- Volume 78, Issue 4, 1997
Volume 78, Issue 4, 1997
- Articles
-
-
-
Molecular characterization of ovine pestiviruses
More LessForty-two ovine pestivirus isolates, collected over a period of 18 years, were compared by phylogenetic analysis. The viruses were mostly field isolates from Britain; two others originated from Sweden and two from New Zealand. RT-PCR products were obtained from two genomic regions, one within the 5′- noncoding (5′-NC) region, and the other encompassing parts of the p20 (Npro) and C coding regions. Direct sequencing of the 5′-NC PCR products, followed by computer-assisted phylogenetic analysis, divided the ovine pestiviruses into three main genotypes. The results demonstrated that sheep may naturally be infected not only with border disease virus (BDV), but also with bovine viral diarrhoea virus (BVDV) types I and II. The BDV isolates segregated into two principal subtypes represented by the Moredun strain from Scotland and the 137/4 strain from England. The BVDV-I group was composed of three clusters, two of them represented by BVDV reference strains NADL and Osloss, respectively, and the third by ovine isolates D1120/1 and D1432/P. The grouping of ovine pestiviruses, based on comparative nucleotide sequence analysis of the 5′-NC region, was confirmed by comparative analysis of the p20 (Npro) and C coding regions, performed both at the nucleotide and at the amino acid level. The presence of three genotypes in sheep, including BVDV-I and BVDV-II, indicates the inadequacy of the current host- species-based nomenclature and classification of pestiviruses.
-
-
-
The entire nucleotide sequences of two GB virus C/hepatitis G virus isolates of distinct genotypes from Japan
Recently, putative viral agents responsible for human non-Ato E hepatitis have been independently reported by two groups of investigators and designated GB virus C (GBV-C) and hepatitis G virus (HGV), respectively. The entire nucleotide sequences were determined for two viral genomes isolated from Japanese blood donors with GBV-C RNA. One of them (GT230) had a total genomic length of 9390 nucleotides (nt) with 5′ and 3′ untranslated regions of 551 and 313 nt, while the other (GT110) had genomic lengths of 9395, 281 and 315 nt, respectively. They both had a single long open reading frame, encoding 2842 amino acids (aa) in GT230 and 2933 aa in GT110. Surprisingly, they both lacked a clearly identifiable core gene, and possessed the E1/E2 gene with only four potential N-linked glycosylation sites. Pairwise comparison and phylogenetic analysis of the entire sequence indicated that the prototype GBV-C and two HGV isolates reported, as well as GT230 and GT110, are the same virus possibly of different genotypes. The five GBV-C/HGV isolates were variable up to 13 8% in the genomic nucleotide sequence, and contained deletions and insertions within the 5 -terminal 518-593 nt, which resulted in four different sizes of predicted polyproteins encoded by genomes of individual isolates. By contrast, the 3′ untranslated region was well conserved. The high degree of sequence conservation within this region would favour it as a target for sensitive detection of GBV- C/HGV RNA.
-
-
-
No evidence for quasispecies populations during persistence of the coronavirus mouse hepatitis virus JHM: sequence conservation within the surface glycoprotein gene S in Lewis rats
More LessThe surface glycoprotein S (spike) of coronaviruses is believed to be an important determinant of virulence and displays extensive genetic polymorphism in cell culture isolates. This led us to consider whether the observed heterogeneity is reflected by a quasispecies distribution of mutated RNA molecules within the infected organ. Corona- virus infection of rodents is a useful model system for investigating the pathogenesis of virus-induced central nervous system (CNS) disease. Here, we investigated whether genetic changes in the S gene occurred during virus persistence in vivo. We analysed the variability of S gene sequences directly from the brain tissue of Lewis rats infected with the coronavirus mouse hepatitis virus (MHV) variant JHM-Pi using RT-PCR amplification methods. The S gene sequence displayed a remarkable genetic stability in vivo. No evidence for a quasispecies distribution was found by sequence analysis of amplified S gene fragments derived from the CNS of Lewis rats. Furthermore, the S gene also remained conserved under the selection pressure of a neutralizing antibody. Only a few mutations predicted to result in amino acid changes were detected in single clones. The changes were not represented in the consensus sequence. These results indicate that to retain functional proteins under the constraints of a persistent infection in vivo, conservation of sequence can be more important than heterogeneity.
-
-
-
Induction of protective immunity against influenza virus in a macaque model: comparison of conventional and iscom vaccines
Cynomolgus macaque monkeys (Macaca fascicu- laris) were immunized twice intramuscularly, either with a conventional non-adjuvanted subunit vaccine or with a candidate immune-stimulating complex (iscom) vaccine, each containing 10 µg envelope glycoprotein of a recent human influenza A(H3N2) virus (A/Netherlands/18/94). In contrast to the macaques vaccinated with the classical subunit vaccine, those immunized with the iscom vaccine developed high titres of specific IgM, IgA and IgG serum antibodies, as well as high titres of haemag- glutination-inhibiting and virus-neutralizing serum antibodies. Also, specific proliferative T cell responses were only found in the iscom-vaccinated monkeys and their levels were similar to those found in monkeys experimentally infected with the homologous virus. Upon intratracheal challenge with the homologous virus, the iscom-vaccinated monkeys were completely protected from detectable virus replication in lungs, pharynx and nose, whereas those vaccinated with the classical subunit vaccines were not, or were only partially protected. The kinetics of specific serum antibody development in the iscom-vaccinated monkeys after challenge were quite similar to those of monkeys after secondary infection with the same virus. In contrast, the post-challenge kinetics of serum antibody development in the monkeys vaccinated with the classical subunit vaccines resembled those of naive monkeys, confirming that these vaccines only provided limited protection in such animals.
-
-
-
Different modes of inhibition by adamantane amine derivatives and natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein
More LessThe influenza virus M2 protein, target of the antiviral drugs amantadine and rimantadine, forms a proton channel which functions during virus uncoating and maturation by modifying the pH in virions as well as in trans-Golgi vesicles. We studied the influence of different ionic gradients on the inhibition of the proton translocation activity of isolated, baculo- virus-expressed M2 protein reconstituted into liposomes. Two distinct patterns of inhibition were observed. A group of amphiphilic amines including amantadine, cyclooctylamine and rimantadine inhibited M2 effectively in the presence of physiological Na concentrations. The 10-fold greater activity of rimantadine over amantadine and the 100-fold stronger effect of cyclooctylamine compared to cyclopentylamine matched the relative activities in influenza virus-infected cells. A com-pletely different inhibitory pattern emerged for the polyamines spermine, spermidine and putrescine. Polyamines have recently been identified as the ‘intrinsic’ rectifiers of a class of potassium channels and shown to interact with acidic amino acid residues lining and flanking the channel pore. In the presence of a physiological Na /K gradient their minimal inhibitory concentrations for influenza virus M2 protein were 100, 400 and 500 µM, polyamine levels reported to exist in oocytes. In conditions depleted for Na , polyamines inhibited M2 at concentrations two to three orders of magnitude lower. The data suggest that influenza virus M2 protein possesses a binding site for polyamines, distinct from the amantadine binding site, which is normally masked by Na and which could be targeted by selective antiviral inhibitors.
-
-
-
Paramyxovirus-induced syncytium cell formation is suppressed by a dominant negative fusion regulatory protein-1 (FRP-1)/CD98 mutated construct: an important role of FRP-1 in virus-induced cell fusion
Syncytium formation and subsequent generalized cell fusion have been reported as potentially important mechanisms of virus-induced cytotoxic effects. We tried to clarify the roles of fusion regulatory factor-1 (FRP-1) in virus-induced cell fusion. Two mutated human FRP-1/CD98 proteins [FRP-1/ HN, in which the cytoplasmic domain was replaced with the cytoplasmic domain of human parainfluenza virus type 2 (HPIV-2) haemagglutinin- neuraminidase (HN), and FRP-1/330 (serine), in which a cysteine at amino acid 330 was mutated to serine], when expressed stably in L929 cells, were lacking in cell-fusion-enhancing activity stimulated by anti-FRP-1 antibodies. Anti-FRP-1 antibodies enhanced Newcastle disease virus (NDV)-mediated polykaryocyte formation in parent HeLa cells, while anti-FRP-1 antibodies showed no/low effect on polykaryocyte formation in NDV-infected HeLa cells constitutively expressing FRP-1/HN (HeLa-FRP-1/ HN cells), indicating that the FRP-1/HN molecule is capable of acting as a dominant negative inhibitor. Furthermore, when HeLa-FRP-1/HN cells were infected with various rubulaviruses (HPIV-2, mumps virus, simian viruses 5 and 41), virus-induced cell fusion was also suppressed, although virus replication was not inhibited in these cells, showing that FRP-1 molecules are required for virus-induced cell fusion. Therefore, FRP-1 is considered to be related to the pathogenesis of paramyxoviruses.
-
-
-
Secretion of a murine retroviral Env associated with resistance to infection
More LessFv4 is an endogenous defective murine leukaemia virus (MuLV) which expresses high levels of an envelope protein (Env) closely related to that of the ecotropic class of MuLVs. Mice bearing the natural Fv4 gene or a transgenic version are resistant to infection by ecotropic MuLVs. Fv4 mice secrete the surface peptide (SU) of the Fv4 Env in their serum and this secreted Env can block infection of NIH3T3 cells. To study the secretion of Fv4, we metabolically labelled cells expressing Fv4 Env or Env from infectious MuLVs and followed synthesis, glycosyl- ation, proteolytic processing and secretion of Env species. We found no difference in the kinetics of synthesis or processing of Fv4 Env compared to the envelopes of infectious MuLVs, but Fv4 Env associated more weakly with its transmembrane anchor and was shed from the surface of cells.
-
-
-
Infectivity of lion and puma lentiviruses for domestic cats
More LessInfection of domestic cats with feline immunodeficiency virus (FIV) causes progressive immunological deterioration similar to that caused by human immunodeficiency virus (HIV). Lentiviruses related to but phylogenetically distinct from FIV have been detected in several non-domestic feline species. Serological cross-reactivity of these viruses raises the question as to whether inter-species transmission may occur. To address this issue, we asked whether lion lentivirus (FIV-Ple) or two strains of puma lentivirus (FIV-Pco) could replicate or cause disease in domestic cats. We found that domestic cats inoculated with FIV-Ple developed persistent cell-associated viraemia, transient cell-free viraemia and antiviral antibody. Clinical disease was not detected throughout a 6 month observation period. Two of four cats inoculated with FIV-Pco developed cell-associated viraemia, seroconverted and exhibited transient lymphadenopathy. No changes in white blood cell parameters or other haematological abnormalities were detected in any of the infected cats. Virus-specific RNA was detected in cocultivated lymphocytes of all infected cats by RT- PCR. These findings reveal that non-domestic cat lentiviruses are infectious for domestic cats and can establish persistent infection in the absence of disease.
-
-
-
Productive replication of caprine arthritis-encephalitis virus is associated with induction of apoptosis
More LessCaprine arthritis-encephalitis virus (CAEV), an ungulate lentivirus, causes a natural infection in goats. The present report demonstrates that in vitro, CAEV infection is associated with apoptosis, characterized by morphological changes such as condensation of chromatin and the appearance of apoptotic bodies. The presence of DNA fragments was documented by the appearance of a DNA ‘ladder’ in agarose gel electrophoresis, as well as by in situ end-labelling of DNA ends. In addition, flow cytometric analyses revealed the presence of a hypodiploid peak that gradually appeared as virus infection progressed.
-
-
-
Insertions, duplications and substitutions in restricted gp90 regions of equine infectious anaemia virus during febrile episodes in an experimentally infected horse
We have studied a horse which exhibited typical clinical signs of disease when experimentally infected with a non-adapted virulent strain of equine infectious anaemia virus (EIAV), designated V70. Five viruses (F1V, F2V, F3V, F4V and F5V) were recovered during periodic febrile episodes. Crossneutralization tests revealed that all of these variants and the parental V70 were antigenically distinct. Sequencing of their full-length env gp90 genes and gp45 5′ sequences revealed novel mutations at a limited number of nucleotide positions, consisting of insertions and duplications in the gp90 principal neutralizing domain (PND) in F1V, F3V and F5V. Parts or all of small units (6, 9 and 12 nucleotides) located just before the insertion site were used for the duplications. Furthermore, amino acid substitutions in the env PND and hypervariable region were also observed in all five viruses. These mutations may contribute to the generation of serial variants. Consequently, the full-length gp90 sequences showed close relationships between V70, F2V and F4V, and between F1V, F3V and F5V. In addition to the two domains (PND and hypervariable region), a comparison of these viruses with the reported env gp90 sequences revealed four additional variable domains, although these four domains were highly conserved among the five variants.
-
-
-
CD8 cytotoxic T lymphocytes of a cynomolgus macaque infected with simian immunodeficiency virus (SIV) mac32H-J5 recognize a nine amino acid epitope in SIV Gag p26
A detailed analysis of simian immunodeficiency virus (SIV)-specific cytotoxic T lymphocyte (CTL) responses and the identification of the proteins and epitopes they target may improve the design of immunotherapeutic interventions and provide insights into AIDS pathogenesis. Here, we identified a new CTL epitope in the SIV Gag protein, recognized by CD8 and MHC class I-restricted CTL clones from a long-term asymptomatic cynomolgus macaque (Macaca fascicularis) infected with SIVmac32H-J5. Using overlapping synthetic peptides, the optimal minimal epitope was characterized as a nine amino acid peptide representing amino acids 242–250 of p26 (SVDEQIQWM). CTL recognition was shown to be abolished by amino acid substitutions observed within homologous human immunodeficiency virus (HIV)-1 and HIV-2 sequences.
-
-
-
The non-immunosuppressive cyclosporin A analogue SDZ NIM 811 inhibits cyclophilin A incorporation into virions and virus replication in human immunodeficiency virus type 1-infected primary and growth-arrested T cells
More LessSDZ NIM 811 is a cyclosporin A (CsA) analogue that is completely devoid of immunosuppressive capacity but exhibits potent and selective antihuman immunodeficiency virus type 1 (HIV-1) activity. Binding to cyclophilin A, the intracellular receptor for cyclosporins, is a prerequisite for HIV-1 inhibition by cyclosporins. Cyclophilin A was demonstrated to bind to HIV-1 p24 gag and this cyclophilin-Gag interaction leads to the incorporation of cyclophilin Ainto HIV-1 virions. SDZ NIM 811 inhibitsthis protein interaction, and this is likely to be the molecular basis for its antiviral activity. Here, we show that in activated primary T cells SDZ NIM 811 interferes with two stages of the virus replication cycle: (i) translocation of pre-integration complexes into the nucleus and (ii) production of infectious virus particles. SDZ NIM 811 not only inhibits translocation of HIV-1 pre-integration complexes in primary T cells, but also in a growth-arrested T cell line. In vivo, most T lymphocytes are quiescent, but serve nevertheless as a major and inducible HIV-1 reservoir in infected individuals. Significant amounts of cyclophilin A were found to be associated with virus particles propagated in primary T cells. SDZ NIM 811 caused a strong reduction in the amount of incorporated cyclophilin A, thereby reducing infectivity. Thus, cyclophilin A seems to be necessary for HIV-1 replication in primary T cells.
-
-
-
Sequence variation of the human immunodeficiency virus primer-binding site suggests the use of an alternative tRNA(Lys) molecule in reverse transcription
More LessRetroviruses use a cellular tRNA molecule as primer for reverse transcription. The complementarity between the 3′ end of this tRNA and a sequence near the 5′ end of the viral RNA, the primer-binding site (PBS), allows the primer to anneal onto the viral RNA. During reverse transcription 18 nucleotides of the tRNA primer are copied into the viral cDNA, thereby regenerating the PBS sequence of the progeny. Thus, the PBS sequence reveals which primer was used. Human immunodeficiency viruses are known to replicate efficiently with tRNALys3 as primer. Examination of the PBS sequence in natural and laboratory isolates indicates that a variant tRNALys is occasionally used as primer. This variant, for which the murine genomic sequence was described previously, was termed tRNALys5 and differs from tRNALys3 at five nucleotide positions. These results suggest that HIV uses both tRNALys3 and tRNALys5 molecules as primer, causing a switch of the PBS sequence.
-
-
-
Human immunodeficiency virus type 1 Vpu modifies viral cytopathic effect through augmented virus release
We characterized two human immunodeficiency virus type 1 (HIV-1) strains, HIVMCK and HIV213, which have different cytopathic effects in infected cells. HIV213 was highly cytopathic, whereas HIVMCK was not. Biological analyses of chimeric viruses from the cloned infectious DNAs of HIVMCK and HIV213 showed that the Vpu region was responsible for the differing cytopathicity of these viruses. Although HIVMCK expressed Vpu protein, HIV213 did not because of a mutation at the start codon for Vpu. The amounts of envelope glycoprotein and virus particles associated with the cell surface were significantly increased on cells infected with Vpu-deficient viruses compared with Vpu-positive viruses. These data suggest that the highly cytopathic effects of HIV213 (Vpu-deficient) are due to an accumulation of envelope glycoprotein at the infected-cell surface, which would be caused by the retention of progeny virions in the absence of Vpu-facilitated virion release.
-
-
-
Infection with AIDS-related herpesviruses in human immunodeficiency virus-negative infants and endemic childhood Kaposi’s sarcoma in Africa
More LessNovel herpesviruses have been described recently. These include human herpesviruses 6, 7 and 8 (HHV-6, -7, -8). HHV-6 has at least two strain groups, variants A and B. The B strains are predominant in the West and can account for over 97% of infections in infants. In contrast, the A strains are rare and the few well-characterized isolates have been from adult African AIDS patients. It is not clear whether the HHV-6 variant A strains are AIDS-related and/or whether they can also be acquired as childhood infections and may reactivate later during adulthood. What contribution geographical variation plays has yet to be assessed. HHV-8 has been associated with AIDS-related epidemic Kaposi’s sarcoma (KS), but has also been identified in endemic KS. In regions of Africa where KS is endemic, the onset of AIDS has led to increased prevalence of KS. In this report, we examine in Zambia, an AIDS epidemic and KS endemic region, infection with these novel herpesviruses during infancy. In blood samples from human immunodeficiency virus-negative infants with first febrile episode, both semi-quantitative PCR and sequence analyses were used to identify HHV-8 in 8% and HHV-6 in 30%, with 44% of these variant A; in childhood endemic KS biopsies HHV-8 was detected in 100% and HHV-6 in none. The high viral-DNA loads in the infant blood samples were consistent with viraemia. This is the first demonstration that HHV-6 variant A and HHV-8 may be acquired as common childhood infections.
-
-
-
A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit
More LessThe herpes simplex virus type 1 (HSV) singlestranded DNA-binding protein (SSB, ICP8) stimulates the viral DNA polymerase (Pol) on an oligonucleotide-primed single-stranded DNA template. This stimulation is non-specific since other SSBs also increase Pol activity. However, only ICP8 was stimulatory when Pol activity was dependent upon priming by the viral helicase-primase complex. ICP8 also specifically stimulated the primer synthesis and ATPase activities of the helicase-primase. The mechanism of stimulation was different from that of Pol; helicase-primase stimulation required much lower amounts of ICP8 than the amount that saturates the DNA and optimally stimulates Pol. Furthermore, ICP8 did not act by removing secondary structure as stimulation also occurred on homopolymer templates. While the UL8 component of the helicase-primase is not required for enzymatic activities by a subassembly of the UL5 and UL52 proteins, only the holoenzyme (UL5/8/52) was stimulated by ICP8. These results identify a unique, functional interaction between the ICP8 SSB and the helicase-primase complex, mediated by the UL8 subunit.
-
-
-
Retention of herpes simplex virus DNA sequences in the nuclei of mouse footpad keratinocytes after recovery from primary infection
More LessPersistence of herpes simplex virus (HSV) DNA in mouse footpad keratinocytes was studied by nonisotopic in situ hybridization. HSV DNA was retained in keratinocyte nuclei for more than 2 weeks after disappearance of infectious virus and viral antigens. The anatomical location of viral DNA became more superficial with increasing time post-infection, reflecting the migration of cells from the basal layer of the epidermis towards the stratum granulosum. Latency-associated transcripts (LATs) were not detected in footpad cells at any of the times studied. In contrast, LATs were detected readily in the nuclei of lumbar ganglionic neurons innervating HSV DNA positive footpads. It was concluded that, after termination of productive infection in the skin, HSV DNA persists transiently in keratinocyte nuclei, in the absence of abundant latency-associated transcription. An implication of these data is that detection of HSV DNA in the skin may reflect recent, but not necessarily current, cutaneous virus replication.
-
-
-
Complete DNA sequence of canine adenovirus type 1
More LessThe complete DNA sequence of a field strain of canine adenovirus type 1 was determined by sequencing random fragments of viral DNA cloned into pBluescript. The virus has a genome of 30536 bp flanked by two identical 161 bp inverted terminal repeats. Thirty ORFs have been identified, based on genomic location or sequence identity with published adenoviruses. These are arranged into similar discrete regions found in the human adenoviruses. ORFs in the late region show greatest identity with published human adenovirus sequences, whereas the E3 and E4 ORFs show little or none.
-
-
-
A cis-acting element 7 bp upstream of the ESF-1-binding motif is involved in E1A 13S autoregulation of the adenovirus 12 TS2 promoter
More LessTranscription of the E1A gene of the highly oncogenic adenovirus 12 (Ad12) initiates at two start sites (TS1 and TS2). We have previously shown that the E2F and ATF motifs distal of TS1 co-operatively participate in E1A autostimulation from the TS1 promoter region. Here we report the identification of a second E2F-like target region (E2FII) immediately upstream of the E1A-stimulating factor 1 binding site (ESF-1), important for 13S-mediated autoactivation from TS2. Reporter constructs lacking distinct TS2 c/s-acting elements were analysed for their levels of CAT expression in the absence and presence of the E1A 13S protein in transient expression assays. In the absence of 13S, full promoter activity was observed only for a construct containing all elements (the E2F-like motif, an E-Box and the TATA element). Promoter activation increased significantly in Ad12 E1A-co-transfected cells. Induction by the 13S protein was also detected for the construct containing a non-functional ESF-1 sequence. Our results indicate that the E2F-like motif is responsible for activation mediated by the 13S protein from TS2, while ESF-1-or TATA-binding protein activity were not involved. Additionally, the TATA sequence appeared to be dispensable for transactivation. Gel-shift experiments using the E2F-like promoter element as a probe indicated the binding of an E2F-5 or E2F-5-like transcription factor to this region. We conclude that transcription through the TS1 as well astheTS2 promoter region is stimulated by the Ad12 13S protein. Moreover, transfection of the construct including bothTS1 and TS2 indicates an E2F-site-mediated synergism between both regions with respect to E1A-induced transactivation.
-
-
-
Role of p53 mutation in polyomavirus-induced tumorigenesis
Small DNA tumour viruses, such as simian virus 40 (SV40), papilloma viruses and adenoviruses, encode proteins that form complexes with and inactivate the p53 and retinoblastoma (RB) proteins. This convergent evolution reflectsthe common need of these viruses to inactivate these two important regulators of cell cycle progression and cell survival. Polyomavirus, a close relative of SV40, is different. Its large T protein complexes only with RB, not with p53. We have examined whether this is compensated by the frequent appearance of p53 mutations in polyomavirus-induced tumours. We tested the p53 status of 15 polyomavirus-induced sarcomas. Two sarcomas were p53-negative while six carried mutant p53. Another six sarcomas expressed low levels of wild-type p53. One tumour expressed high levels of wild-type p53 protein as shown by DNA sequencing and immunofluorescence staining. MDM2 amplification was not detected in any of the tumours, but Northern blotting showed that MDM2 was overexpressed in at least two tumours that expressed wild-type p53 and in one tumour that expressed both wild-type and mutant p53. Treatment with the DNA-damaging agent mitomycin C caused p53 protein accumulation followed by induction of MDM2 and WAF1/p21 mRNA in four of the tumours expressing wild-type p53, indicating that p53-mediated transcriptional activation was unaltered in these tumours. However, p53-mediated transactivation of WAF1/p21 was impaired in the wild-type p53-expressing tumours that expressed elevated levels of MDM2. These results demonstrate that p53 mutation and inactivation are frequently but not invariably involved in polyomavirus-induced tumorigenesis.
-
-
-
Analysis of the interaction between human papillomavirus type 16 E7 and the TATA-binding protein, TBP
More LessThe E7 protein encoded by human papillomavirus type 16 shows transforming and immortalizing activities which are mediated, in part, through the interaction of the viral oncoprotein with the pRB protein family. This interaction is not solely responsible for E7 function, however, and other properties of E7, such as the interaction with basal transcription factors such as TBP, are likely to be of importance. We show here that three regions of the viral protein contribute to the interaction between E7 and TBP; the pRB-binding domain, the casein kinase II phosphorylation region and the C-terminal dimerization domain. Mutations within each region reduced the interaction of E7 with TBP in vitro, and simultaneous alterations within each of these regions completely abrogated binding. Unlike the pRB interaction, the association of E7 with TBP was enhanced following phosphorylation of E7 by casein kinase II, demonstrating a functional significance for phosphorylation of the viral protein.
-
-
-
Characterization of the DNA-binding activity of the E1 and E2 proteins and the E1/E2 complex of human papillomavirus type 33
More LessThe E1 and E2 proteins of papillomaviruses are essential for the initiation of viral DNA replication. We have purified the E2 protein of human papillomavirus type 33 (HPV-33) by immunoaffinity chromatography. The purified E2 protein bound with high affinity to all four consensus binding sites of HPV-33 (Kd ≌ 2×10 10 M). A putative E2 binding site differing at one position in the second stem of the palindrome was not bound by E2. The E1 protein of HPV-33 purified by affinity chromatography using glutathione S-transferase as tag displayed specific DNA-binding activity in footprint analyses protecting HPV-33 nucleotides 7896 to 7909/1 to 18 from DNasel digestion. Hypersensitive sites at position 6 on the sense and position 1 on the antisense strand were observed in the middle of the protected region. An E1/E2 complex protected the E1 binding site and E2 binding sites from DNasel digestion suggesting that both proteins retain DNA-binding activity in the complex.
-
-
-
Serological and T-helper cell responses to human papillomavirus type 16 L1 in women with cervical dysplasia or cervical carcinoma and in healthy controls
More LessIn a cross-sectional study we have investigated serological and T-helper (Th) cell responses to human papillomavirus type 16 (HPV-16) L1 in women with HPV-16 related diseases and related them to cervical histology and HPV DNA status. Using a virus-like particle (VLP) based ELISA to detect antibodies to the HPV-16 L1 capsid protein, 45% (33/73) of women with cervical dysplasia, 40% (2/5) of women with cervical cancer, 36% (4/11) of healthy adult female controls and 6% (2/35) of healthy children were found to be seropositive. Amongst women with cervical dysplasia, the highest levels of seropositivity were found in those who were HPV-16 DNA positive (60%, 15/25) or positive for any of the ‘high-risk’ HPV types, 16/18/33 (58%, 18/31), when compared with those with HPVtype ‘X’ (25%, 5/20) or with healthy children (6%, 2/35; P < 0·05 for all comparisons). There was a trend for women with cervical dysplasia to show an increased level of seropositivity with increasing grade of lesion. There was no direct correlation found between seropositivityand Th cell responses in all groups studied. However, a combined analysis of each individual’s Th and B cell responses suggests that a Th1 pattern of response is predominant amongst healthy adult controls (80% of responders) but reduced in women with cervical dysplasia (55% of responders). A trend towards a decrease in Th1 type responses was also noted with increasing grade of dysplastic lesion. These findings provide further evidence for the importance of the Th response in the control of genital HPV infections.
-
-
-
Tropic determinant for canine parvovirus and feline panleukopenia virus functions through the capsid protein VP2
More LessCanine parvovirus (CPV) can productively infect canine and feline cell lines whereas feline panleukopenia virus (FPV) is restricted to the latter. The major determinants of tropism are two amino acids in the sequence shared by the capsid proteins, VP1 and VP2. We have shown that a rodent parvovirus-derived transducing genome, containing the luciferase reporter, can be packaged by VP1 and VP2 from separate helper sources. Canine A72 cells and feline CFK cells were transduced with recombinant virions generated using VP1 and VP2 combinations from CPV and FPV. Both VP1 and VP2 were necessary for production of transducing virions. Efficient transduction of A72 cells required VP2 of CPV. Therefore, the capsid determinants of tropism for CPV and FPV are in VP2, although a source of VP1 is also necessary to produce infectious particles. The results extend similar observations on the tropic determinants of different strains of minute virus of mice.
-
-
-
Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3
More LessStrains of peanut stunt cucumovirus (PSV) were classified into two distinct subgroups, I and II, based on Western and Northern blot analyses using antisera and cloned cDNA probes to strains PSV-ER and PSV-W. These results were corroborated by nucleotide sequence analyses of full-length cDNA clones of RNA3 from representative strains of the two subgroups. Whereas the percentage nucleotide sequence identity between PSV-ER (or PSV-J) and PSV-W RNA3s was determined to be 80%, the corresponding value between strains ER and J was 91%, confirming that strains ER and J belong to the same subgroup (subgroup I) whereas strain W belongs to a separate subgroup (subgroup II). PSV-W and PSV-ER RNA3s are 2173 and 2188 nucleotides long, respectively. Each is dicistronic, encoding a putative movement protein (3a protein) and a coat protein (CP). The intercistronic and 5′ untranslated region (UTR) sequences of PSV strains, unlike those of cucumber mosaic cucumovirus (CMV) strains, are highly conserved and thus not useful for distinguishing the two subgroups. However, the 3′ UTR sequences of PSV strains, like those of CMV strains, can discriminate between the two subgroups since strains within the same subgroup are 95% identical in their 3′ UTRs whereas those in different subgroups are only 74–78% identical. PSV-W and PSV-ER RNA4s were determined to be 994 and 1006 nucleotides long, respectively. PSV 3a and CP genes have higher percentage nucleotide sequence identities to those of tomato aspermy cucumovirus than to those of CMV.
-
-
-
Formation of multimers of cucumber mosaic virus satellite RNA
More LessDouble-stranded RNA multimers of cucumber mosaic virus (CMV) satellite RNA were detected in CMV-infected plants. RT-PCR showed that plussense and minus-sense monomers and plus-sense multimers of satellite RNA were present. Multimeric minus-sense RNA was not present except in the form of multimeric dsRNA. Sequence analysis of 52 cloned junction regions in head-to-tail repeats of unit-length satellite RNA indicated that about 35% of the junction sequences were precise fusions of monomer units, 56% lacked sequence of the 5′ component, and 10% lacked sequence of both 3′ and 5′ components. No junction contained additional nucleotides. Deletions at the junction regions may have accumulated during CMV multiplication in inoculated plants. These data suggest that replicase is not released from the template during synthesis of multimeric molecules of satellite RNA.
-
-
-
Analysis of the infectivity of monomeric clones of pepper huasteco virus
The infectivity of several monomeric clones of pepper huasteco virus was investigated. All clones were infectious when inoculated excised from the plasmid DNA. However, only certain clones were infectious when inoculated in the non-excised form. Constructs in which the cloning site lies inside regions or genes involved in replication (e.g. Repbinding site, rep and AC2-AC3 genes) were not infectious, whereas constructs in which the site was located inside the CP or BC1 genes were infectious. A clone that interrupts the BV1 gene was not infectious suggesting an early role of BV1 during the establishment of the infection. Linear viral clones containing different DNA fragments at both extremes were also infectious although with a lower efficiency. Analysis of the progeny suggested a precise excision mechanism since in most cases only wild type virus was recovered. The results suggest that excision could be linked to replication through a very specific recombination process.
-
-
-
Baculovirus-mediated trans-epithelial transport of proteins in infected caterpillars
More LessBaculovirus-mediated abnormal protein trafficking was studied in infected caterpillars by using heterologous proteins. The gene for human complement C1r was expressed in larvae of Mamestra brassicae by a recombinant Autographa californica nuclear polyhedrosis virus (AcMNPV) vector. By following the time-course of recombinant C1r distribution among various tissues, cell types and cell organelles, we concluded that the dominant site of recombinant protein synthesis was the fat body, although some production in the haemocytes and midgut was also observed. Only about 4% of the cells of the infected organs expressed recombinant C1r, which was then secreted into the haemolymph. The tracheal and integumental cuticle was rich in recombinant protein from the fourth day after infection although epidermal cells did not synthesize recombinant C1r. The morphological picture suggested that the accumulation was a consequence of a trans-epithelial transport. This transport process was checked by following the fate of the 49 kDa haemolymph protein and injected ovalbumin in AcMNPV-infected Mamestra brassicae and in Lymantria dispar nuclear polyhedrosis virus- infected Lymantria dispar larvae. Both proteins were able to pass the basal membrane of the epidermis and accumulated in the cuticle, while in control larvae neither was transported. The observed transepithelial transport points to the role of baculo- viruses in directing recombinant, endo- and exogenous proteins to cuticulated tissues. Based on these results we conclude that the permeability of basal membranes undergoes a characteristic change during the course of baculovirus infection.
-
-
-
Comprehensive physical map of the Cydia pomonella granulovirus genome and sequence analysis of the granulin gene region
More LessA cloned strain of Cydia pomonella granulovirus, CpGV-M1, was obtained using successive rounds of an in vivo limiting dilution method. A detailed physical map of the genome was constructed using 11 restriction enzymes. The region containing the granulin gene and an open reading frame immediately upstream of the granulin gene was sequenced. This region showed a high degree of homology to the equivalent region from Cryptophlebia leucotreta granulovirus with 98% amino acid identity for the granulins and 68% identity for the putative polypeptides encoded by the upstream ORFs. These latter polypeptides contained two zinc finger-like motifs and showed a low degree of homology to ME53 from Autographa californica nucleopoly- hedrovirus (AcMNPV). Evidence is presented for a similar upstream ORF in Artogeia rapae GV also. Hybridization studies showed that the CpGV genome had a similar overall organization to the Artogeia rapae GV genome. Hybridization between CpGV and AcMNPV was limited to fragments spanning about 15% of each genome suggesting that very few genes are highly conserved between GVs and NPVs.
-
-
-
PrP genotypes and experimental scrapie in orally inoculated Suffolk sheep in the United States
One-hundred and three United States Suffolk sheep were inoculated orally with a scrapie agent preparation and monitored for clinical disease and histopathological lesions characteristic of scrapie. A retrospective study of the polymorphisms at codon 171 of the prion protein (PrP) gene was performed on these sheep. All 63 sheep that developed scrapie during the observation period were homozygous for the glutamine 171 (171-QQ) PrP allele. Twelve 171-QQ sheep failed to develop disease. All 5 sheep homozygous for arginine (171-RR) and all 23 heterozygous (171-QR) sheep remained free of scrapie.
-
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)