- Volume 75, Issue 7, 1994

Cytoplasmic DNA viruses encode a DNA-dependent RNA polymerase (DdRP) that is essential for transcription of viral genes. The amino acid sequences of known large subunits of DdRPs contain highly conserved regions. Oligonucleotide primers, deduced from two conserved domains [RQP(T/S)LH and NADFDG- DE] were used in PCR experiments for the detection of the corresponding gene of the genome of insect iridescent virus type 6, also known as Chilo iridescent virus (CIV). A specific DNA product of about 150 bp could be amplified and was used as a hybridization probe against the CIV gene library to identify the corresponding gene. The gene encoding the DdRP was identified within the EcoRI fragments M (7099 bp) and L (7400 bp) of CIV DNA, between map units 0·310 and 0·347 (7990 bp). The DNA nucleotide sequence (3153 bp) of the gene encoding the largest subunit of DdRP (RPO1) was determined. Northern blot hybridization revealed the presence of a 3·4 kb RNA transcript in CIV-infected cells that hybridized to the CIV DdRP gene. This predicted viral protein consists of 1051 amino acid residues (120K) and showed considerably higher similarity to the largest subunit of eukaryotic RNA polymerase II than to the homologous proteins of vaccinia virus and African swine fever virus. Phylogenetic analysis suggested that the putative RPO1 of CIV could have evolved from RNA polymerase II after the divergence of the three types of eukaryotic RNA polymerases. The putative RPO1 of CIV lacked the C-terminal domain that is conserved in eukaryotic, eubacterial and other viral RNA polymerases and in this respect was analogous to the RNA polymerases of Archaea. It is hypothesized that the equivalent of the C-terminal domain may reside in another subunit of CIV DdRP encoded by an unidentified viral gene.

Nucleotide sequence data reported in this paper are deposited in the GenBank data library under accession no. M81388.

We constructed two recombinant Autographa califomica nuclear polyhedrosis baculoviruses. Spodoptera frugi-perda (Sf9) cells containing these constructs produce carboxy-terminally truncated envelope E proteins representing dengue (DEN) virus serotypes 2 and 3. The two recombinant proteins contained their homologous signal sequences at the N terminus and were truncated by 71 and 74 amino acids at the C terminus, respectively. This allowed the translocation of the recombinant proteins to the endoplasmic reticulum followed by glycosylation processing and secretion into the extracellular medium. An additional unglycosylated form which was not secreted was detected inside the infected Sf9 cells. Sera from Swiss mice immunized with the infected Sf9 cell lysates gave a DEN cross-reactive response in ELISA and substantial amounts of neutralizing antibodies to the homologous virus. Similar antibody titres were obtained when the two recombinant proteins were inoculated concomitantly. BALB/c mice were vaccinated with three doses of the recombinant E proteins, taken as monovalent or bivalent immunogens, and challenged with mouse-adapted DEN-2 virus. DEN-2 E protein induced a good protection (90 %) against lethal encephalitis and recombinant DEN-3 E protein gave a substantial cross-protection (54%). Eighty-two percent of the mice immunized with a mixture of both recombinant E proteins survived the DEN-2 virus challenge.

Viral haemorrhagic septicaemia (VHS) is a fish rhabdo- virus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a Safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The M r of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculo- virus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV.

Maedi-visna virus (MVV)-specific cytotoxic T lymphocytes (CTL) were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in the efferent lymph and peripheral blood of sheep chronically infected with MVV. Cytotoxicity was mediated by CD8+ lymphocytes and was specific for particular strains of MVV. These precursor CTL were detected in the blood between day 23 and day 100 after infection via the skin. In one out of seven persistently infected sheep MVV-specific cytotoxicity was seen in uncultured peripheral blood cells. Again the effector population consisted of CD8+ lymphocytes. The only other viral infections in which CTL have been detected in peripheral blood mononuclear cells prior to secondary stimulation are those caused by the simian and human immunodeficiency viruses.

The tropism of human immunodeficiency virus (HIV) for macrophages (mø)is a well recognized phenomenon, but the range and distribution of m𝜙-tropic phenotypes have not been defined by quantitative means. This study uses a PCR-based infectivity assay to derive an index of m𝜙 tropism for several common strains of HIV. The results show that m𝜙 tropism varies over about six orders of magnitude and that the most m𝜙-tropic strains have a higher infectivity for m𝜙 than for peripheral blood lymphocytes. Strains were distributed throughout this range, suggesting that m𝜙 tropism is a continuously variable phenotypic property. Although the degree of tropism was strongly influenced by the mode of isolation and propagation of virus strains, there was no evidence for the existence of distinct m𝜙-tropic or non-m𝜙-tropic phenotypes. Finally, the tropism of two selected strains was found to be determined by an early step in replication, probably virus entry.

We have studied the presence and significance of retroviral genome-derived DNA in the core of human immunodeficiency virus (HIV) particles produced from transfections of HXB2 expression vectors in COS-7 cells and from HIV type 1 IIIB chronically infected H9 cells. Viruses purified by sucrose cushion centrifugation and treated with DNase I contained 1000-fold more viral RNA than DNA. However protease-defective viruses that contained only p160 gag−pol had less than 100 times the amount of DNA in their cores than wild-type viruses suggesting that the p66/p51 form of reverse transcriptase was responsible for DNA transcription. Viruses produced by transfections in the presence of 3′-azido-3′-deoxythymidine (AZT) contained the viral RNA genome but only DNA of premature length because of the chain terminating effects of AZT. However such viruses were as infectious for CD4+ cells as wild-type virus. We conclude that retrovirus-derived DNA in HIV-1 particles is not required for infection and does not play a significant role in this process.

PCR has been used to amplify a 540 base pair fragment of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat region encompassing the U5/R/U3 regions from proviral HIV DNA that was present in the lymph nodes and peripheral blood mononuclear cells of three HIV-infected individuals. The resulting PCR products were cloned and the DNA sequence of multiple clones from each reaction was determined to enable the distribution and phylogenetic relationship between the variants in each body site to be assessed. The results of bootstrapped parsimony phylogenetic analyses showed that a distinct polarization was evident between peripheral blood and lymph node variants in the patient who possessed a histologically intact lymph node. In contrast, similar analyses conducted on samples from two patients with extensive lymph node disruption showed similar variants in lymph node and peripheral blood. In the patient with an intact lymph node, lymph node variants were significantly more heterogeneous than those present in blood samples taken either simultaneously or 11 months later. No differences in heterogeneity between lymph node and peripheral blood were observed in patients with disrupted lymph nodes. The significance of these results for our understanding of HIV pathogenesis is discussed.

Human Jurkat T cells containing a stably integrated human T cell leukaemia virus type 1 (HTLV-1) long terminal repeat (LTR) reporter gene construct were used to study the role of calcium-dependent cellular activation pathways in LTR trans-activation. Treatment of these cells with the calcium ionophore ionomycin resulted in a reduced basal response of the LTR and reduced responses to 12-O-tetradecanoylphorbol-13-acetate- and Tax-mediated trans-activation. This effect was also observed for virus production in the HTLV-1-producing T cell line MT-2. Experiments designed to determine the events underlying this inhibition, using inhibitors of calcium-related events, revealed that the ionomycin- induced repression of the LTR was alleviated in all cases by cyclosporin A. This compound was also effective in preventing the ionomycin-induced reduction in virus production in MT-2 cells. These results suggest a role for calcium-related events in the down-regulation of HTLV-1 expression.

We have characterized the temporal appearance and accumulation of minute virus of mice (MVM) replicative forms (RF) in highly synchronized single rounds of infection using a combination of restriction endonuclease analysis and two-dimensional agarose gel electrophoresis. Between 4 and 12 h after release of infected cells into the S-phase, both monomer (mRF) and dimer RF (dRF) increased exponentially at similar rates such that the ratio of mRF relative to dRF remained unchanged. These DNA forms accumulated at a faster rate than MVM RNAs, suggesting that the number of DNA templates available for replication is limiting, not the expression of MVM gene products, and that the majority of DNA templates are likely to be destined for DNA amplification rather than transcription and further gene expression. During this exponential DNA amplification phase, approximately 65 % of mRF were in a fully extended form, whereas most of the remaining mRF were covalently closed in the left end and extended in the right end. Although MVM replication presumably generates right-hand turn-around mRF, only a low level of this form persists (5 to 10 % of total mRF) at all times examined, suggesting that this form must be quickly converted to the extended form. Greater than 90% of dRF, which have right-hand palindromes on both ends of the molecule, were extended on both ends. A significant proportion of dRF and higher concatemers are nicked in the left-hand palindrome, suggesting that resolution of dRF into two mRFs may occur via single-stranded nicks rather than a double-stranded cut. An additional replicative form, previously termed band X, has been identified as an RNA-DNA duplex. This band is formed predominantly intracellularly, before cell lysis but its biological significance remains unclear. Our results provide direct experimental support for many of the predictions of the current models of parvovirus replication and suggest that the kinetic hairpin transfer model should be adjusted to include a strand-transfer or similar mechanism for the resolution of dRF to account adequately for the production of left-end turn-around forms.

We used a capture ELISA to quantify the E7 protein of human papillomavirus type 18 (HPV-18). In HeLa cells, which express low levels of immunoreactive E7 protein (iE7), iE7 had a mean half-life of 13·5 min. In HPV-18 E7 recombinant baculovirus (E7rec BV)-infected Sf21 cells, which express higher levels of E7, the half-life of iE7 was much longer (90 min and > 24 h, with two different E7rec BVs). For two transformed human cervical cell lines expressing HPV-18 E7, exposure of the cells to hydrocortisone resulted in a twofold increase in steady-state levels of the E7 protein: no similar effect was observed with progesterone, oestrogen or testosterone. The half-life of iE7 was unaltered by hydrocortisone or progesterone exposure. An immunoassay which distinguished Ser33-phosphorylated E7 from E7 not phosphorylated at this residue (Ser33dephospho-E7), showed that in HeLa and Sf21 cells the majority of E7 was phosphorylated : the half-life of both species of E7 was similar in HeLa cells, but the half-life of Ser33dephospho-E7 was much shorter (90 min) in Sf21 cells than that of Ser33phospho-E7 (> 24 h). A HeLa- fibroblast fusion cell line with tumorigenic potential (CGL-1) had a similar ratio of dephospho-E7 to total E7 (0·06), as a similar fusion cell line (CGL-4) with no tumorigenic potential (0·03). We conclude that E7 is a labile phosphoprotein, and that the expression and steady-state level of the E7 protein in eukaryotic cells may be influenced by the hormonal environment of the cells.

The nucleotide sequence data in this paper will appear in the EMBL database under the accession no. X71982.

The nucleotide sequence of a 55098 bp region from the right end of the genome of a virulent African swine fever virus (ASFV) isolate (Malawi LIL20/1) has been determined. Translation of the sequence identi fied 67 major open reading frames (ORFs) which are closely spaced and read from both DNA strands. At six positions intergenic tandem repeat arrays are found. Comparison of the predicted amino acid sequences of encoded proteins with protein sequence databases identified a number of homologies. These include three subunits of RNA polymerase, a protein with homology to transcription factor SII (TFSII), a DNA ligase, two subunits of mRNA capping enzyme, a DNA topo- isomerase type II, a dUTPase, a protein kinase, three helicases, a ubiquitin-conjugating enzyme, a protein with homology to the nif S and nif S-like proteins identified in some bacteria and Saccharomyces cerevisiae, a protein with homology to both a myeloid differentiation primary response antigen (MyD116) and to a herpes simplex virus-encoded neurovirulence-associated protein (ICP34.5), a protein with homology to the ASFV-encoded structural protein p22, two proteins with homology to copies of the ASFV-encoded multigene family 360 and one protein with homology to the ASFV- encoded multigene family 110. Four genes encode proteins which have homology to each other and constitute a new multigene family (MGF100). Nine ORFs encode proteins which contain predicted transmembrane domains. The possible functions of these predicted ASFV-encoded proteins are discussed and the evolutionary relationship of ASFV to other viruses are considered. Despite the similarities in genome structure and replication strategy of ASFV with poxviruses, sequence similarity between them is low and the organization of ASFV-encoded genes is not colinear with that of the orthopoxviruses.

The ras gene family encodes 2IK proteins that reside on the inner face of the plasma membrane and bind GTP and GDP with an equally high affinity. Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a viral Harvey-raj (v-Ha-ras) cDNA, together with a plasmid (pCMVCAT) carrying the immediate early (IE) enhancer of the murine cytomegalovirus (MCMV) linked to the chloramphenicol acetyltrans- ferase (CAT) reporter gene strongly stimulated CAT activity. Basal levels of pCMVCAT expression as well as trans-activation by v-ras plasmid were both inhibited by cotransfection of an expression vector containing the dominant inhibitory mutant gene Ha-ras Asn-17. This indicates that the p21 ras protein is responsible for these activities. High pCMVCAT activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. To define the cis-acting DNA elements in the MCMV IE enhancer responsible for this trans-activation by p21 ras protein, we constructed several plasmids containing the CAT gene under control of MCMV IE enhancers that were deleted in different regions. The CAT assays demonstrated that several sequences were responsive to p21 ras protein. These sequences are scattered throughout the IE enhancer, upstream of the transcription start site, and contain responsive elements that are homologous to the binding sites for cellular transcription factors such as NFkB, API, ATF and SP1. Activation of the p2l ras protein may thus be one of the signals that regulate IE genes transcription during MCMV infection.

The sequence coding for the 5′ untranslated region (UTR) of ICP22 mRNA of herpes simplex virus type 1 has been tested for its ability to regulate gene expression. This sequence was placed in frame with the chloramphenicol acetyltransferase (CAT) coding sequence and under the control of the simian virus 40 early promoter-enhancer. Under these conditions, the sequence coding for the 5 UTR led to an increase of about 13-fold in CAT activity, measured during transient expression. The use of mutants with progressive deletions within the sequence coding for the 5′UTR allowed localization of the sequence responsible for the enhancement of gene expression to the first exon of the ICP22 gene. Precise quantification of hybrid ICP22-CAT mRNA showed that the sequence coding for the 5′UTR induced an increase in the amounts of transcripts, which resulted in a parallel increase in CAT activity. This increase in the level of hybrid ICP22-CAT mRNA is not the result of an increase in mRNA stability, nor is it due to more efficient nucleo-cytoplasmic transport of the transcripts. Moreover, the distribution of hybrid mRNA in the different ribosomal populations indicates that the 5′UTR of ICP22 mRNA does not induce a preferential recruitment of the transcripts by the translational apparatus. Taken together, these results indicate that a cis-acting element located in the sequence coding for the 5′UTR of ICP22 mRNA can mediate a high level of gene expression independently of the viral promoter and of viral trans-acting factors.

Kinetic analysis of the two divergent immediate early (IE) transcription units of bovine herpesvirus 1 (BHV-1) revealed an unexpected behaviour. The IE1.7 promoter was not turned off at the end of the IE period but acted as a late promoter, unlike the adjacent IE4.2/2.9 promoter which was active only under IE conditions. The genome region specifying the IE 1.7 gene was sequenced (0·814 to 0·839 map units). The IE1.7 promoter was found to overlap with duplicated sequence elements bearing close similarity to herpesvirus origins of replication, which may explain the biphasic transcription kinetics. Exons 1 and 2 of the spliced IE1.7 transcript were non-coding. Exon 3 was found to contain a single open reading frame encoding a protein of 300 amino acids that was designated BICP22 because of its homology to ICP22 (Vmw68) of herpes simplex virus type 1 and related proteins from other herpesviruses. The protein probably represents IEP-55, the most abundant BHV-1 phosphoprotein observed under IE conditions.

Libraries of cDNA were generated from polyadenylated RNAs derived from Marek’s disease virus (MDV)- transformed cell lines by directional cloning of oligo- (dT)-primed cDNAs in lambda gt22A. Analysis of the libraries for viral sequences showed that a number of cDNA clones originated from transcripts mapping in the BamHl A region of the MDV genome. Sequencing and line mapping of these cDNAs suggested that the RNA transcripts expressed from this region were either in the sense or antisense direction with respect to the MDV homologue of the ICP4 gene of herpes simplex virus. The longest cDNA clone from antisense transcripts was 2756 bp long and partially overlapped the 5′ end of the coding region of the ICP4 gene. The cDNA clone contained at least four introns, shown by comparison of its sequence with the sequence of the ICP4 gene. The presence of introns was confirmed by PCR analysis. All the introns have the consensus splice donor and acceptor signals at their 5′ and 3′ ends respectively. Northern blot analysis showed that the ICP4 gene homologue of MDV was abundantly transcribed only in lytically infected fibroblasts, whereas transcripts complementary to the ICP4 gene were the major transcripts in MDV- transformed cell lines and lymphomas. The transcripts complementary to ICP4 consist of two major RNA species approximately 15 kb and 1·32 kb long. The results suggest that there might be an inverse relationship between the abundance of ICP4 transcripts and their complementary transcripts in MDV-infected and transformed cells.

Herpesvirus envelope glycoproteins play important roles in mediating infection initiation and also represent major immunogens. We recently showed that a pseudorabies virus (PrV) mutant lacking the essential glycoprotein gD (gp50), after phenotypic complementation by propagation on genetically engineered PrV gD- expressing cell lines was able to infect primary target cells and spread exclusively by means of direct cell-to- cell transmission. Virions released from non-complementing cells that lacked gD were not infectious because of a defect in penetration and so free infectious virions did not arise after infection of animals by phenotypically complemented gD-negative PrV. This formed the basis for the development of novel non-spreading live herpesvirus vaccines. However, the gD-negative PrV mutant still retained a residual level of virulence, which prevented its use as vaccine, and the need to propagate the gD-negative PrV mutant on trans-complementing cell lines resulted in the appearance of wild-type revertants, rescued by the resident gene in the cell line. To overcome these problems we isolated a PrV mutant designated PrV(376) that, in addition to gD, also lacked the non-essential glycoproteins gG, gl and gE. PrV(376), because of the lack of gD, was also dependent on gD- expressing cells for productive replication. Non-complementing cells infected by phenotypically gD-comple- mented PrV(376) produced non-infectious particles lacking glycoproteins gD and gE as shown by immuno- electron microscopy. Owing to the absence of any homologous sequences between the viral genome and the viral genes resident in the complementing cell line, rescue by homologous recombination was impossible. In cell culture, plaques of PrV(376) were significantly smaller than those of either wild-type, or gD- or gE- deleted mutants, indicating an additive or synergistic effect of the combined deletion on viral cell-to-cell spread capability. Intranasal or intramuscular infection of pigs with phenotypically gD-complemented PrV(376) showed a complete attenuation of viral virulence, with an expected lack of shedding of infectious virus. The PrV(376)-vaccinated pigs exhibited a significant level of protection against challenge infection, measured by survival and weight loss. In summary, PrV(376) represents a novel type of herpesvirus vaccine that combines innocuity, efficacy and biological safety.

A murine intranasal infection model for equine herpesvirus type 1 (EHV-1) was used to evaluate immune responses following immunization with insect cells infected by baculoviruses that express EHV-1 glycoproteins. Baculovirus recombinant glycoprotein D (gD) and gH both induced serum antibodies to EHV-1 when measured by ELISA. The gD recombinant also produced a neutralizing antibody response. Protective immunity, determined by accelerated clearance of virus from the target organs in the respiratory tract, was demonstrated in mice immunized with a baculovirus recombinant expressing gD. In addition to the serological response, evidence is presented which shows that cell-mediated responses also play an important role in protection. Both recombinants induced delayed-type hypersensitivity and lymphoproliferation to EHV-1 antigen. The protective effects of T cells were confirmed by adoptive transfer of spleen cells from baculovirus gD- immunized donors to recipients that were challenged with live EHV-1. Depletion of either CD4- or CD8- bearing cells from the gD-immunized donors reduced the ability of the recipients to clear virus from the target organs, although depletion of CD4 cells had a more marked effect.

The mechanism responsible for the decreased sensitivity of a clinical herpes simplex virus type 1 (HSV-1) isolate, HSV-145, to (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) was examined. Measurements of 50% inhibitory doses of several drugs demonstrated that although HSV-145 was sensitive to phosphonoacetic acid, adenine arabinoside and acyclovir, its sensitivity to BVDU and 5-(2-chloroethyl)-2′-deoxyuridine was significantly less than that normally observed for HSV-1. Analysis of the thymidylate kinase (TMP-K) activity of HSV-145 thymidine kinase (TK) demonstrated a decreased level of TMP-K activity when compared to HSV-1 TK. The TMP-K activity of HSV-145 resembled that observed for HSV-2 and the TK-deficient strain HSV-1 TK−7. When the nucleotide sequence of the HSV-145 TK gene was compared to that of the HSV-1 strains Cl(101) and SC16 a single nucleotide substitution (G changed to A at base position 502) was detected which would result in the substitution of threonine at amino acid position 168 for alanine. The substitution is the same as that for the laboratory- derived BVDU-resistant virus HSV-1 SC16B3. Collectively, these studies highlight the importance of amino acid conservation at position 168 of the HSV-1 TK in conferring efficient TMP-K activity and BVDU sensitivity.

The fine structure of light (L) particles of herpes simplex virus type 1 was examined by cryo-electron microscopy and compared to that of virions. The L particles appeared to be spherical entities with significant variation in size, on average smaller in diameter than virions (140 nm compared to 180 nm). The technique confirmed that L particles are composed of an outer envelope, i.e. a bilaminar membrane with protruding glycoprotein spikes, and a uniformly granular tegument, but lack any nucleocapsid. In addition it revealed the presence of one or occasionally more spherical objects, termed inclusion vesicles (IVs), embedded in the tegument of a large proportion of L particles but not observed in virions, suggesting that presence of IVs is unique to the L particles. The IVs vary in size and appear to be composed of a bilaminar membrane without surface projections and filled with material of relatively low electron density, suggesting that the composition of IVs is distinct from that of the envelope and tegument of the L particles.