- Volume 78, Issue 10, 1997
Volume 78, Issue 10, 1997
- Articles
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Antigenic structure of the human respiratory syncytial virus G glycoprotein and relevance of hypermutation events for the generation of antigenic variants
More LessA set of monoclonal antibodies (MAbs) specific for the attachment (G) glycoprotein of a recently isolated strain of human respiratory syncytial virus (HRSV) is described. Antibody reactivity with a series of HRSV isolates belonging to antigenic groups A and B identified three epitope categories: (i) strain- specific or variable epitopes that were present in a limited set of viruses from the same antigenic group, (ii) group-specific epitopes shared by viruses from the same antigenic group and (iii) conserved epitopes present in all HRSV isolates. Sequence analysis of escape mutants was used to map relevant antigenic sites of the G glycoprotein. Strain-specific epitopes were located preferentially in the variable C-terminal third of the G polypeptide, in agreement with previous studies of the Long strain. However, a new strain-specific epitope was mapped into another variable region, N-terminal to the cluster of cysteines in the G protein ectodomain. In contrast, the group-specific and conserved epitopes were located in the central conserved region of the G protein primary structure. These results, together with previous analysis of the Long strain, provide a detailed antigenic map of the HRSV attachment protein. Some mutants selected with group-specific antibodies contain multiple A-G substitutions (hypermutations) and lack one or two of the four cysteines which are conserved in all HRSV isolates. The genetic mechanism implicated in the generation of the hypermutated viruses and its relevance for the natural history of HRSV are discussed.
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Variations in the neutralizing and haemagglutination-inhibiting activities of five influenza A virus-specific IgGs and their antibody fragments
More LessNeutralization and haemagglutination-inhibition (HI) of a type A influenza virus by a panel of five monoclonal IgGs, their F(ab′)2s, Fabs and Fabs anti-mouse Fab were compared. The MAbs were specific for antigenic sites A, B and D of the haemagglutinin. Activities of the IgGs varied by up to 6-fold on a molar basis, apart from the HI activity of HC58 which was > 100-fold lower. This was not due to low functional affinity as HC58 had the second highest value (nM) as determined by an equilibrium method with whole virions. Conversion to the F(ab′)2 reduced neutralization and HI by only 2- to 6-fold, indicating that the Fc region had little involvement in these processes. However, all Fabs had low neutralization and HI activity compared with their IgGs, neutralization being reduced by 86 to > 1912-fold, and HI by 13 to > 69-fold. Although decreased, their affinities remained high, in the nM range. Neutralization and HI by three of the Fabs (HC2, HC3W and HC61) were restored by the addition of anti-Fab IgG; however, HC10 Fab antiFab IgG still had no detectable neutralization activity but gave HI, and HC58 FabManti-Fab IgG had no detectable HI activity but neutralized to the same extent as its IgG. The different properties of the antibodies are discussed in the light of their known mechanisms of action: HI by steric blocking of attachment of virus to the red cell receptor, and neutralization by the inhibition of post-attachment events (HC2, HC10 and HC61). The data demonstrate just how variable are the antiviral properties of individual IgGs.
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High and low efficiency neutralization epitopes on the haemagglutinin of type A influenza virus
More LessThe relationship between the efficiency of the neutralization process and the affinity of five monoclonal IgG antibodies specific for the haemagglutinin of type A influenza virus has been investigated by determining their neutralization rate constants (Kneut.) and affinities (Kdissoc). We addressed the hypothesis that if antibody affinity alone determined the efficiency of neutralization, then the Kneut.:Kdissoc. ratio would be the same for all antibodies. However, we found that the Kneut.:Kdissoc. ratio varied by up to 125-fold, suggesting that properties unique to the epitope are of major importance in determining the efficiency of neutralization. These data suggest that vaccines should preferentially stimulate antibodies to epitopes that mediate the most efficient neutralization, and that a high Kdissoc. should be an important but secondary consideration.
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Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo.
More LessThe interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immuno- precipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled- down in the NSI-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.
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Mx1 sensitivity: Batken virus is an orthomyxovirus closely related to Dhori virus.
More LessBatken virus, isolated from mosquitoes and ticks, was tentatively classified as a member of the family Bunyaviridae. Here we show that Batken virus is inhibited by the interferon-induced Mx1 protein of mice which selectively blocks the growth of orthomyxoviruses, including Thogoto and Dhori viruses. Furthermore, we show that Batken virus multiplication is characterized by accumulation of viral proteins in the nucleus and by budding of viral particles from the cell surface. Serological crossreactions between Batken and Dhori viruses revealed a phylogenetic relationship of these viruses, as previously also proposed by D. K. Lvov. Fragments of the Batken virus glycoprotein and nucleo- protein genes were amplified by RT-PCR. The deduced amino acid sequences were similar to the corresponding Dhori virus sequences. Therefore, Batken virus should be classified into the newly established genus Thogotovirus of the family Ortho- myxoviridae. Finally, our results demonstrate that Mx1 susceptibility of orthomyxoviruses is a reliable marker in the hunt for new family members.
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Detection of a novel Borna disease virus-encoded 10 kDa protein in infected cells and tissues.
More LessBorna disease (BD) is a transmissible, progressive polioencephalomyelitis primarily of horses and sheep. The genomes of two cell-adapted strains of Borna disease virus (BDV), the aetiological agent of BD, have been cloned and sequenced. According to the structural characterization achieved so far, BDV contains a non-segmented negative-sense 8·9 kb single-stranded RNA genome. In this paper we report the expression, purification and intracellular tracing of a novel non-glycosylated BDV-specific protein with a molecular mass of approximately 10 kDa (BDV p10 protein). The successful isolation of the corresponding mRNA from infected cells, amplification of the genetic region by RT-PCR and its efficient expression as a glutathione S-trans- ferase (GST) fusion protein demonstrated that antibodies specific for the BDV p10 protein are induced in infected animals. In addition, we have produced monospecific antisera against the GST-p10 fusion protein in rabbits. This monospecific antiserum recognized the BDV p10 protein in brain cells of naturally and experimentally infected animals as well as in persistently BDV-infected cells. Antibody- mediated affinity-chromatography using the anti- p10 serum could successfully be applied to purify a ca. 10 kDa antigen from infected animal cells to such an extent that glycosylation of this component could be ruled out.
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Establishment of persistent hepatitis C virus infection and replication in vitro.
Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNAin infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A stabilizing effect on HCV propagation was observed for 50 days in a serum-free medium with stimulation of the low-density lipoprotein (LDL) receptor expression by lovastatin. Using partially serum-free culture conditions, long-term persistence of HCV in cells and release of virions into supernatant was achieved for up to 130 days. Infectivity of released virions in supernatants after long-term culturing (day 30–80) was shown by successful infection of fresh cells. In conclusion, supplementation with PEG, DMSO and lovastatin during inoculation did not enhance virus replication substantially, but continued stimulation of LDL-receptor expression resulted in infections which persisted for over 4 months. These data support the hypothesis of an LDL-receptor mediated uptake of HCV into cells in vitro.
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Evolutionary analysis of the 5′-terminal region of hepatitis G virus isolated from different regions in China.
More LessWe have determined the nucleotide sequence of the 5′ -terminal region of the hepatitis G virus (HGV) genome in 11 hepatitis patients from three cities in China. Phylogenetic analyses revealed that the Chinese isolates were genetically distinct from previously described West African isolates (type 1) and American, European and East African isolates (type 2), with a mean sequence divergence of approximately 10 %. The mean divergence between isolates from Lanzhou, in the northwest of China, and those from Shanghai and Nanjing, on the east coast of China, was 5 % (range 3–7 %). The isolates from Shanghai and Nanjing were closely related to a common strain in Japan, while some of those from Lanzhou were closely related to a southeast Asian type 3 isolate. Thus, the Chinese isolates belong to the type 3 variant of HGV.
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In vivo induction of interferon-α in pig by non-infectious coronavirus: tissue localization and in situ phenotypic characterization of interferon-α-producing cells.
More LessA low frequency peripheral blood mononuclear cell (PBMC) subpopulation, referred to as natural interferon-producing (NIP) cells, is described as producing interferon-α (IFN-α) following contact with non-infectious viral structures, namely viral glycoproteins. These cells are characterized in vitro as non-T, non-B, MHC class II and CD4 cells. In this study, NIP cells were analysed in vivo after an intravenous injection of UV-inactivated transmissible gastroenteritis virus in newborn piglets, which resulted in strong serum IFN-α production. Spleno-cytes, but not PBMC, were the IFN-α producers in vivo. Using double immunohistochemical labelling for both IFN-α and leukocyte markers, we established that splenic NIP cells were not T or B cells. The majority were MHC class II and only a minority expressed a macrophage marker. NIP cells were localized in contact with MHC class II-expressing cells and T cells, which suggested that NIP cells might modulate the antiviral immune response.
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Inhibition of human immunodeficiency virus type 1 particle formation by alterations of defined amino acids within the C terminus of the capsid protein.
More LessIn previous studies, we demonstrated that the substitution of amino acid triplets for alanines in the carboxy-terminal portion (amino acids 341–352: ATL EEM MTA CQC) of the capsid protein domain (p24) of human immunodeficiency virus type 1 (HIV- 1) partly led to an inhibitory effect on the capacity to form virus-like particles (VLPs). In these experiments, the uncleaved Pr55gag precursor protein was expressed by recombinant vaccinia viruses. We have now investigated the effects of these mutations with respect to a replication-competent HI-provirus system. substitution of amino acids 344–346 (EEM) for alanines, which was previously shown to lead to an inhibition of VLP formation, completely blocked assembly and release of HIV. A substantial reduction of HIV synthesis was also observed in the proviral system after exchange of amino acids 347–348 [MT(A)] which, in contrast, was formerly shown to result in an increased formation of VLPs. Western blot analysis of lysates of cells transfected with these mutated proviral constructs revealed an abnormal intracellular processing pattern of the Pr55gag precursor molecules. Further analyses suggest a structural aberration of these altered polyproteins as the basis for the observed block of virus formation.
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Generation of infectious virus particles by transient co-expression of human immunodeficiency virus type 1 gag mutants.
More LessWe have demonstrated that COS7 cells transiently co-expressing myristylation-defective (Myr ) and protease-defective (PR ) human immunodeficiency virus (HIV) mutants can release infectious virions when co-transfected with an amphotropic murine leukaemia virus envelope protein expression plasmid (SV-A-MLV-env). In contrast, no infectious virions were detected when a PR, noninfectious HIV gag mutant was co-expressed with the Myr mutant, although the Myr mutant could still process the immature core particles in trans. This result indicates that generation of functionally normal Gag proteins is required for virus infectivity in our complementation system. A mutant with a 56- amino-acid deletion in the N-terminal region of the capsid (CA) domain could still complement the PR mutant to generate infectious virions, suggesting that the deletion mutant could provide a functional protease for processing in the PR mutant. This result is consistent with the concept that mutations within the N-terminal region of the CA domain have no major effects on Gag-Pol incorporation into particles.
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Expression of naturally occurring antisense RNA inhibits human immunodeficiency virus type 1 heterologous strain replication.
More LessRecently, the presence of human immunodeficiency virus type 1 (HIV-1) RNA transcripts with negative- strand polarity has been shown in tissue culture models of acute and persistently infected cells. One of these transcripts encodes a 189 amino acid open reading frame. This highly conserved antisense sequence is complementary to the structured Rev-responsive element and extends through the cleavage site of the Env protein. We tested the ability of this antisense RNA to modulate HIV-1 replication and the mRNA profile when expressed stably or transiently in several cell types. Different cell lines and PBLs were transduced by retroviral vectors producing antisense RNA and were then challenged by HIV infection. We have shown that the endogenously expressed antisense RNA containing the natural open reading frame inhibits HIV-1 IIIB and HIV-1ndk replication in these cells. The level of inhibition varied according to the cells, but was significant in all cases. The production of HIV-1 (BRU, IIIB, NDK) mRNAs was also significantly decreased. HIV-2 replication was not inhibited by expression of the antisense RNA. Our results also suggest that this inhibitory effect is due to the antisense RNA and not to the protein which is encoded by this sequence.
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Syncytium formation induced by human immunodeficiency virus type 1 isolates correlates with affinity for CD4.
More LessDifferent strains of human immunodeficiency virus type 1 (HIV-1) show considerable divergence in genetic content and biological properties. One property that has been closely correlated with clinical prognosis is the ability to induce syncytia formation in susceptible cells. This ability had been correlated with the V3 loop sequence of major envelope glycoprotein, gp120, but recent reports have questioned this connection. We investigated the contributions of different regions of the env gene to syncytia induction using chimeric viruses that contain part of the genome of a strain that lacks this ability (HIV-1Ba-L) within the genome of a virus that can form syncytia (HIV-1HXB-2). When tested in two cell lines susceptible to both parental viruses, as well as in primary cells, these chimeric viruses demonstrated that the ability to induce syncytia formation was determined by regions of env outside the V3 loop, which encompass residues that contribute to the binding of CD4 by gp120. Further investigation failed to show any difference in the expression of gp120 on the cell surface or cell adhesion molecules by cells infected with SI or NSI variants that would explain the observed differences in the ability to form syncytia. Assays of relative affinity for CD4 indicated that gp120 from SI variants showed a significantly higher affinity for CD4 than gp120 from NSI variants. These observations suggest that areas of the HIV-1 env gene contributing to the CD4 binding site may also contribute to the determination of syncytium- inducing (SI) and non-syncytium-inducing (NSI) phenotypes.
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No reactivation of attenuated immunodeficiency viruses in rhesus macaques after vaccinia virus-induced immune activation.
More LessLive-attenuated simian immunodeficiency virus (SIV) protects macaques against challenge with pathogenic SIV. To evaluate the safety of such vaccines, an investigation of whether or not nef- deleted SIV could be reactivated in vivo by immune activation of the host was conducted. In addition, monkeys infected with apathogenic SIV/HIV-1 chimeric viruses, and two control monkeys that had suppressed replication of pathogenic SIV were examined. During the infection virus became undetectable or persisted at a low level of replication in all monkeys. At this time-point 11 monkeys were immune-activated by a vaccinia virus (VV) superinfection. After VV infection up to 80% of their lymphocytes showed expression of the activation markers CD25 and CD69 over 2 weeks. However, only the two non-progressing monkeys infected with pathogenic SIV showed a noticeable but transient enhancement of SIV replication and increased SIV antibody titres. By contrast, in monkeys infected with apathogenic immunodeficiency viruses no change in virus load was observed. Therefore, attenuated immunodeficiency viruses cannot be reactivated in vivo by a VV-induced immune activation.
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Live attenuated simian immunodeficiency virus prevents super-infection by cloned SIVmac251 in cynomolgus monkeys.
The ability of a live attenuated simian immunodeficiency virus (SIV) to protect against challenge with cloned SIVmac251/BK28 was evaluated in four cynomolgus macaques. The intravenous infection of the C8 variant of the SIVmac251/32H virus, carrying an in-frame 12 bp deletion in the nef gene, did not affect the CD4 and CD8 cell counts, and a persistent infection associated with an extremely low virus burden in peripheral blood mononuclear cells (PBMCs) was established. After 40 weeks, these monkeys were challenged intravenously with a 50 MID50 dose of SIVmac251/BK28 virus grown on macaque cells. Four naive monkeys were infected as controls. Monkeys were monitored for 62 weeks following challenge. Attempts to rescue virus from either PBMCs or bone marrow from the C8-vaccin- ated monkeys were unsuccessful, but in two cases virus was re-isolated from lymph node cells. The presence of the SIV provirus with the C8 variant genotype maintaining its original nef deletion was shown by differential PCR in PBMCs, lymph nodes and bone marrow. Furthermore, in contrast to the control monkeys, the vaccinated monkeys showed normal levels for CD4M and CD8M cells, minimal lymphoid hyperplasia and no clinical signs of infection. Our results confirm that vaccination with live attenuated virus can confer protection. This appears to be dependent on the ability of the C8 variant to establish a persistent but attenuated infection which is necessary for inducing an immune response, as suggested by the persistence of a strong immune B cell memory and by the over-expression of interleukin (IL)-2, interferon-γ and IL-15 mRNAs in PBMCs of C8-vaccinated monkeys but not in those of control monkeys.
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Elevation of cytokines associated with the thrombocytopenia of equine infectious anaemia.
More LessThrombocytopenia is a common finding in infection with equine infectious anaemia virus (EIAV), a lentivirus with some homology to human immunodeficiency virus (HIV). The thrombocytopenia of EIA, like that in some HIV patients, appears to have a multifactorial pathogenesis. To investigate the decreased platelet production seen in experimental EIA, the levels of three potential negative regulators of platelet production - tumour necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interferon-α (IFN-α) - were measured in serum and bone marrow of six severe combined immunodefi- cient (SCID) foals and ten immunocompetent EIAV- infected foals. Levels of cytokines in pre-infection foal sera and bone marrow were compared with levels observed during clinical EIA. Mean serum levels of TNF-α and IFN-α were significantly higher (P < 0·05) on days 4 to 0 of thrombocytopenia than before infection. Serum TGF-β was significantly elevated on all days except day 1 of thrombocytopenia. Bone marrow TNF-α levels were significantly increased in infected foals just before clinical thrombocytopenia. TGF-β activity was not different in pre-infection and pre-thrombocytopenia bone marrows, but levels of TGF-β protein as determined by immunohistochemical staining were significantly higher in pre-thrombocytopenia bone marrow. IFN-α activity in bone marrow increased just before thrombocytopenia, but the difference was not significant at P < 0·05. Serum TNF-α levels were 2–2·5 times higher in SCID foals on three of the days prior to thrombocytopenia than in immunocompetent foals. No significant differences were found between the levels in SCID and immunocompetent foals of serum and bone marrow TGF-β or IFN-α at any of the times examined.
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Comparison of the complete sequence of feline spumavirus with those of the primate spumaviruses reveals a shorter gag gene.
C R Helps and D A HarbourThe complete nucleotide sequence of the provirus of feline foamy virus (FeFV), strain F-17, was determined, and compared to the available data for human and simian spumaviruses. In addition to the usual retroviral gag, pol and env genes, two open reading frames are present between the env gene and the 3′-LTR, as in the simian spumaviruses, the first being the putative transactivator. The gag gene is predicted to encode a precursor protein of only 53 kDa compared to 70 kDa for simian spumaviruses and a doublet of 70/74 kDa for human spumavirus. The gag gene contains conserved splice acceptor and donor sites suggesting that, like human foamy virus, FeFV expresses its pol gene using a spliced mRNA.The pol and env genes showed greater sequence similarity to their counterparts in the primate spumaviruses than the gag gene and additional open reading frames.
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Interleukin 4 stimulates infection and temporary growth of human neonatal lymphocytes exposed in vitro to human T-lymphotropic virus type I, but fails to substitute for interleukin 2 in the immortalization of infected cultures
It has been shown that interleukin 4 (IL-4) stimulates the proliferation of cells from patients affected by adult T-cell leukaemia, the haematological malignancy aetiologically associated with human T-lymphotropic virus type I (HTLV-I). In the present study, human neonatal lymphocytes were exposed to HTLV-I in vitro in the presence of IL-4. The results showed that: (i) cultures exposed to HTLV-I in the presence of either IL-4 or IL-2 bound IL-4; (ii) IL-4 did not substitute for IL-2 as a growth factor in cell lines previously infected and maintained in IL-2; (iii) cultures exposed to HTLV-I and maintained in IL-4 or IL-2 became infected; and (iv) IL-4 sustained the growth of HTLV-I-infected cultures for a maximum of 14 weeks. Moreover, HTLV-I-infected cultures grown in IL-4 showed upregulation of the IL-4 message and lower expression of HLA-DR and CD25 when compared with counterpart cultures maintained in IL-2. These results suggest that continuous growth of T-lymphocytes induced in vitro by HTLV-I infection, at least temporarily, requires signals specifically provided by IL-2 and not by IL-4.
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Spliced human endogenous retroviral HERV-H env transcripts in T-cell leukaemia cell lines and normal leukocytes: alternative splicing pattern of HERV-H transcripts
More LessThe majority of human endogenous retroviral HERV-H elements in the human genome have large deletions in pol and lack most of env, 5–10% are more or less complete with a potentially immunosuppressive transmembrane protein-encoding env region. Spliced HERV-H env transcripts were detected in T-cell leukaemia cell lines and lymphocytes from healthy blood donors by using RT-PCR. The transcripts all contained a splice donor in the leader region downstream from the primer-binding site and a previously unreported splice acceptor in the integrase-encoding region of pol, absent in the HERV-H deletion elements. In singly spliced transcripts the leader and integrase regions were joined directly whereas in multiply spliced transcripts they were joined with an alternative exon from the protease-encoding region located between the two regions. env transcripts from three different HERV-H elements were identified: one element similar to a HERV-H consensus sequence was primarily amplified from the T-cell leukaemia cell lines and two other more defective elements were amplified from normal lymphocytes. One of these elements was shown to be a reintegrated spliced transcript where the protease and integrase regions were joined, removing most of pol but leaving gag intact. Other spliced transcripts, joining the protease region and the 3′-LTR, were also amplified. The fact that HERV-H elements with an intact env splice acceptor also use the splice sites in the protease-encoding region suggests that this unusual multiple splice pattern could have a biological function in the intact HERV-H.
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Inhibition of Moloney murine leukaemia virus by a retroviral vector, LNL6, carrying ribozymes targeted to the 5′ non-coding sequence
More LessThe packaging region ψ is the RNA sequence which directs the inclusion of genomic viral RNA into virions. As such it represents an apparently accessible site for ribozyme action. Ribozymes directed against the ψ site of Moloney murine leukaemia virus (MoMLV) were delivered to target cells using a related retroviral vector, LNL6. LNL6 is a vector predominantly derived from MoMLV and, like all retroviruses, requires the ψ region to be packaged. By exploiting the heterogeneity between nucleotide sequences of the MoMLV target and LNL6 vector in the ψ packaging region, two ribozymes were designed and shown to selectively cleave the target but not the vector sequence. Clonally derived cell lines expressing ribozymes showed inhibition of virus replication. These results show the utility of catalytic RNA as specific antiviral agents.
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Inhibition of murine leukaemia virus retrotranscription by the intracellular expression of a phage-derived anti-reverse transcriptase antibody fragment
More LessThe intracellular targeting of recombinant antibodies is an experimental strategy to interfere with the function of selected molecules that is being utilized in a variety of different systems for research and medical applications. Since recombinant antibodies are increasingly being derived from phage display libraries, we have exploited phage technology to isolate, from a large combinatorial library, human antibody fragments directed against human immunodeficiency virustype 1 reverse transcriptase (HIV-1 RT). We describe in this paper the in vitro and in vivo properties of a neutralizing anti-RT antibody fragment. We demonstrate that the heavy chain domain (VH-CH1) of the phage-derived antibody is able to inhibit the retroviral enzyme, in that it neutralizes the RNA-dependent DNA polymerase activity of HIV-1 RT. TheVH-CH1 antibody fragment also neutralizes the activity of RT of drug-resistant HIV-1 mutants as well as that of murine retrovirus RT. To confirm the broad reactivity of the synthetic antibody fragment, we have assessed the ability of the intracellularly expressed VH-CH1 protein to interfere with murine retroviral infection. To this end, we developed an in vivo selection procedure based on the antibody-mediated resistance to a cytotoxic retrovirus and used this selection procedure to rescue, from a heterogeneous population, cells expressing the VH-CH1 antibody fragment. We finally demonstrate that the intracellular expression of the recombinant heavy chain antibody fragment leads to an efficient inhibition of viral retrotranscription by murine-based retrovirus.
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Cytokeratin patterns of expression in human epithelial cell lines correlate with transcriptional activity of the human papillomavirus type 16 upstream regulatory region
More LessA comparison of the CAT reporter activity of a plasmid which contains the 232 bp epithelial specific enhancer alone with that of plasmids which contain additional sequences from the human papillomavirus type 16 (HPV-16) upstream regulatory region (URR) revealed two markedly different patterns, in an analysis of six human epithelial cell lines. In HeLa, C33A and SiHa, the CAT reporter activities of all the constructs were comparable. In contrast, in CaSki, HK2bE6-E7 and HaCaT we detected very low levels of CAT reporter activity using the constructs with the additional HPV-16 URR sequences. The ability of HPV-16 E2 to transactivate a construct with 2 E2 binding sites also differed markedly and showed the same pattern. Cytokeratin staining revealed a correlation between cytokeratin 10 and 14 expression and the transcriptional differences observed. We also found alterations in the activity of one of the constructs on altering the growth conditions of the HaCaT cell line.
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Human papillomavirus type 16 E7 binds to the conserved carboxy-terminal region of the TATA box binding protein and this contributes to E7 transforming activity
More LessWe have previously shown that the human papillomavirus E7 proteins bind to the cellular TATA box binding protein (TBP). In this paper we showthatthe HPV-18 E6 and the HPV-16 E2 proteins will also bind TBP in vitro. This feature of virus proteins is conserved across many viral types and we were interested in determining whether these HPV proteins interacted with the same conserved region of the TBP molecule. A series of deletions was introduced into the TBP protein and its binding to these HPV proteins was measured. The previously well-characterized interaction between p53 and TBP was used for comparison. All four proteins were found to interact with the carboxy-terminal domain of the TBP protein, although the precise residues involved and the relative strengths of association differed between the different HPV proteins. Mutational analysis of HPV-16 E7 protein identified a stretch of four amino acids responsible for the binding to TBP. This mutant E7 protein possessed wild-type levels of transcriptional activity on the adenovirus E2 promoter but exhibited reduced transforming activity in cooperation with EJ-ras. These results demonstrate that the mechanisms of interaction between diverse viral proteins and TBP are similar and that, in the case of E7, this interaction may contribute to its transforming activity.
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Identification of a cytotoxic T-lymphocyte epitope in the human papillomavirus type 16 E2 protein
More LessPersistent infection with oncogenic types of human papillomaviruses (HPV) is the major cause of cervical cancer precursor lesions. Cellular immune responses are considered important in the elimination of HPV infection, but the targets are not well defined. HPV E1 and E2 proteins form a replicative complex necessary for viral genome maintenance. To investigate whether epitopes in the E1 or E2 proteins can serve as targets for cytotoxic T-lymphocyte (CTL)-mediated killing, we identified peptides containing the human leukocyte antigen (HLA)-A*0201 binding motif in the deduced amino acid sequences of the HPV-16 E1 and E2 genes. Binding affinity of the peptides was measured by HLA-A*0201 upregulation on T2 cells. Peptides with high binding-affinity were tested for their ability to elicit peptide-specific CTLs from healthy blood donors. We found one peptide from the E1 and one from the E2 protein sequence that were capable of eliciting peptide-specific CTLs. The E2-specific CTLs lysed an HPV-16-transfected cervical carcinoma cell line, but not the untransfected HPV-negative parental cell line, indicating that the identified E2 epitope can be presented to CTLs in HPV-positive epithelial cells. These findings might have potentially important implications for studies of the natural history of HPV infection in relation to cervical carcinogenesis.
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Human cytomegalovirus late-phase maturation is blocked by stably expressed UL32 antisense mRNA in astrocytoma cells
Human cytomegalovirus (HCMV) open reading frame UL32 codes for the basic phosphoprotein pp150 (ppUL32), an abundant constituent of the virion tegument. In order to study its potential role in the assembly and/or transport of progeny particles, astrocytoma cell lines (U373MG) were generated, stably expressing a 2·1 kb 5′ fragment of UL32 in antisense orientation under the control of the HCMV major immediate early promoter. The steady-state level of the UL32 sense mRNA and pp150 synthesis were strongly reduced in infected antisense cell lines. Neither immediate early and early gene expression, nor viral DNA replication, was inhibited; the expression of the late gene product gB (gpUL55) was also reduced, but mainly at the level of translation. Control experiments indicated that this differential effect of UL32 antisense expression on the synthesis of viral products was specific. As a consequence of the inhibitory effect, virus yield was significantly reduced in antisense mRNA cell lines. Ultrastructural comparison of control and antisense cells revealed no difference in nucleocapsid forms in the nucleus. However, in the cytoplasm of antisense cells, DNA-containing C capsids and virions were absent and abnormal forms of non-infectious enveloped particles were observed. The data suggest the involvement of pp150 either in the transport of DNA-containing particles through the nuclear envelope or in the stabilization of capsids in the cytoplasm. Thus, UL32 antisense mRNA appears to interfere strongly with virus maturation during the late phase of the infectious cycle.
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Characterization of the vaccinia virus F8L protein
More LessVaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly of actin stress fibres and formation of actin tails which propel the virus intra- and intercellularly. The viral factors responsible for these actin rearrangements remain unknown. Sequence analysis reveals significant homology between the vaccinia F8L ORF and the proline repeats of iActA, the protein which initiates actin tail assembly and motility in the bacterial pathogen Listeria ivanovii. We characterized the F8L gene product to examine its possible role in vaccinia rearrangements of the host actin cytoskeleton. F8L is a ~ 8 kDa protein expressed early during infection and is found throughout the cytoplasm, with no discernible association with viral or cellular structures. Furthermore, the F8L deletion strain, WR∆F8L, forms particles and actin tails indistinguishable from WR. Our observations demonstrate that F8L is not required for vaccinia virus morphogenesis or the actin rearrangements observed during infection.
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Altered antigenicity of 'a' determinant variants of hepatitis B virus
More LessThe ‘a’ determinant of hepatitis B virus (HBV) surface antigen (HBsAg) is the most important target for diagnosis and immunoprophylaxis. Several HBV variants with point mutations within the ‘a’ determinant have been identified among fully vaccinated children in Taiwan. We investigated the effect of each of these mutations on the antigenic nature of the S protein by cloning and expression of the mutant S antigens in Pichia pastoris. Four variants, Ser-126, His-129, Arg-129 and Arg-145, all exhibited various degrees of altered binding of HBsAg to several monoclonal antibodies. Arg-145, a well-characterized immune escape mutant, and Arg-129 had the lowest binding capacities to all monoclonal antibodies as compared with other variants. Similar to Arg-145, the Arg-129 variant could be isolated from both vaccinated children and unvaccinated adults, thus representing a naturally occurring mutant with an altered ‘a’ determinant. Whether these ‘a’ determinant variants with altered antigenicity might gradually become major circulating strains, as a consequence of the immune pressure against the wild-type HBV created by vaccination, remains to be monitored.
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The minute virus of mice (MVM) nonstructural protein NS1 induces nicking of MVM DNA at a unique site of the right-end telomere in both hairpin and duplex conformations in vitro
The right-end telomere of replicative form (RF) DNA of the autonomous parvovirus minute virus of mice (MVM) consists of a sequence that is self-complementary except for a three nucleotide loop around the axis of symmetry and an interior bulge of three unpaired nucleotides on one strand (designated the right-end ‘bubble ’). This right-end inverted repeat can exist in the form of a folded-back strand (hairpin conformation) or in an extended form, base-paired to a copy strand (duplex conformation). We recently reported that the right-end telomere is processed in an A9 cell extract supplemented with the MVM nonstructural protein NS1. This processing is shown here to result from the NS1-dependent nicking of the complementary strand at a unique position 21 nt inboard of the folded-back genomic 5′ end. DNA species terminating in duplex or hairpin con-figurations, or in a mutated structure that has lost the right-end bulge, are all cleaved in the presence of NS1, indicating that features distinguishing these structures are not prerequisites for nicking under the in vitro conditionstested. Cleavage of the hairpin structure is followed by strand-displacement synthesis, generating the right-end duplex conformation, while processing of the duplex structure leads to the release of free right-end telomeres. In the majority of molecules, displacement synthesis at the right terminus stops a few nucleotides before reaching the end of the template strand, possibly due to NS1 which is covalently bound to this end. A fraction of the right-end duplex product undergoes melting and re-folding into hairpin structures (formation of a ‘rabbit-ear’ structure).
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Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors
A baculovirus (Autographa californica nucleopoly- hedrovirus) vector containing a strong promoter, the CAG promoter, was developed to introduce foreign genes into mammalian cells. Recombinant baculoviruses carrying a reporter gene under the control of the CAG promoter were inoculated into various mammalian cell lines. High-level expression was observed not only in hepatocytes but also in other non-hepatic cell lines tested. Expression of the reporter gene was detected even 14 days after infection. The infectious titre of the recovered baculoviruses decreased significantly after infection, indicating that the baculoviruses did not replicate in mammalian cells. We then compared the efficiencies of gene expression by the baculovirus vector with that of a replication-defective adenovirus vector by using the same expression unit. The same level of expression was observed in HepG2, HeLa and COS7 cells by both vectors. Efficient expression and proper processing were observed in mammalian cells infected with baculoviruses carrying genes coding for structural regions of hepatitis C virus. These results suggest that the baculovirus vector is a good tool for gene delivery into various mammalian cells in order to study the function of foreign genes.
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The gene encoding the capsid protein P82 of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus: sequencing, transcription and characterization by immunoblot analysis
More LessA gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV). The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71·4 kDa. Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.). Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL- c2, and the fusion protein was used to generate antibodies in rabbits. It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i. in infected cells. The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus. Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV. Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.
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Nucleotide sequence evidence for the occurrence of three distinct whitefly-transmitted, Sida-infecting bipartite geminiviruses in Central America
More LessThe nucleotide sequences of two S/da-infecting geminiviruses from Honduras were determined. The symptoms of both viruses are identical in S/da rhomb/fol/a but different in N/cot/ana bentham/ana. An additional symptom of one virus was yellow vein clearing on infected N. bentham/ana leaves. Both Sida golden mosaic viruses (SiGMV-Ho and SiGMV- Hoyv) have bipartite genomes (DNAs A and B). From the SiGMV-Hoyv-infected S. rhombifolia plant two different DNA B molecules were isolated and cloned. They differ in length by 24 nucleotides [SiGMV-Hoyv B1 (2593 nt) and B2 (2569 nt)] and at eight nucleotide positions. Both proteins encoded by DNA B(BV1 and BC1) are affected by these substitutions. Computer analysis shows that the bipartite genomes resemble those of other whitefly-transmitted geminiviruses. From homology analyses we conclude that both viruses are closely related but distinct. Comparison with a Sida-infecting virus from Costa Rica (SiGMV-Co) showed that the two viruses from Honduras are more similar to each other than either of them are to SiGMV-Co. Exchange of SiGMV-Ho and SiGMV-Hoyv genomic components resulted in viable pseudorecombinant viruses. SiGMV-Ho DNA A was able to produce a viable pseudorecombinant with SiGMV-Co DNA B while the reciprocal exchange was not infectious in N. bentham/ana. SiGMV-Ho^ DNA A and SiGMV-Co DNA B produced a viable pseudorecombinant virus whereas only pseudorecombination of SiGMV-Co DNA A with SiGMV-Hoyv DNA B2, and not with DNA B1, was infectious in N. bentham/ana.
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Long-term association of tomato yellow leaf curl virus with its whitefly vector Bemisia tabaci: effect on the insect transmission capacity, longevity and fecundity
More LessThe association between tomato yellow leaf curl geminivirus (TYLCV, Israeli isolate) and its insect vector, the whitefly Bemisia tabaci, was investigated. Insects that emerged during a 24 h period were caged with TYLCV-infected plants for a 48 h acquisition access period, then with egg-plants-a TYLCV non-host-for the rest of their lives. While TYLCV DNA was associated with the whiteflies during their entire adult life, the amount of capsid protein rapidly decreased and was not detectable in the insect after approximately 12 days of age. The ability of the infected whiteflies to transmit TYLCV to tomato test plants steadily decreased with age but did not disappear completely. Transmission by viruliferous insects decreased from 100% to 10–20% during their adult lifetime, compared with a decrease from 100% to 50% for non-viruliferous insects. The association of TYLCV with adult B. tabaci led to a reduction of 17–23% in their life expectancy compared with insects that had not acquired the virus, and to a 40–50% decrease in the mean number of eggs laid. These results suggest that TYLCV has some features reminiscent of an insect pathogen.
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Cap-independent leaky scanning as the mechanism of translation initiation of a plant viral genomic RNA
More LessThe genome of plum pox virus contains a single open reading frame that is translated into a large polyprotein. Although the open reading frame starts at nucleotide 36 (36AUG), it is translated from the second, 147AUG, which is in a more favourable context for translation initiation. We have carried out in vitro translation and transient expression analysis in protoplasts of a nested set of substitution and deletion mutants, and the results show that no internal structure in the 5′ noncoding region of plum pox virus is necessary for efficient translation initiation. On the other hand, when the cryptic 36AUG was placed in a favourable context, it turned into an efficient initiation codon in vitro. Furthermore, AUGs that were placed in a favourable context, initiating short intraleader open reading frames, repressed translation initiation from the 147AUG in vitro and in vivo. These results point to leaky scanning as the mechanism of translation initiation of plum poxvirus RNA. Nevertheless, it is a peculiar leaky scanning where the initiation of translation does not require a cap structure at the 5′ end. This fact is congruent with the experimentally predicted absence of a stable secondary structure at the 5′ noncoding region.
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Intracellular ingestion and salivation by aphids may cause the acquisition and inoculation of non-persistently transmitted plant viruses
More LessTransmission of non-persistent plant viruses is related to aphid behaviour during superficial brief probes. A widely accepted hypothesis postulates that virus acquisition occurs during ingestion of plant cell contents, and inoculation during egestion or regurgitation of previously ingested sap. Although conceptually attractive, this ingestion- egestion hypothesis has not been clearly demonstrated. Furthermore, it overlooks the anatomy of the tips of the stylets (mouthparts) and, consequently, the potential role of salivation in the inoculation process. Here, we used the electrical penetration graph (EPG) technique to investigate aphid-stylet activities associated with uptake (acquisition) and release (inoculation) of two nonpersistently transmitted viruses. Our results show that acquisition occurs primarily during the last sub-phase (II-3) of intracellular stylet punctures, whereas inoculation is achieved during the first sub-phase (II-1). An alternative mechanism to the ingestion-egestion hypothesis is proposed on the basis of our findings.
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Severely combined immunodeficient (SCID) mice resist infection with bovine spongiform encephalopathy
More LessFollowing combined intraperitoneal and intracerebral injection with bovine spongiform encephalopathy (BSE) cow brain homogenate, SCID mice show a resistance to infection in comparison with immunocompetent CB20 mice. BSE occurred in only five out of 22 challenged SCID mice, with a mean incubation period of 573 days, whereas all the CB20 mice developed the disease with a mean incubation period of 456 days. In contrast, previous studies have shown that intracerebral infection of SCID mice with a mouse-passaged scrapie strain, ME7, produces 100 % incidence of disease but no replication of infectivity in spleen. The results with BSE suggest that there is little or no direct infection of the CNS in interspecies transmissions, but that processing or replication of infectivity in peripheral lymphoid tissues may facilitate subsequent spread of infection to the CNS.
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Volumes and issues
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Volume 105 (2024)
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