- Volume 73, Issue 7, 1992
Volume 73, Issue 7, 1992
- Animal
-
-
-
Physical map of the Cryptophlebia leucotreta granulosis virus genome and its relationship to the genome of Cydia pomonella granulosis virus
More LessA physical map of the genome of Cryptophlebia leucotreta granulosis virus (ClGV) was constructed for the restriction enzymes BamHI, EcoRI, KpnI, NdeI, NruI, SacI and XhoI using hybridization techniques. The size of the viral genome was determined to be 112.4 kbp. A restriction fragment library covering almost the entire genome of ClGV was constructed, and the position of the granulin gene was identified by cross-hybridization with granulin coding fragments of Cydia pomonella granulosis virus (CpGV). Two further regions of intergenomic similarity between ClGV and CpGV were mapped. These regions were aligned and show a collinear arrangement.
-
-
-
-
Persistence and expression of Microplitis demolitor polydnavirus in Pseudoplusia includens
More LessPersistence and expression of Microplitis demolitor polydnavirus (MdPDV) was examined in parasitized and virus-injected Pseudoplusia includens larvae. Viral DNA persisted in P. includens larvae for 6 days, but no increase in the amount of viral DNA present was detected. Viral transcripts were observed in parasitized and virus-injected larvae 4 h post-parasitism and expression continued for 6 days. When specific host tissues were examined, more viral DNA and RNA was detected in haemocytes than in the gut, nervous system and fat body. 32P-labelled MdPDV DNA hybridized to approximately six different size classes of mRNAs on Northern blots of RNA from haemocytes of parasitized larvae. MdPDV transcription was first detected in haemocytes at 4 h post-parasitism and continued for 6 days. Similar transcripts were observed in haemocytes from larvae that had been injected with calyx fluid or MdPDV plus venom. First-strand cDNA probes of haemocyte-specific MdPDV transcripts hybridized to only certain MdPDV viral DNAs, suggesting that only part of the MdPDV genome is expressed in this host cell type.
-
-
-
Scrapie in the central nervous system: neuroanatomical spread of infection and Sinc control of pathogenesis
More LessFollowing bilateral intraocular (i.o.) infection of Sinc s7 mice with ME7 scrapie, sequential tissue pools were taken from retina, optic nerve, superior colliculus (SC), dorsal lateral geniculate nucleus (dLGN), visual cortex and cerebellum. The infectivity levels in these pools were estimated by intracerebral (i.c.) assay in C57BL/FaBtDk mice. Infectivity was first detected in retina at 35 days post-injection (as an increase above residual injected inoculum), SC at 56 days, dLGN at 77 days and in optic nerve, visual cortex and cerebellum at 98 days. Pathological lesions were shown to develop in the same sequence later in the incubation period. Comparison of sequential retina and SC assays in congenic mice, which differ only in the vicinity of the Sinc locus, revealed a difference in the initial detection and progression of i.o. infection of between 60 and 100 days, indicating that Sinc acts by delaying the initiation of replication. Higher levels of infectivity were found in retina and SC of mice infected with 79A scrapie, which destroys the photoreceptor layer in the retina, than with ME7 scrapie, which does not. Retrograde transport of infection was indicated by the levels of infectivity in the retina after i.c. infection with ME7 or 79A scrapie. These results indicate that scrapie spread within the central nervous system is restricted to neuroanatomical pathways, and that Sinc controls the initiation, but not the rate of replication.
-
-
-
Variations in prion protein and glial fibrillary acidic protein mRNAs in the brain of scrapie-infected newborn mouse
More LessTo begin to understand the molecular basis of cases of Creutzfeldt-Jakob disease recently described in young children, the expression of prion protein and glial fibrillary acidic protein (GFAP) mRNAs was investigated during the development of the brain of scrapie-infected newborn mice. Changes in the time course of expression were identified by Northern blot quantification between days 1 and 172. Although scrapie-infected and control animals showed no detectable changes in brain development (first 56 days of life), GFAP mRNAs were found to increase significantly as early as day 84. A 10-fold increase in the level of GFAP mRNA was observed in brain between day 112 and death (day 172).
-
-
-
The right end of the unique region of the genome of human herpesvirus 6 U1102 contains a candidate immediate early gene enhancer and a homologue of the human cytomegalovirus US22 gene family
More LessThe nucleotide sequence of a 12 kbp HindIII fragment (HindIII C) from the right end of the unique component of the genome of human herpesvirus 6 (HHV-6) (strain U1102) was determined. The sequence has a mean G+C content of 42% and contains approximately 28 copies of a tandemly repeated 104 to 107 bp element, which, with a single exception, contain a cleavage site for KpnI (the KpnI repeats). Each of these elements contains potential binding sites for transcription factors NF-κB and AP2. The KpnI repeats lie immediately upstream of a region previously identified as a candidate immediate early (IE) gene locus and therefore may constitute an IE gene enhancer element. One incomplete and six complete open reading frames (ORFs) were identified in the unique sequence of the HindIII C fragment. The predicted products of these ORFs do not include homologues of proteins encoded by members of the alpha- or gammaherpesvirus subfamily. However, the HindIII C fragment does contain a homologue of the US22 gene family, previously found only in the betaherpesvirus human cytomegalovirus (HCMV). These findings provide evidence that the close phylogenetic relationship between HHV-6 and HCMV is not confined to the betaherpesvirus-specific arrangement of conserved replicative and structural genes which has been demonstrated previously.
-
-
-
Identification of homologues to the human cytomegalovirus US22 gene family in human herpesvirus 6
More LessThe sequence of 10079 bp corresponding to the overlapping SalI H and SmaI G restriction fragments of the genome of human herpesvirus 6 (HHV-6) strain U1102 was determined. The sequence contains six complete open reading frames (ORFs) and two incomplete ORFs located at the 5′ and 3′ ends of the SalI H and SmaI G fragments respectively. Seven of these ORFs have recognizable homologues only in the betaherpesvirus human cytomegalovirus (HCMV), no obvious counterparts being detectable in the genomes of the human alphaherpesviruses, varicella-zoster virus and herpes simplex virus type 1 or the gammaherpesvirus Epstein-Barr virus. The DNA sequenced is located proximal to the left repeat of the HHV-6 genome outside the well recognized region encompassing conserved herpesvirus gene blocks. A close collinear relationship is evident between the HHV-6 ORFs identified in this study and their counterparts in HCMV, ORFs UL23, UL24 and UL27 to UL31. Four of the HHV-6 ORFs, SHL1, SHL2, SFL1 and SSL2, are related to members of the HCMV US22 family of proteins, which are themselves tandemly arranged and located predominantly within the unique short and the left end of the unique long region of the prototype HCMV strain AD169 genome. Two adjacent HHV-6 ORFs, SSL1 and SHL3, are related to HCMV UL27. The identification of this gene set in addition to the HHV-6 ORFs with amino acid sequence similarity to the HCMV US22 family indicates a particularly close relationship between these two human herpesviruses, and suggests that the clustering of these related tandemly arranged genes may be a general feature of betaherpesvirus-type genomes.
-
-
-
Experimental infection of cynomolgus and African green monkeys with human herpesvirus 6
Cynomolgus and African green monkeys were inoculated with human herpesvirus 6 (HHV-6). An antibody response was first observed 10 days and 5 days after inoculation of cynomolgus monkeys and African green monkeys, respectively, and was detectable for the duration of the experiment (33 days). HHV-6 DNA was first detected by the polymerase chain reaction in mononuclear cells of one cynomolgus monkey and one African green monkey 10 days after virus inoculation, and in a total of three of four cynomolgus monkeys (75%) and four of five African green monkeys (80%) later after inoculation. Furthermore, HHV-6 DNA was detected in the lymph nodes and spleen of monkeys killed 33 days after virus inoculation. A rash was observed on the trunk of one African green monkey 13 days after virus inoculation, otherwise the infection was asymptomatic. When mononuclear cells from both groups of monkeys were cultured in medium containing concanavalin A and interleukin 2, and infected with HHV-6 in vitro, virus replication was observed. The data suggest that HHV-6 infects these species of monkey and that this system could be useful as an animal model of HHV-6 infection.
-
-
-
Simian virus 40 (SV40) large T antigen-dependent amplification of an Epstein-Barr virus-SV40 hybrid shuttle vector integrated into the human HeLa cell genome
More LessWe analysed the DNA rearrangements that occurred during the integration and amplification of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector in human cells. The human HeLa cell line was episomally transformed with the EBV-SV40 p205-GTI plasmid. After a 2 month culture in a selective medium, a HeLa cell-derived population (H-G1 cells) was obtained in which the p205-GTI vector was integrated as a single intact copy deleted in the EBV latent origin of replication (OriP). Sequencing data showed that the endpoints of the plasmid sequences, at the plasmid-cell DNA junctions, are located within the two essential elements of EBV OriP, which may form several secondary structures. This result suggests that a specific DNA sequence (OriP) or palindromic structures could play a role in this integration process. This represents the first fully characterized site of integration of an EBV vector in human cells. The transient expression of the SV40 large T antigen in H-G1 cells leads to the appearance of episomal molecules with an extremely heterogeneous size pattern. Individual analysis of these episomes after rescue in bacteria indicated that they retained sequences of both the p205-GTI plasmid and cellular DNA. Comparison of the structure of these circular DNAs with those of the integrated p205-GTI copy indicated that large T antigen expression in human cells leads to the amplification of the integrated shuttle vector according to the ‘onion skin’ model developed for transformed rodent cells. Indeed, amplified sequences were collinear with the integrated p205-GTI copy and its surrounding cellular sequences, distributed almost equally around the SV40 replication origin, and circularized by illegitimate recombination which did not involve specific nucleotide sequences. This system is of interest in that it enables easy recovery of individual recombined molecules in host bacteria. Each isolated clone contains a unique recombination junction which is easily and rapidly characterized and sequenced.
-
-
-
RNA polymerase III-transcribed EBER 1 and 2 transcription units are expressed and hypomethylated in the major Epstein-Barr virus-carrying cell types
More LessThe genome of Epstein-Barr virus (EBV) codes for two non-translated small RNA molecules, EBER 1 and 2. We found that both EBERs are expressed in the major EBV-carrying cell types, group I and III Burkitt’s lymphoma (BL) cell lines, lymphoblastoid cell lines (LCLs) and in two nude mouse-passaged nasopharyngeal carcinoma (NPC) tumours. The relative amount of EBER 1 and EBER 2 varied in different host cells but did not correlate with the cellular phenotype. The EBER coding and flanking sequences were predominantly hypomethylated at HpaII sites not only in LCLs which usually carry hypomethylated EBV genomes but also in BL and NPC cell lines harbouring EBV episomes that are highly methylated in other regions. Thus, the EBER trancription units, actively transcribed by RNA polymerase III in the major EBV-carrying cell types, represent a methylation-free region in the EBV genome similarly to regulatory sequences of the latent membrane protein gene when the latter is transcribed by RNA polymerase II.
-
-
-
Identification and characterization of a 30K glycoprotein of herpes simplex virus type 2
More LessA herpes simplex virus type 2 (HSV-2) type-specific monoclonal antibody (MAb), CH-A9, precipitated a glycoprotein with an M r of approximately 30000 (g30K) from extracts of HSV-2-infected BHK cells labelled with [3H]leucine, [14C]fructose or [3H]glucosamine. The M r of this glycoprotein is lower than those of other HSV glycoproteins. Immunoassays of BHK cells infected with HSV-1-HSV-2 intertypic recombinants localized the gene encoding the target antigen of MAb CH-A9 to the unique long (UL) region at map units 0.490 to 0.564. Tunicamycin effectively inhibits N-linked glycosylation of g30K, which suggests that g30K may be modified by addition of N-linked oligosaccharides and that the amino acid sequence may contain Asn-X-Ser or Asn-X-Thr. The g30K was also purified on an immunoadsorbent column consisting of MAb CH-A9 linked to Sepharose 4B and was shown to be an HSV-2 type-specific antigen by indirect ELISA. The glycoprotein could induce HSV-2 type-specific neutralizing antibody in BALB/c mice. This evidence suggests that g30K may be a novel glycoprotein of HSV-2.
-
-
-
A leucine zipper structure present in the measles virus fusion protein is not required for its tetramerization but is essential for fusion
More LessThe biological role of a leucine zipper motif present in the measles virus fusion (F) protein has been investigated. This motif is present in all paramyxovirus F proteins, all coronavirus spike proteins and many if not all retrovirus envelope proteins. By analogy to its role in certain transcription factors, it has been suggested that the motif may be responsible for the oligomerization of these viral membrane proteins. In this study, one, two or four heptadic leucines in the motif were substituted using site-directed mutagenesis. We found that fusion is prevented when all four heptadic leucines present in the motif are mutated whereas cellular transport and the oligomeric state of the F protein are unaffected.
-
-
-
Sequence analysis of the 22K, SH and G genes of turkey rhinotracheitis virus and their intergenic regions reveals a gene order different from that of other pneumoviruses
More LessThe nucleotide and deduced amino acid sequences of three genes of turkey rhinotracheitis virus (TRTV) together with the nucleotide sequences of the relevant intergenic regions were determined. The deduced amino acid sequence of one of the genes shows significant identity (42%) to that of the 22K protein of human respiratory syncytial virus (RSV). The TRTV 22K gene, like that of RSV, has a second open reading frame, although the amino acid sequence deduced from this reading frame does not show any similarity to the equivalent predicted RSV protein. The other two genes and their deduced amino acid sequences do not show any sequence similarity to the genes of other pneumoviruses. However, the hydrophobicity profiles of the predicted proteins do show similarities to those of the small hydrophobic (SH) and attachment protein (G) genes of RSV. The TRTV G gene is 1193 nucleotides in length and encodes a protein of 391 amino acids (M r 42984), which is rather larger than the RSV G protein (predicted M r 36000). The TRTV SH gene is 589 nucleotides in length, encoding a protein of 174 amino acids (M r 18797), which is considerably larger than the size of the RSV SH protein (M r 7500). The sequences of the intergenic regions derived from clones of polycistronic mRNAs and polymerase chain reaction products obtained with primers from different genes reveal the order on the virus genome to be 3′ F-22K-SH-G 5′. This differs from the gene order of paramyxoviruses and morbilliviruses, which lack a 22K gene (and in some cases a SH gene), and the pneumoviruses RSV and pneumonia virus of mice, which have the F and 22K genes located after the G gene.
-
-
-
Sequence analysis of the gene encoding the fusion glycoprotein of pneumonia virus of mice suggests possible conserved secondary structure elements in paramyxovirus fusion glycoproteins
More LessThe gene encoding the fusion (F) glycoprotein of pneumonia virus of mice consists of 1657 bases and contains an open reading frame encoding 537 amino acids which is more similar to the F proteins of pneumoviruses than to those of other paramyxoviruses. Computer-assisted sequence analyses can be combined with data on the antigenicity of various F proteins to suggest a possible arrangement of secondary structure elements common to all pneumovirus and paramyxovirus F proteins.
-
-
-
Bovine coronavirus peplomer glycoproteins: detailed antigenic analyses of S1, S2 and HE
More LessForty-four monoclonal antibodies (MAbs) to the G110 isolate of bovine enteric coronavirus were used for the characterization of the peplomer proteins S and HE. Fourteen of these MAbs reacted with HE and the remaining 30 with the products of the S gene, S1 (19 MAbs), S2 (six MAbs) and gp200 (five MAbs). S1 and HE were found to carry major neutralization determinants, and S1 appeared to elicit the production of the MAbs displaying the highest neutralizing activity. The topography of the epitopes was assessed by means of a competitive binding assay; the 44 MAbs defined four independent antigenic domains on S1, two on S2, one on gp200 and two on HE. All the neutralizing anti-S1 MAbs mapped in antigenic sites A and B and all the neutralizing anti-HE MAbs in HE-B. Antigenic site S1-B was further subdivided into four subsites. Functional mapping was performed by testing a library of neutralization-resistant mutants against the neutralizing MAbs. Analysis of their reactivity in a neutralization test confirmed the overall distribution of epitopes in S1-B and HE-B.
-
-
-
Compartmentalization of subviral particles during poliovirus eclipse in HeLa cells
More LessHeLa cells were preincubated with radiolabelled poliovirus type 1 at 26 °C, such that the 160S virions were internalized, but not altered structurally. The temperature was then shifted to 37 °C to study the intracellular redistribution of the virions and the modifications they undergo at that temperature. Using subcellular fractionation in isoosmotic Nycodenz gradients, we obtained evidence for the rapid loss of virions from the plasma membrane and from a vesicular fraction, as well as for the formation of two populations of intracellular 135S particles. The first population was associated with lysosomes and was slowly converted to (RNA-containing) 110S particles. In the presence of the lysosomotropic agent chloroquine, the lysosomal 135S population was converted to 80S empty capsids. The second 135S population, which was not associated with any organelle, was converted to 80S empty capsids. Similar observations were made during unsynchronized infection at 37 °C. We propose a model for infection in which 135S particles cross a membrane barrier, and are uncoated in the cytosol.
-
-
-
Nucleotide sequence and coding strategy of the Uukuniemi virus L RNA segment
More LessThe complete nucleotide sequence of the L RNA segment of Uukuniemi virus has been determined from cloned cDNA. The L RNA is 6423 nucleotides in length, and is of negative polarity. The viral-complementary RNA contains a single large open reading frame of 2104 codons which corresponds to the L protein (M r 241039). Comparison with the L protein sequences of other members of the Bunyaviridae showed homology with the Rift Valley fever phlebovirus L protein (38% amino acid identity), but no detectable similarity with bunyavirus, hantavirus or tospovirus L proteins. These data lend further support for the recent reclassification of uukuviruses and phleboviruses into the same genus, Phlebovirus, in the family Bunyaviridae. The L RNA sequence completes the determination of the Uukuniemi virus genome: since the M RNA segment is 3229 and the S RNA segment 1720 nucleotides, the whole genome comprises 11372 nucleotides.
-
-
-
Activation of integrated human immunodeficiency virus type 1 in human neuroblastoma cells by the cytokines tumour necrosis factor alpha and interleukin-6
More LessHuman immunodeficiency virus type 1 (HIV-1) infection was studied in two different human neuroblastoma cell lines, SK-N-MC and SH-SY5Y. Results from immunofluorescence analysis indicate that SK-N-MC cells express a 68K neurofilament, and SH-SY5Y cells express additionally a 160K to 200K neurofilament complex and thus represent a more differentiated state. HIV-1 infection in these cell lines was demonstrated by nested polymerase chain reaction and further characterized by in situ hybridization, which showed that about 50% of SK-N-MC cells and 20% of SH-SY5Y cells were infected by HIV-1 and contained integrated proviral HIV-1 DNA. Among the cytokines and growth factors studied, tumour necrosis factor alpha (TNF-α) enhanced virus production in both cell lines, but to a differing extent, according to our mRNA and p24 antigen capture assay. In SK-N-MC cells the enhancement of HIV-1 mRNA was detected after 24 h of stimulation, and declined to the control level by 48 h. In SH-SY5Y cells a clear-cut stimulation was seen at both time points. By contrast, interleukin-6 (IL-6) enhanced the virus replication only in SK-N-MC cells, as shown at the mRNA level. Immunochemical staining showed no differences in the proportion of HIV-1-positive cells after 48 h of stimulation by TNF-α or IL-6 when compared to the control cells. In addition, based on a thymidine incorporation assay, TNF-α inhibited, but IL-6 strongly increased, the DNA synthesis in SK-N-MC cells, whereas in the SH-SY5Y cell line no such differences were seen. We discuss the possibility that developing, less-differentiated neurons may be more readily infected by HIV-1 than fully differentiated neurons, and that cytokines such as TNF-α and IL-6, which are elevated in HIV-1-infected individuals, may enhance HIV production.
-
-
-
Human immunodeficiency virus type 1 (HIV-1) superinfection of a cell clone converting it from production of defective to infectious HIV-1 is mediated predominantly by CD4 regions other than the major binding site for HIV-1 glycoproteins
A cell clone, L-2, which produces non-infectious doughnut-shaped human immunodeficiency virus type 1 (HIV-1) particles, was permissive for HIV-1 superinfection, which resulted in the production of infectious particles. The superinfection showed slow kinetics compared with primary HIV-1 infection of M10 cells, the parent of the L-2 cells using several CD4-related reagents showed that the CD4 molecule was an essential component of the receptor for superinfection. Strong inhibitory effects were obtained using CD4 peptides such as CD4(68–130), which includes a portion homologous to the immunoglobulin third complementarity-determining region (CDR3), as well as recombinant soluble CD4. In contrast, a CD4(45-60) peptide, which includes most of the CDR2-related region, was not effective, although the Leu-3a monoclonal antibody (MAb), which recognizes a site near the CDR2-related region, did slightly, but significantly, delay the superinfection kinetics. Comparative flow cytometry of L-2 and M10 cells revealed that the cell surface of L-2 cells despite expressing HIV-1 env protein, reacted slightly with OKT4 or anti-CD4(68-130) MAb, but not with Leu-3a or OKT4A MAb. In contrast, no reaction was detected with any of these anti-CD4 MAbs on the surface of another HIV-1 superinfection-resistant cell clone, MOLT-#8IIIB-14, which expresses HIV-1 env proteins but does not produce infectious HIV-1 particles. These results strongly suggest that expression of the CD4 major receptor site for primary HIV-1 infection is preferentially decreased on the surface of L-2 cells, but that the OKT4 epitope and the nearby region corresponding to immunoglobulin CDR3 remain exposed on the cell surface. Consequently, the CD4 CDR3-related region could play a major role as the receptor for the superinfection reported here.
-
-
-
Functional mapping of the rev-responsive element of human immunodeficiency virus type 2 (HIV-2): influence of HIV-2 envelope-encoding sequences on HIV-1 gp120 expression in the presence or absence of Rev
More LessThe human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev stimulates expression of structural viral proteins via a target response element (RRE) located within gag-pol and env mRNAs. To analyse the HIV-2 Rev trans-activation effect on the expression of the envelope protein, we cloned a functionally active HIV-2 rev cDNA and showed that it contained four exons. Using transient expression assays, we mapped a 353 bp RRE fragment within the env gene of HIV-2 on which both HIV-1 and HIV-2 Rev could act. Interestingly, smaller fragments suppressed the use of additional splice sites within the env gene and caused envelope protein expression independent of Rev.
-
-
-
Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA
We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high M r DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.
-
-
-
Infection of terminally differentiated myotubes with Rous sarcoma virus: reduced synthesis of env and v-src proteins
More LessWe studied Rous sarcoma virus (RSV) protein synthesis in RSV-infected, terminally differentiated chicken myotubes (‘late-infected’ myotubes), in which no viral DNA integration takes place but all three viral mRNAs (38S, 28S and 21S) are transcribed normally. With the use of specific anti-RSV protein antisera, we found that only the viral gag and pol proteins were synthesized at levels similar to those synthesized in RSV-transformed fibroblasts; the synthesis of env and v-src proteins was significantly reduced in these infected myotubes. We concluded that the viral RNA transcribed from the unintegrated RSV DNA was functional but that genes at the 3′ end of the RSV genome were translated at a lower level. By contrast, when mononucleated replicating chicken myoblasts were infected with a mutant (tsNY68) carrying a temperature-sensitive v-src gene and maintained at the non-permissive temperature for this gene, they developed into myotubes with viral DNA integrated in their chromosomal DNA. These ‘early-infected’ myotubes expressed all four viral genes (gag, pol, env and v-src) at a level similar to that in infected fibroblasts. This result ruled out the possible presence of specific factor(s) in myotubes that preferentially inhibit the 3′ genes of RSV, and suggested other translational control(s) of viral gene expression in late-infected myotubes.
-
-
-
Differential inhibitory effects of TIBO derivatives on different strains of simian immunodeficiency virus
Recently, several classes of compounds have been shown to be extremely selective inhibitors of human immunodeficiency virus type 1 (HIV-1) replication in vitro. These include the tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-one and -thione (TIBO), 1-(2-hydroxyethoxymethyl)-6-(phenylthio)-thymine (HEPT), dipyridodiazepinone, pyridinone and bis(heteroaryl)piperazine derivatives. The hallmark of these new antiviral compounds is a specific interaction with reverse transcriptase (RT) of HIV-1. They are inactive against HIV-2 and any other viruses tested. Here we describe that, in addition to the HIV-1 strains, two simian immunodeficiency virus (SIV) strains from African green monkeys (SIVagm3 and SIVagmTYO-1) are also sensitive to the TIBO class of compounds. TIBO and HEPT derivatives block the replication of SIVagm in cell culture at micromolar concentrations. Kinetics of inhibition of SIVagm RT by TIBO are competitive with respect to the natural substrate (dGTP). Amino acid alignments and site-directed mutagenesis point to the critical role of amino acid residues Y181 and Y188 in the sensitivity of HIV-1 RT and SIVagm RT to inhibition by the TIBO derivatives. Antiviral efficacy studies with this range of compounds and using sensitive SIV strains are now feasible in monkeys.
-
-
-
Failure to isolate human T cell lymphotropic virus type I and to detect variant-specific genomic sequences by polymerase chain reaction in Melanesians with indeterminate Western immunoblot
The controversy over the endemicity of human T cell lymphotropic virus type I (HTLV-I) in Melanesia has been settled recently by the isolation of genetically distinct, highly divergent sequence variants of HTLV-I from unrelated inhabitants of Papua New Guinea and the Solomon Islands. Still at issue, however, is the significance of the high frequency of indeterminate HTLV-I Western blots (defined as reactivity to only gag-encoded proteins) among Melanesians. To investigate whether this indeterminate seroreactivity reflects specific reactivity to the Melanesian HTLV-I variants, 27 seroindeterminate Melanesians from Papua New Guinea and the Solomon Islands were studied for evidence of HTLV-I infection. Although antibodies against Melanesian variant-specific env gene products and variant-specific env gene sequences were detected by Western blot analysis and polymerase chain reaction, respectively, in all 11 HTLV-I Western blot-positive Melanesians, none of the 27 seroindeterminate Melanesians had such variant-specific antibodies or HTLV-I proviral sequences. In addition, attempts to isolate HTLV-I from seroindeterminate individuals were unsuccessful. These data indicate that HTLV-I infection is not the cause of the indeterminate Western blot reactivity seen in Melanesia.
-
-
-
The use of feline herpesvirus and baculovirus as vaccine vectors for the gag and env genes of feline leukaemia virus
More LessThe env and gag genes from feline leukaemia virus were expressed in a thymidine kinase-negative feline herpesvirus and a baculovirus. Cats were vaccinated with various combinations of these recombinant viruses and 100% protection against feline leukaemia virus challenge was achieved using an immunization schedule which utilized both env and gag products delivered at both a mucosal and systemic site.
-
-
-
Expression of feline leukaemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression vector system
More LessIn order to test components of feline leukaemia virus (FeLV) as subunit vaccines, we have constructed recombinant baculoviruses that express the FeLV envelope glycoprotein gp85 [Autographa californica nuclear polyhedrosis virus (AcNPV)-gp85] and the structural protein, gag (AcNPVgag). The gag protein is expressed and shed into the medium of infected cells as particles which have a buoyant density on sucrose gradients and appearance by electron microscopy similar to those of authentic FeLV virions. The gag precursor protein within the particles is not fully processed and appears to be a result of partial cleavage of the gag polypeptide. Insect cells that are coinfected with AcNPVgag and AcNPVgp85 shed particles that contain both the gag protein and the gp85 glycoprotein.
-
-
-
Identification of two novel human immunodeficiency virus type 1 splice acceptor sites in infected T cell lines
More LessThe regulatory and accessory proteins of human immunodeficiency virus type 1 (HIV-1) are produced from singly or multiply spliced mRNAs. We have used HIV-1-specific oligonucleotide primer pairs in a polymerase chain reaction procedure on RNA from lymphocyte cell lines infected with HIV-1228200. The amplified products were analysed by hybridization with splice junction-specific oligonucleotide probes to determine splice donor/splice acceptor combination utilization, subcloned into a plasmid vector and the nucleotide sequences were obtained. Two novel splice acceptor sites have been identified.
-
-
-
Geographical dependence of sequence variation in the E7 gene of human papillomavirus type 16
We have determined nucleotide sequences of the E7 open reading frame (ORF) of human papillomavirus type 16 (HPV-16) isolates obtained from 32 genital tumours and two HPV-16-transformed human keratinocyte cell lines. In comparison to the prototype HPV-16 isolated from a German cervical cancer biopsy, no sequence variations were noticed in either the two cell lines or the 10 biopsies that were obtained from German patients. In contrast only three of 22 (13.6%) of Tanzanian isolates showed the prototype sequence. In 18 of these biopsies two alterations (T to C and T to G) not affecting the amino acid sequence were found within the HPV-16 E7 ORF (nucleotide positions 789 and 795) but eight of these isolates contained an additional change (nucleotide position 647) coding for serine instead of asparagine (amino acid position 29). One tumour harbours HPV-16 DNA with a mutation (C to T) at nucleotide 790 changing the E7 amino acid sequence (arginine to cysteine) at position 76. Our findings suggest that clustering of E7 sequence variants may occur in different geographical regions of the world.
-
-
-
Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers
Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1^E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.
-
-
-
The small non-structural protein NS2 of the autonomous parvovirus minute virus of mice is required for virus growth in murine cells
More LessMutants of the autonomous parvovirus minute virus of mice (MVM) strains MVM(p) and MVM(i) that either fail to produce or produce a truncated NS2 protein, were deficient in the production of infectious virus and attained lower levels of viral DNA synthesis than wildtype virus following infection of a series of normal and transformed murine cell lines. Mutant virus growth and the levels of DNA replication were similar to those of wild-type virus in the rat, hamster and human lines tested. These results suggest that the requirement of NS2 for the growth of MVM is murine species-specific.
-
-
-
Herpes simplex virus type 1 infection of mouse astrocytes treated with basic fibroblast growth factor
More LessWe explored a possible role for the basic fibroblast growth factor (FGF) receptor during herpes simplex virus type 1 (HSV-1) infection of primary mouse astrocytes, glial cells of the central nervous system known to express FGF receptors. Plaque reduction experiments showed that treatment of astrocyte monolayers with human recombinant basic FGF failed to inhibit HSV-1 infectivity, although treatment with either heparin or poly-l-lysine reduced it by approximately 100%. Identical results were obtained when monolayers of human embryonic lung fibroblasts or African green monkey kidney cells were treated with FGF, heparin or poly-l-lysine prior to HSV-1 infection. We conclude that the basic FGF receptor is not involved in the uptake of HSV-1 during productive infection of astrocytes. Our findings suggest that this molecule is not the predominant cellular receptor for HSV-1 among vertebrate cells and plays no role in defining HSV-1 neurotropism in vivo.
-
-
-
Identification of the glycoprotein H gene of murine cytomegalovirus
More LessPartial sequencing of the HindIII C fragment of murine cytomegalovirus (MCMV) revealed an open reading frame of 2172 nucleotides in length encoding a 724 amino acid protein with a predicted M r of 80.4K. Analysis of the predicted amino acid sequence revealed homology with glycoprotein H (gH) from a number of other herpesviruses. MCMV gH showed strongest amino acid identity with human (H) CMV and human herpesvirus 6 gH, and less identity with the gH protein sequences of Epstein-Barr virus, varicella-zoster virus and herpes simplex virus type 1. The greatest identity between MCMV and HCMV gH occurs in the C-terminal region. The MCMV gH is characterized by having a 14 amino acid signal sequence, a 23 amino acid transmembrane region, a seven amino acid positively charged cytoplasmic anchor sequence and eight putative N-linked glycosylation sites. Comparison of MCMV gH with that of HCMV indicates that there are 12 conserved cysteine residues and three conserved potential N-linked glycosylation sites.
-
-
-
In vitro reconstitution of active influenza virus ribonucleoprotein complexes using viral proteins purified from infected cells
More LessA procedure to obtain RNA-free preparations containing the nucleoprotein (NP) and polymerase (P) proteins from influenza virus-infected cell extracts has been developed. The influenza virus P proteins present in these preparations copied small synthetic RNA molecules derived from plasmid sequences. In addition, RNA molecules encapsidated with the NP and P proteins were amplified and packaged into virus particles when transfected into influenza virus-infected cells. Thus, the preparations of NP and P proteins display features similar to those isolated from purified influenza virions, and represent an alternative for the preparation of active influenza virus P protein.
-
-
-
The role of CD8+T cells in the acute and chronic phases of Theiler’s murine encephalomyelitis virus-induced disease in mice
More LessThe technique of in vivo depletion with T cell subset-specific monoclonal antibodies was used to study the involvement of CD8+ T cells in protection/pathogenesis during the acute and chronic demyelinating phases of Theiler’s murine encephalomyelitis virus (TMEV)-induced disease. Mice rendered CD8-deficient prior to infection with TMEV were less efficient at clearing virus from the central nervous system compared to intact animals and also suffered demyelinating disease of earlier onset and increased severity. This indicates that CD8+ cells have a protective role in virus clearance at early times post-infection, and may also be involved in downregulating the severity of the chronic demyelinating disease. How CD8+ T cells function to produce these effects is discussed.
-
-
-
Baculovirus expression of pestivirus non-structural proteins
More LessBovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3′-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.
-
-
-
Rotavirus 993/83, isolated from calf faeces, closely resembles an avian rotavirus
More LessPolypeptides from purified virions of the calf rotavirus (RV) isolate 993/83 and those from the pigeon RV isolate PO-13 comigrated on SDS-polyacrylamide gels. Two polypeptides of 45K and 47K were detected at the position of VP6. Both proteins behaved like authentic VP6 protein with EDTA and heat treatment. RV 993/83 and PO-13 showed identical one-dimensional peptide maps for VP2, and the 45K and 47K proteins. More than 70% of sera from German cattle older than 1 year showed neutralizing serum antibodies to RV 993/83 and RV PO-13.
-
-
-
Foreign gene expression by a baculovirus vector with an expanded host range
A nuclear polyhedrosis virus (NPV) (Baculoviridae)-based gene expression system was improved by DNA recombination. The BmN cell line established from Bombyx mori and the Sf21 cell line established from Spodoptera frugiperda are non-permissive for Autographa californica multicapsid NPV (AcMNPV) and B. mori NPV (BmNPV) replication, respectively. After cotransfection of AcMNPV DNA and BamHI-digested BmNPV DNA into Sf21 cells, progeny viruses were isolated by plaque purification on BmN cell monolayers and the host specificity of one viral isolate was analysed. The virus had a wider host range, and replicated and produced polyhedra in Sf21 cells, BmN cells and larvae of the silkworm, B. mori. DNA restriction endonuclease analysis showed that the isolate was a hybrid of AcMNPV and BmNPV. Using the AcMNPV transfer vector pAcYM1 a portion of the polyhedrin gene of the hybrid virus was replaced with the coding region of the firefly luciferase gene, producing a recombinant virus. The latter expressed firefly luciferase in both cell lines and in silkworm larvae under the control of the polyhedrin promoter.
-
-
-
Characterization of two variants of Panolis flammea multiple nucleocapsid nuclear polyhedrosis virus
More LessThe baculovirus Panolis flammea multiple nucleocapsid nuclear polyhedrosis virus (PfMNPV) was originally isolated from a natural virus epizootic and shown to consist of a mixture of variants. Two subclasses of variants (PfMNPV A and B) were identified by Southern blot hybridization, their polyhedrin genes being located on different restriction fragments. The proportion of the A and B variants changed according to the larval host in which the virus was propagated; PfMNPV(B) predominated in P. flammea but PfMNPV(A) was predominant in Mamestra brassicae. Bioassays of the two pure virus variants in M. brassicae larvae have shown the LD50 values to be 4610 polyhedron inclusion bodies (pibs) for PfMNPV(A) and 5937 pibs for PfMNPV(B). Genomic DNA from the two variants was compared using restriction endonuclease analysis, and dot blot and Southern blot hybridization. Reciprocal quantitative dot blot hybridization analysis in 50% formamide showed PfMNPV(A) and PfMNPV(B) to be only distantly related to Autographa californica MNPV (less than 1%) and more closely related to M. brassicae MNPV (21 to 26%). The two PfMNPV variants exhibited a very high degree of identity to each other (nearly 100%) and therefore are very closely related. This was confirmed by physical mapping of the virus genomes. The nucleotide sequence of the polyhedrin gene of PfMNPV(B) was determined and compared with the published DNA sequences of other polyhedrin genes.
-
- Plant
-
-
-
Complete sequence of maize stripe virus RNA4 and mapping of its subgenomic RNAs
More LessThe complete nucleotide sequence of maize stripe virus RNA4 was determined and found to consist of 2227 nucleotides containing two significant open reading frames. One, in the 5′ end of the viral RNA, encodes the major non-capsid protein of M r 19815. The other is located in the 5′ end of the viral complementary RNA and could encode a protein of M r 31900. This protein has not been identified previously and has been designated NS4, a non-structural protein. RNA-RNA hybridization detected subgenomic RNAs encoding these proteins, a characteristic of RNA possessing an ambisense gene organization.
-
-
-
-
Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo
More LessThe synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M r 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.
-
-
-
Early embryo invasion as a determinant in pea of the seed transmission of pea seed-borne mosaic virus
More LessSeed transmission of an isolate of pea seed-borne mosaic virus (PSbMV) in several pea genotypes has been studied. Cross-pollination experiments showed that pollen transmission of PSbMV did not occur and accordingly, virus was not detected in pollen grains by ELISA or electron microscopy. Comparative studies between two pea cultivars, one with a high incidence of seed transmission and one with none, showed that PSbMV infected the floral tissues (sepals, petals, anther and carpel) of both cultivars, but was not detected in ovules prior to fertilization. Virus was detected equally well in seed coats of the progeny in both cultivars. Analysis of virus incidence and concentration in pea seeds of different developmental stages demonstrated that in the cultivar with a high incidence of seed transmission, PSbMV directly invaded immature embryos, multiplied in the embryonic tissues and persisted during seed maturation. In contrast, the cultivar without seed transmission did not show invasion of immature embryos by the virus; there was no evidence for virus multiplication or persistence during embryo development and seed maturation. Hence seed transmission of PSbMV resulted from direct invasion of immature pea embryos by the virus and the block to seed transmission in the nonpermissive cultivar probably occurred at this step.
-
-
-
Partial nucleotide sequence of poplar mosaic virus RNA confirms its classification as a carlavirus
More LessThe nucleotide sequence of the 3′-proximal 1328 nucleotides of poplar mosaic virus (PMV) was determined and shown to contain two large open reading frames (ORFs). The ORF nearer to the 3′ terminus of the RNA is capable of encoding a polypeptide of 14K with a ‘zinc-finger’ motif, and is homologous to sequences in corresponding positions in five other carlaviruses. The other ORF encodes a protein of 36K which includes two sequences of amino acids identified in tryptic digests as virion capsid protein, and has amino acid sequences in common with both carlaviruses and potexviruses.
-
Volumes and issues
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)