrocytes. Thus, cytokines, particularly TNF-α and perhaps IL-6,from infiltrating cells and resident glial cells may have a role both in virus clearance and in normal homeostatic mechanisms in the nervous system such as repair and protection of neurons from damage.

Herpes simplex virus type 1 (HSV-1) immediate early protein IE63, an essential nuclear protein, is pleiotropic in function and, at the post-transcriptional level, inhibits RNA splicing, interacts with cellular splicing small nuclear ribonucleoprotein particles (snRNPs), binds RNA and prevents the nucleocytoplasmic transport of intron-containing mRNAs. Here it is reported that IE63 is a nucleo-cytoplasmic shuttle protein able to travel from snRNP-and RNA-rich nuclear foci to the cytoplasm, where it accumulates during actinomycin D treatment. This newly identified property suggests that IE63 facilitates nuclear export of HSV-1 transcripts, in addition to retaining intron-containing transcripts in the nucleus. The mechanism by which IE63 controls RNA export has yet to be defined.

Herpes simplex virus (HSV) mutants lacking the gene encoding infected cell protein (ICP) 34.5 exhibit an attenuated phenotype in models of pathogenesis and have been used for experimental cancer therapy. Recently it was shown that the HSV ICP 34.5 protein functions to prevent the host cell- induced double-stranded RNA-activated protein kinase (PKR)-dependent translational block that normally occurs during virus infection. We now report that an HSV ICP 34.5 mutant called HSV- 1716 is unable to replicate in the simian kidney cell- derived line CV-1, due to a translational block. Moreover, we find that this block can be overcome by simian virus 40 (SV40). This has been shown directly by infecting CV-1 cells with SV40 and HSV- 1716 simultaneously, and indirectly via HSV-1716 infection of COS-1 cells (CV-1 cells transformed by an origin-defective mutant of SV40 that codes for wild-type T antigen). The translational block is restored when infections are done in the presence of the phosphatase inhibitor okadaic acid. These results support, but do not directly prove, contentions that HSV ICP 34.5 interacts with the PKR pathway to restore translation in non-permissive cells, and that SV40 large T antigen has a similar functional role, but acts downstream of the site of ICP 34.5 interaction (eIF2α) in the pathway. Study of this CV-1/COS-1 system should allow further clarification of the virus-host interactions that underlie the restricted replication of HSV-1 ICP 34.5 gene null mutants.

Small DNA viruses (adenoviruses, simian virus 40, or human papillomaviruses) induce S-phase progression but prevent cell division to provide precursors for viral DNA replication. Herpes simplex viruses types 1 or 2 (HSV-1 or HSV-2) contain genes which encode DNA-metabolizing enzymes, for example, ribonucleotide reductase, thymidine kinase and dUTPase, suggesting that S-phase factors are not required for an efficient infection. However, several studies indicated that HSV induces some events that occur during cell-cycle progression. To determine if HSV-2 induces S-phase entry, we examined serum-arrested African green monkey kidney cells (CV-1) after infection. Two hours after infection steady-state levels of the S-phase-specific cyclin, cyclin A, increased. S-phase cyclin-dependent kinase activity (CDK2) was stimulated 10-fold 8 h after infection but decreased at 16 or 24 h after infection. Mitotic CDK activity (CDC2) was not activated after infection, in part due to decreases in CDC2 protein levels and inactivation of enzymatic activity resulting from tyrosine phosphorylation of CDC2. Furthermore, CDK4 activity was not dramatically affected by infection. These studies indicate that HSV-2 infection selectively activates CDK2 after infection but cell-cycle progression does not occur. We hypothesize that infection activates certain components of the cell cycle which enhance viral gene expression and DNA replication.

The well-characterized human oligodendroglioma (HOG) cell line, cells of which resemble immature oligodendrocytes, was used to investigate the level of permissiveness to human cytomegalovirus (CMV) infection. Expression of CMV genes was incomplete following exposure of HOG cells to CMV, in contrast with results observed with the astroglioma cell line U373-MG (used as a positive control). However, treatment with phorbol 12-myristate 13-acetate (PMA) or with dibutryl cAMP (dbcAMP) plus the phosphatase inhibitor 1-isobutyl-3,3-methyl xanthine (IBMX) rendered the HOG cells fully permissive to CMV; down-regulation of HLA class I and production of virions were only observed under these conditions. In contrast to the findings seen with the HOG cell line, treatment of U373-MG cells with dbcAMP/IBMX or PMA did not interfere with CMV- induced down-regulation of HLA class I. However, these chemical stimulators reduced virus production in U373-MG cells by 30 and 70%, respectively.

In this study we have compared the use of PCR and conventional tissue culture methods to detect Epstein-Barr virus (EBV) in peripheral blood mononuclear cells and throat wash samples. The study population included 29 healthy adult and 20 immunocompromised EBV-seropositive donors. The results show significantly higher EBV detection rates by PCR than the tissue culture methods in throat wash samples from both donor groups (P < 0·01 in healthy donors and P < 0·009 in the immunocompromised donors) and in peripheral blood from the immunocompromised but not from the healthy donors (P < 0·008). Furthermore, when EBV DNA detection rates in throat wash cell pellet and supernatant fluid were compared, a higher positive result was obtained with the cell pellets which reached statistical significanceinthe immunocompromised group (P < 0·02). No correlation was found between positivity in throat wash and peripheral blood from the same donors.