Biomedical subjects

M Lobigs

Publications and source records attributed to M Lobigs.

36 records · Page 2Linked to original sources

PubMed1996

Flavivirus-induced up-regulation of MHC class I antigens; implications for the induction of CD8+ T-cell-mediated autoimmunity.

Infection of a wide variety of cells of human, mouse and other species' origin by flaviviruses such as WNV, YF, Den, MVE, KUN and JE, increases the cell-surface expression of MHC class I. This MHC class I up-regulation is not due to increased MHC class I synthesis per se, but the result of increased peptide availability in the ER for MHC class I assembly. This is most likely due to the interaction of the viral polyprotein with the ER membrane during viral replication. Flavivirus infection can overcome peptide deficiency in TAP-deficient or non-permissive cell lines such as RMA-S and Syrian hamster cells, BHK and NIL-2. The consequence of this increased MHC class I expression manifests itself in reduced susceptibility to NK cells and augmented lysis by Tc cells. In mice, long-term flavivirus-immune Tc cell memory formation is impaired, following the appearance of strong anti-self Tc cell reactivity observed in in vitro cultures from splenocytes of flavivirus-primed animals. We hypothesize that flavivirus-induced MHC class I up-regulation leads to transient T-cell autoimmunity, followed by down-regulation of both autoimmunity and virus-specific Tc cell memory. Furthermore, we speculate that flavivirus infections of humans in the tropics may be responsible for the observed lower incidence of overt autoimmunity in these geographic regions than in temperate climates where flaviviruses are not endemic.

Animals↗

PubMed1995

Polymorphic peptide transporters in MHC class I monomorphic Syrian hamster.

We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TAP1 and TAP2 is detectable (Lobigs and Müllbacher 1993). Investigating the antigen-presentation machinery of the class I MHC monomorphic Syrian hamster using mouse MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (Kk, Kd, Kb, Dd) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed Dd and Kb in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not Kk and Kd. However, Kd was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recognition. Kk is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtp1 and VV-mtp2 rendered both cell lines, after infection with either VV-Kk and VV-Kd, susceptible to lysis by either Kk- or Kd-restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters. The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Up-regulation of MHC class I by flavivirus-induced peptide translocation into the endoplasmic reticulum.

Flavivirus infection of mammalian cells increases the cell surface expression of major histocompatibility complex (MHC) class I molecules, the recognition elements for cytotoxic T cells. Here, we show that the mechanism for flavivirus-induced up-regulation of class I MHC involves an increase in peptide supply to the endoplasmic reticulum. Flavivirus-mediated peptide supply for MHC class I assembly is independent of the peptide transporters for class I antigen presentation, since infection of class I MHC peptide transport-deficient cell lines with flaviviruses results in the cell surface expression of biologically functional class I MHC peptide complexes. The flavivirus-induced supply of antigenic peptides to the endoplasmic reticulum is not restricted to flavivirus-encoded peptides and independent of interferon. The data imply that peptide availability regulates surface expression of class I MHC restriction elements and suggests a mechanism for flavivirus-induced immunopathology.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Recombinant expression of vaccinia virus-encoded TAP1 and TAP2 promotes MHC class I-restricted antigen presentation in a Syrian hamster cell line.

Transporter associated with antigen presentation (TAP) molecules supply small peptides to the lumen of the endoplasmic reticulum for assembly with MHC class I glycoproteins. Here we report that a Syrian hamster cell line, BHK, which is commonly used in cell biological and virological investigations, is deficient in antigen presentation in a xenogeneic model. Thus when the mouse MHC class I restriction element, Kd, is expressed in BHK cells via a vaccinia virus (VV) recombinant, no significant recognition by VV-immune and Kd-restricted cytotoxic T cells is induced. This defect is apparently a consequence of a deficiency in TAP-dependent peptide transport and can be repaired when rat TAP1 and TAP2 are provided by recombinant VV expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Posttranslational signal peptidase cleavage at the flavivirus C-prM junction in vitro.

We have investigated the cleavages at the flavivirus capsid-prM protein junction in vitro. When expressed in the absence of the flavivirus proteinase, capsid and prM, which are separated by an internal signal sequence, exist as a membrane-spanning precursor protein. Here we show the induction of posttranslational signal peptidase cleavage of prM by trypsin cleavage of a cytoplasmic region of this precursor protein.

Animals↗

PubMed1994

The flavivirus nonstructural protein NS3 is a dominant source of cytotoxic T cell peptide determinants.

Vaccinia virus recombinants encoding regions of the Murray Valley encephalitis virus (MVE) genome, which together cover the entire viral coding region, were employed to identify the MVE protein which is the dominant source of CD8+, cytotoxic, T cell antigenic determinant(s) presented by the mouse H-2Kk major histocompatibility antigen. MVE and West Nile virus-immune, H-2k-restricted, effector cells recognized peptides derived from the MVE nonstructural polyprotein segment, and in this region the immunodominant determinant mapped to protein NS3. Interestingly, mapping of cytotoxic T cell antigenic determinants of other flaviviruses also identified the NS3 protein as the dominant source of antigenic peptides (A. B. Hill, A. Müllbacher, C. Parrish, G. Coia, E. G. Westaway, and R. V. Blanden, 1992, J. Gen. Virol. 73, 1115-1123; A. L. Rothman, I. Kurane, C.-J. Lai, M. Bray, B. Falgout, R. Men, and F. A. Ennis, 1993, J. Virol. 67, 801-806). Using an allele-specific peptide motiff for H-2Kk, we predicted 12 peptides in the MVE NS3 protein as ligands for the restriction element and identified three peptides which were recognized in association with H-2Kk by MVE-immune cytotoxic T cells. We also examined the effect of proteolytic processing in the MVE nonstructural polyprotein segment mediated by the viral proteinase NS3 on antigen processing and presentation of the MVE H-2Kk-restricted T cell determinant. Processing of the MVE polyprotein by the viral proteinase did not markedly influence the availability of this peptide determinant.

Alleles↗

PubMed1993

Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3.

Flavivirus protein biosynthesis involves the proteolytic processing of a single polyprotein precursor by host- and virus-encoded proteinases. In this study, the requirement for the proteolytic function of the viral proteinase NS3 for correct processing of a polyprotein segment encompassing the Murray Valley encephalitis virus structural proteins is shown. The NS3-mediated cleavage in the structural polyprotein region presumably releases the capsid protein from its membrane anchor and triggers the appearance of the premembrane (prM) protein. This suggests that cleavage of prM by signal peptidase in the lumen of the endoplasmic reticulum is under control of a cytoplasmic cleavage catalyzed by a viral proteinase. The function of the viral proteinase is also essential for secretion of flaviviral spike proteins when expressed from cDNA via vaccinia virus recombinants or in COS cell transfections. This has important implications for the design of flavivirus subunit vaccines.

Animals↗

PubMed1993

Recognition of vaccinia virus-encoded major histocompatibility complex class I antigens by virus immune cytotoxic T cells is independent of the polymorphism of the peptide transporters.

In the cytotoxic T-cell response to viruses, peptide antigens of cytoplasmic origin are presented at the cell surface by the highly polymorphic major histocompatibility complex (MHC) class I molecules to CD8+ T-lymphocyte receptors. Peptide transporter molecules and other MHC-linked gene products have been implicated in the generation and import of antigenic peptides into the lumen of the endoplasmic reticulum for assembly with MHC class I glycoproteins. These accessory molecules in the antigen-presentation pathway map to a polymorphic region in the class II MHC, and the possibility of their allele-specific selectivity in antigen presentation has been raised. Here we show that additional, functionally polymorphic components are not apparent in an in vitro mouse MHC class I-restricted cytotoxic T-cell response to vaccinia and influenza viruses. When the mouse H-2Kd molecule was expressed via a recombinant vaccinia virus in target cells of different mouse MHC haplotypes or cells of rat, Syrian hamster, monkey, and human origin, efficient Kd-restricted and vaccinia virus-specific lysis was observed as measured with bulk effectors and at the clonal level. In addition, human transporters efficiently processed peptides originating from influenza virus nucleoprotein and hemagglutinin antigens as recognized by mouse influenza immune cytotoxic T cells.

Animals↗

PubMed1992

Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry.

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.

Animals↗

PubMed1992

Proteolytic processing of a Murray Valley encephalitis virus non-structural polyprotein segment containing the viral proteinase: accumulation of a NS3-4A precursor which requires mature NS3 for efficient processing.

The proteolytic processing of a non-structural polyprotein segment from the cytoplasmic domain of NS2A to the C terminus of NS5 of Murray Valley encephalitis (MVE) virus was examined, when expressed from cDNA via a vaccinia virus recombinant, in transiently transfected COS cells, or synthesized by cell-free translation. Cleavages mediated by the virus-encoded proteinase domain in NS3 at the junctions of NS2A-2B, NS2B-3 and NS4B-5 were catalysed efficiently. However, the cleavage at the NS3-4A junction, also mediated by the NS3 proteinase, was greatly delayed. Little or no NS3 was found, but an 85K precursor molecule accumulated; this was identified as NS3-4A. Termination codons were introduced by site-directed mutagenesis at the junctions of the NS3-4A, NS4A-4B and NS4B-5 genes to generate C-terminal truncations of the MVE virus polyprotein segment. In expression studies of these constructs the predicted NS3-mediated proteolytic cleavages were catalysed, except for that at the NS3-4A junction. In co-infections and co-transfections with constructs encoding the MVE virus nonstructural polyprotein region truncated at the C termini of NS3 or NS4A, efficient processing at the NS3-4A site was induced. Thus it appears that the MVE virus polyprotein is cleaved inefficiently in cis at the NS3-4A junction, whereas the site is processed efficiently in trans by mature NS3. The NS3-4A precursor is also seen in flavivirus-infected cells. Its function remains to be determined, but it could play a role in the replication of flavivirus, in view of the importance of polyprotein processing in the regulation of gene expression of positive-stranded RNA viruses, the modulation of processing at the NS3-4A site by NS3 or NS3-containing precursors described in the present study and the importance of NS3 as an integral part of the viral polymerase complex.

Animals↗

PubMed1990

Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse virulence.

We have passaged the prototype strain of Murray Valley encephalitis virus in SW13 (human) cells, sequenced the E and M genes, and examined the virulence of the passaged virus for 21-day-old mice following intracranial and intraperitoneal inoculation. Six independent passage series were carried out: four in the presence of mouse hyperimmune ascitic fluid and two without antibody. Changes were observed in the E protein deduced amino acid sequence for each of the six 10th passage stocks sequenced. Eleven changes were observed in total for the six stocks sequenced; these were at residues 117, 118, 390, 423, and 460. Nine of the changes were nonconservative. Five of the six passaged variants were altered at Asp 390 which is part of an Arg-Gly-Asp (RGD) sequence. This change resulted from adaptation to SW13 cells rather than from antibody selection. The RGD sequence (and residue 423) falls within a region which is highly conserved between flaviviruses and is strongly hydrophilic. All five variants which were altered at Asp 390 were attenuated in 21-day-old mice following i.p. inoculation. We propose that the domain of E encompassing the RGD sequence is an important determinant of flavivirus pathogenicity.

Adenocarcinoma↗

PubMed1990

Spike protein oligomerization control of Semliki Forest virus fusion.

We have recently shown, using cleavage-deficient mutants of the p62-E1 membrane protein complex of Semliki Forest virus that p62 cleavage to E2 is necessary for the activation of the fusion function of the complex at pH 5.8 (a pH optimal for virus fusion) (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990). In this study, we show that the mutant precursor complexes can be induced to activate membrane fusion when treated with more acidic buffers (pH 5.0 and 4.5), which also appear to dissociate most of the p62-E1 complexes and change the conformation of the E1 subunit (the supposed fusion protein of Semliki Forest virus into a form which is resistant to trypsin digestion. These data suggest that p62 cleavage is not essential for membrane fusion per se but that the crucial event activating this process seems to be the apparent dissociation of the heterodimer, which in turn is facilitated by the spike precursor cleavage.

Animals↗

PubMed1990

Fusion function of the Semliki Forest virus spike is activated by proteolytic cleavage of the envelope glycoprotein precursor p62.

The precursor protein p62 of the prototype alphavirus Semliki Forest virus (SFV) undergoes during transport to the cell surface a proteolytic cleavage to form the mature envelope glycoprotein E2. To investigate the biological significance of this cleavage event, single amino acid substitutions were introduced at the cleavages site through mutagenesis of cDNA corresponding to the structural region of the SFV genome. The phenotypes of the cleavage site mutants were studied in BHK cells by using recombinant vaccinia virus vectors. Nonconservative substitutions completely abolished p62 cleavage. Uncleaved p62 was transported with normal kinetics to the cell surface, where it became accessible to low concentrations of exogenous trypsin. The proteolytic cleavage of envelope glycoprotein precursors has been shown to activate the membrane fusion potential of viral spikes in several virus families. Here we demonstrate that the fusion function of the SFV spike is activated by the cleavage of p62. Cleavage-deficient p62 expressed at the cell surface did not function in low-pH-triggered (pH 5.5) cell-cell membrane fusion; however, cleavage of the mutated p62 with exogenous trypsin restored the fusion function. We discuss a model for SFV assembly and fusion where p62 cleavage plays a crucial role in the stability of the multimeric association of the viral envelope glycoproteins.

Animals↗

PubMed1990

Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1.

The Semliki Forest virus spike glycoproteins E1 and p62 form a heterodimeric complex in the endoplasmic reticulum (ER) and are transported as such to the cell surface. In the mature virus particle, the heterodimeric association of E1 and E2 (the cleavage product of p62) is maintained, but as a more labile and acid-sensitive oligomer than the E1-p62 complex. The E3 peptide forms the N-terminal part of the p62 precursor and carries the signal for the translocation of p62 into the lumen of the ER. The question of whether E3 is also important in the formation and stabilization of the E1-p62 heterodimer has been addressed here with the aid of an E3 deletion mutant cDNA. In this construct, the entire E3 was replaced with a cleavable, artificial signal sequence which preserved the membrane topology of an authentic E2. The E3 deletion, when expressed via a recombinant vaccinia virus, abolished heterodimerization of the spike proteins. It also resulted in the complete retention of E1 in the ER and almost total inhibition of E2 transport to the plasma membrane. The oligomerization and transport defect of E1 expressed from the E3 deletion mutant could be complemented with a wild-type p62 provided from a separate coding unit in double infections. These results point to a central role of E3 in complex formation and transport of the viral structural components to the site of budding. In conjunction with earlier work (M. Lobigs and H. Garoff, J. Virol. 64:1233-1240, 1990; J. Wahlberg, W. A. M. Boere, and H. Garoff, J. Virol. 63:4991-4997, 1989), the data support a model of spike protein oligomerization control of Semliki Forest virus assembly and disassembly which may be mediated by the presence of E3 in the uncleaved p62 precursor and release of E3 after cleavage.

Animals↗

PubMed1988

Murray Valley encephalitis virus field strains from Australia and Papua New Guinea: studies on the sequence of the major envelope protein gene and virulence for mice.

We have compared the nucleotide sequence of the gene encoding the major envelope (E) protein of a number of Murray Valley encephalitis virus (MVE) isolates from Australia and Papua New Guinea (PNG). The isolates, from widely separated geographic regions, were from four fatal human cases, a heron, and six mosquito pools and covered a period of 25 years. The sequences of the Australian strains were notable for their similarity, showing not more than 1.7% nucleotide sequence divergence in pairwise comparisons. There was 6.8% divergence in the E gene between the two available strains from PNG, and 9-10% divergence between each of the PNG strains and the Australian prototype. These data are consistent with previous conclusions based on HaeIII restriction digest analysis of cDNA to virion RNA (M. Lobigs, I. D. Marshall, R. C. Weir, and L. Dalgarno, 1986, Aust. J. Exp. Biol. Med. Sci. 64, 571-585). We conclude that a single MVE genetic type exists in Australia. Separate foci of MVE evolution appear to exist in PNG, generating greater strain variation. For all MVE isolates the deduced length of the E protein was 501 amino acids. The E protein differed at no more than three positions between any two Australian strains. The PNG strains differed from the Australian strains at 6-11 residues depending on the virus pair. Differences in amino acid sequence did not occur at a position corresponding to a previously demonstrated neutralization determinant in yellow fever virus (M. Lobigs, L. Dalgarno, J. J. Schlesinger, and R. C. Weir, 1987, Virology 161, 474-478). Thus selection for neutralization resistance may not be a major evolutionary pressure in the field situation. In comparisons between the E protein amino acid sequence of the prototype strain and those of a number of other MVE strains, 7 out of 14 differences were at residues seen at the corresponding position for Japanese encephalitis virus (JE), consistent with the close serological relationship of MVE and JE. Five Australian MVE strains and two from PNG were tested for virulence by comparing LD50 values after intraperitoneal and intracranial inoculation of 21-day-old mice; all strains were virulent by this test.

Amino Acid Sequence↗

PubMed1987

Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain).

The location of a major antigenic determinant involved in the neutralization of a flavivirus, yellow fever virus (YF), has been defined in terms of its position in the amino acid sequence of the E protein. Neutralization escape variants of the 17D vaccine strain of YF were selected with two neutralizing monoclonal antibodies. Nucleotide sequencing of the envelope protein genes (E and M) of the variants showed that in each variant there was a single nucleotide change in the E gene leading to a nonconservative amino acid substitution in the E protein at position 71 or 72. The changes are in a region of the E protein which is hydrophilic, rich in cysteine residues, and not conserved between flavivirus subgroups. Since the selecting monoclonal antibodies neutralize attenuated 17D and virulent Asibi strains of YF with equal efficiency (J. J. Schlesinger, M. W. Brandriss, and T. P. Monath, 1983, Virology 125, 8-17), it can be concluded that the neutralization determinant defined for 17D YF is also present in Asibi YF.

Amino Acid Sequence↗

PubMed1986

Genetic analysis of Kunjin virus isolates using HaeIII and TaqI restriction digests of single-stranded cDNA to virion RNA.

The genotypic relatedness of 15 Kunjin (KUN) virus isolates from widely separated geographic regions in Australia was examined at the molecular level. HaeIII and TaqI restriction digest profiles of cDNA transcribed from virion RNA revealed a close genetic similarity between all isolates. We estimate that the nucleotide sequence divergence between any pair of KUN isolates is probably less than 1%. We conclude that a single KUN genetic type has existed in enzootic and epizootic areas of virus activity over an extended time span. The epidemiological implications of these results are discussed.

Animals↗

PubMed1986

Genetic differentiation of Murray Valley encephalitis virus in Australia and Papua New Guinea.

The genetic relatedness of ten Murray Valley encephalitis virus (MVE) isolates from Australia has been examined by comparing HaeIII and TaqI restriction digest profiles of cDNA to virion RNA. The isolates were from the Murray Valley region of south-eastern Australia and from the Ord River region of Western Australia and spanned a period of 23 years (1951-1974). The isolates generated closely similar restriction digest profiles. The extent of similarity suggested that the level of nucleotide sequence divergence between any pair of Australian MVE isolates is probably around 1%. The genetic homogeneity of the MVE isolates contrasts with results obtained for Ross River virus, an alphavirus, using an identical methodological approach; we propose that this difference results from the important role of birds in the life cycle of MVE. Four MVE isolates from three fatal human cases showed small genetic differences one from the other. These isolates did not have a common restriction digest profile which distinguished them from strains obtained from other sources (e.g., from mosquitoes or a heron). The data do not support the view that clinical cases of MVE infection in humans are due to a particular strain of virus although this has not been rigorously excluded. The two available MVE isolates from Papua New Guinea (PNG) were from the Sepik and Port Moresby regions. They generated HaeIII and TaqI restriction digest profiles which were different both from each other and from those of the Australian type. Genetic divergence between the two PNG isolates was estimated to be approximately 6%; divergence between either of the PNG isolates and the Australian type was greater than 6%. Our data suggest that the evolution of MVE in Australia and PNG has proceeded independently and that circulating Australian MVE strains are not systematically re-seeded from regions of endemicity in PNG. Studies on the relatedness of MVE and two close antigenic relatives, Japanese encephalitis virus (JE) and Alfuy virus (ALF), showed that the genetic relatedness between any MVE isolate and JE or ALF is less than that between the most divergent of the MVE isolates, including those from Papua New Guinea.

Animals↗