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M D Baron

Publications and source records attributed to M D Baron.

At least 19 recordsLinked to original sources
PubMed2001

Rinderpest virus C and V proteins interact with the major (L) component of the viral polymerase.

Rinderpest virus, like other Morbilliviruses, expresses three proteins from the single P gene. In addition to the P protein, which interacts both with the viral polymerase (L) and the nucleocapsid (N) protein, the virus expresses a C and a V protein from the same gene. The functions of these two proteins in the viral life cycle are not clear. Although both C and V proteins are dispensable, in that viable viruses can be made that express neither, each seems to play a role in optimum viral replication. We have used the yeast-two hybrid system, binding to coexpressed fusions of C and V to glutathione-S-transferase, and studies of the native size of these proteins to investigate interactions of the rinderpest virus C and V proteins with other virus-encoded proteins. The V protein was found to interact with both the N and L proteins, while the C protein was found to bind to the L protein, and to self-associate in high-molecular-weight aggregates.

Animals↗
PubMed2000

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.

Animals↗
PubMed2000

Development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein.

In order to effectively control and eliminate rinderpest, a method is required to allow serological differentiation between animals that have been vaccinated and those which have recovered from natural infection. One way of doing this would be to engineer the normal vaccine to produce a genetically marked rinderpest virus (RPV) vaccine. We constructed two modified cDNA clones of the RPV RBOK vaccine strain with the coding sequence of the green fluorescent protein (GFP) gene inserted as a potential genetic marker. RPVINS-GFP virus was designed to produce independent and high level expression of GFP inside infected cells, whilst the GFP expressed by RPVSIG-GFP virus was designed to be efficiently secreted. Infectious recombinant virus was rescued in cell culture from both constructs. The effectiveness of these viruses in stimulating protective immunity and antibody responses to the marker protein was tested by vaccination of cattle and goats. All of the vaccinated animals were completely protected when challenged with virulent virus: RPV in cattle or peste-des-petits ruminants virus in the goats. ELISA showed that all of the animals produced good levels of anti-RPV antibodies. Three of the four cattle and the two goats vaccinated with RPVSIG-GFP produced detectable levels of anti-GFP antibodies. In contrast, no anti-GFP antibodies were produced in the four cattle and two goats vaccinated with RPVINS-GFP. Therefore, secretion of the GFP marker protein was absolutely required to elicit an effective humoral antibody response to the marker protein.

Animals↗
PubMed2000

Recovery and characterization of a chimeric rinderpest virus with the glycoproteins of peste-des-petits-ruminants virus: homologous F and H proteins are required for virus viability.

Rinderpest (RP) and peste-des-petits-ruminants (PPR) are two important diseases of domestic ruminants. To improve on currently available vaccines against PPR, we have created cDNA copies of the RP virus genome in which either the fusion (F) or hemagglutinin (H) gene, or both, was replaced with the corresponding gene from PPR virus. It was necessary to develop a modified rescue system in which the T7 RNA polymerase was provided by a recombinant fowlpox virus and the entire rescue procedure took place in Vero cells before we could obtain live virus from these chimeric constructs. No virus was recovered when only one of the glycoprotein genes was changed, but a chimeric virus containing both F and H genes from PPR virus was reproducibly rescued from cDNA, indicating that a virus-specific functional interaction takes place between the F and H proteins. The rescued virus expressing the PPR glycoproteins grew more slowly in tissue culture than either parental virus and formed abnormally large syncytia. Goats infected with the chimera showed no adverse reaction, as assessed by clinical signs, temperature, leukocyte count, virus isolation, and serology, and were protected from subsequent challenge with wild-type PPR virus.

Animals↗
PubMed2000

Recombinant rinderpest vaccines expressing membrane-anchored proteins as genetic markers: evidence of exclusion of marker protein from the virus envelope.

Rinderpest virus (RPV) causes a severe disease of cattle resulting in serious economic losses in parts of the developing world. Effective control and elimination of this disease require a genetically marked rinderpest vaccine that allows serological differentiation between animals that have been vaccinated against rinderpest and those which have recovered from natural infection. We have constructed two modified cDNA clones of the vaccine strain RNA genome of the virus, with the coding sequence of either a receptor site mutant form of the influenza virus hemagglutinin (HA) gene or a membrane-anchored form of the green fluorescent protein (GFP) gene (ANC-GFP), inserted as a potential genetic marker. Infectious recombinant virus was rescued in cell culture from both constructs. The RPVINS-HA and RPVANC-GFP viruses were designed to express either the HA or ANC-GFP protein on the surface of virus-infected cells with the aim of stimulating a strong humoral antibody response to the marker protein. In vitro studies showed that the marker proteins were expressed on the surface of virus-infected cells, although to different extents, but neither was incorporated into the envelope of the virus particles. RPVINS-HA- or RPVANC-GFP-vaccinated cattle produced normal levels of humoral anti-RPV antibodies and significant levels of anti-HA or anti-GFP antibodies, respectively. Both viruses were effective in stimulating protective immunity against RPV and antibody responses to the marker protein in all animals when tested in a cattle vaccination trial.

Animals↗
PubMed2000

Rinderpest viruses lacking the C and V proteins show specific defects in growth and transcription of viral RNAs.

Rinderpest virus is a morbillivirus and the causative agent of an important disease of cattle and wild bovids. The P genes of all morbilliviruses give rise to two proteins in addition to the P protein itself: use of an alternate start translation site, in a second open reading frame, gives rise to the C protein, while cotranscriptional insertion of an extra base gives rise to the V protein, a fusion of the amino-terminal half of P to a short, highly conserved, cysteine-rich zinc binding domain. Little is known about the function of either of these two proteins in the rinderpest virus life cycle. We have constructed recombinant rinderpest viruses in which the expression of these proteins has been suppressed, individually and together, and studied the replication of these viruses in tissue culture. We show that the absence of the V protein has little effect on the replication rate of the virus but does lead to an increase in synthesis of genome and antigenome RNAs and a change in cytopathic effect to a more syncytium-forming phenotype. Virus that does not express the C protein, on the other hand, is clearly defective in growth in all cell lines tested, and this defect appears to be related to a decreased transcription of mRNA from viral genes. The phenotypes of both individual mutant virus types are both expressed in the double mutant expressing neither V nor C.

Animals↗
PubMed1998

Morbillivirus downregulation of CD46.

There is evidence that CD46 (membrane cofactor protein) is a cellular receptor for vaccine and laboratory-passaged strains of measles virus (MV). Following infection with these MV strains, CD46 is downregulated from the cell surface, and consequent complement-mediated lysis has been shown to occur upon infection of a human monocytic cell line. The MV hemagglutinin (H) protein alone is capable of inducing this downregulation. Some wild-type strains of MV fail to downregulate CD46, despite infection being prevented by anti-CD46 antibodies. In this study we show that CD46 is also downregulated to the same extent by wild-type, vaccine, and laboratory-passaged strains of rinderpest virus (RPV), although CD46 did not appear to be the receptor for RPV. Expression of the RPV H protein by a nonreplicating adenovirus vector was also found to cause this downregulation. A vaccine strain of peste des petits ruminants virus caused slight downregulation of CD46 in infected Vero cells, while wild-type and vaccine strains of canine distemper virus and a wild-type strain of dolphin morbillivirus failed to downregulate CD46. Downregulation of CD46 can, therefore, be a function independent of the use of this protein as a virus receptor.

Animals↗
PubMed1997

Rescue of rinderpest virus from cloned cDNA.

Rinderpest virus is a morbillivirus and is the causative agent of a widespread and important disease of cattle. The viral genome is a single strand of RNA in the negative sense. We have constructed plasmids containing cDNA copies of the 5' and 3' termini of the virus separated by a reporter gene and have shown that antigenome-sense RNA transcripts of these model genomes can be replicated, transcribed, and packaged by helper virus, both rinderpest virus and the related measles virus. Further, these genome analogs can be replicated and transcribed by viral proteins expressed from cDNA clones by using a recombinant vaccinia virus expressing T7 RNA polymerase (MVA-T7). Using this latter system, we have rescued live rinderpest virus from a full-length cDNA copy of the genome of the RBOK vaccine strain. The recombinant virus appears to grow in tissue culture identically to the original virus.

Animals↗
PubMed1996

The genome sequence of the virulent Kabete 'O' strain of rinderpest virus: comparison with the derived vaccine.

We have compared the complete genome sequences of the vaccine strain of rinderpest virus and the virulent strain from which it was derived. Only 87 bases differed between the two genomes (0.55%). Possibly significant differences in amino acid sequence were found in the N, P, F, H and L proteins. A number of differences were also found in the leader region (3' end of the genome), whilst the trailer region appears to be more conserved. In addition, the length of the genome was found in both cases to be 15882, an exact multiple of six, fulfilling predictions made earlier based on work with Sendai and measles viruses.

Base Sequence↗
PubMed1995

The sequence of the N and L genes of rinderpest virus, and the 5' and 3' extra-genic sequences: the completion of the genome sequence of the virus.

We have sequenced the nucleocapsid (N) and polymerase (L) genes of the vaccine strain of rinderpest, and the 5' and 3' terminal domains of the genome. Together with previously published data, this completes the sequence of the entire genome of rinderpest virus. The L gene is identical in length to that of measles virus, encoding a 2183 amino acid protein with a calculated molecular weight of 248,100. The L protein sequence of three morbilliviruses is highly conserved, greater than 76% of residues being identical or conserved in all sequences. The N protein was, as for other sequenced genes, essentially identical to that of the virulent parent. The viral genome is 15,881 bases in length, similar to that of measles virus and slightly longer than that of canine distemper virus. The terminal sequences of the genome and those at the gene boundaries were compared to the analogous regions of other morbilliviruses and representatives of related groups of paramyxoviruses.

Amino Acid Sequence↗
PubMed1995

Editing of morbillivirus P gene transcripts in infected animals.

RNA editing in the Morbillivirus genus in vivo was investigated by applying a polymerase chain reaction-based primer extension technique to measure the edited and non-edited mRNA transcripts. In this genus of the Paramyxoviridae the P gene transcript is altered by the co-transcriptional addition of one extra G residue to produce the mRNA for the V non-structural protein. Using tissues of phocine distemper virus (PDV) infected seals, canine distemper virus (CDV) infected dogs and rinderpest virus (RPV) infected cattle, it was demonstrated that editing occurs in vivo. The P:V mRNA ratios were generally similar to those found in tissue culture infections with the same virus and a minor fraction of transcripts had 2-4 extra G residues. In one seal brain infected with PDV the ratio of P:V mRNA was reversed but no differences were found in the levels of mRNA editing in different tissues from the same animal infected with CDV or RPV. However, variation was seen between animals infected with different isolates of RPV and even between animals infected with the same isolate of RPV.

Animals↗
PubMed1995

Sequencing and analysis of the nucleocapsid (N) and polymerase (L) genes and the terminal extragenic domains of the vaccine strain of rinderpest virus.

The nucleocapsid (N) and polymerase (L) genes of the vaccine strain of rinderpest, and the 5' and 3' terminal domains of the genome have been sequenced. Together with previously published data, this completes the sequence of the entire genome of rinderpest virus. The viral genome is 15,881 bases in length, similar to that of measles virus and slightly longer than that of canine distemper virus. The L gene is identical in length to that of measles virus, encoding a 2183 amino acid protein with a calculated M(r) of 248,100. The L protein sequence of morbilliviruses is highly conserved, more than 75% of residues being identical or conserved in all three sequences currently available. The N protein was, as for the other sequenced genes where comparison is possible, essentially identical to that of the virulent parent. In addition, we have determined the terminal sequences of two virulent strains of rinderpest and compared the sequences of virulent and non-virulent strains.

Amino Acid Sequence↗
PubMed1994

Cloning and sequence analysis of the matrix (M) protein gene of rinderpest virus and evidence for another bovine morbillivirus.

We have cloned and sequenced the entire M gene of the vaccine strain of rinderpest virus and that of the virulent Kabete "O" strain from which it was derived. The sequences of these two genes are essentially identical (99% at the nucleotide level), but were very different from a previously published Kabete O M gene sequence (M. Limo and T. Yilma, 1990, Virology 175, 323-327). Inspection of the nucleotide and deduced amino acid sequences of known morbillivirus M genes showed that the earlier sequence was clearly from a morbillivirus, but neither from rinderpest virus nor from peste des petits ruminants virus.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence comparisons of the fusion protein gene from virulent and attenuated strains of rinderpest virus.

We have cloned and sequenced the entire fusion (F) protein gene of the RBOK vaccine strain of rinderpest virus and the coding regions for the F genes of two mild field isolates of the virus from Africa. Analysis of the nucleotide and the predicted amino acid sequences showed that the vaccine virus was more than 99% identical in the protein coding region to the virulent Kabete O strain from which it was derived, whereas the field isolates differed by 10 to 12% from each other and from the vaccine strain. No changes were found in the F protein which could explain attenuation of the vaccine; however, each of the mild field isolates had amino acid changes in important functional areas which may be related to their attenuated phenotype.

Africa↗
PubMed1993

Cloning and sequence analysis of the phosphoprotein gene of rinderpest virus.

We have cloned several cDNAs derived from the P gene of rinderpest virus. One of these, derived from a bicistronic N-P mRNA, has been sequenced in its entirety. Sequencing of a section of the others, and comparison with the genome sequence, showed that P gene transcripts, as for other morbilliviruses, were variable; non-templated Gs could be added at a site resembling the normal stop transcription site. Primer extension analysis showed that about half the transcripts were edited. Sequences of the P, C and V proteins encoded by the normal and edited transcripts were compared with those of other morbilliviruses and with those of the more distantly related paramyxoviruses.

Amino Acid Sequence↗
PubMed1992

Intracellular transport of rubella virus structural proteins expressed from cloned cDNA.

The structural proteins of rubella virus consist of a nucleocapsid protein (C) and two membrane-embedded spike glycoproteins (E1 and E2). Since many reports have suggested that rubella virus buds intracellularly, we have examined the intracellular transport of the structural proteins in the absence of virion formation, particularly whether the membrane glycoproteins are retained inside the cell or are transported to the cell surface. We have expressed the structural proteins from cloned cDNA either alone or in different combinations, have examined the intracellular location of the proteins by immunofluorescence and using biochemical methods, and have looked for plasma membrane-localized E1 or E2 using a cell surface biotinylation assay. The C protein was found in the Golgi complex when expressed with E2 and E1; without the membrane glycoproteins, C appeared to remain in the endoplasmic reticulum (ER). When expressed alone, E1 was retained in a pre-Golgi compartment, and was not detected at the cell surface in any cell line. When E2 was expressed alone a small fraction could be detected at the cell surface, but the majority was retained intracellularly, apparently in the ER and the Golgi. Both proteins were transported to the surface when they were expressed together, albeit with low efficiencies in all cell lines. These data suggest that, although neither glycoprotein carries a dominant intracellular retention signal, E2 and E1 are largely retained in the Golgi even when present as a transport-competent heterodimer.

Amino Acid Sequence↗
PubMed1991

Oligomerization of the structural proteins of rubella virus.

Rubella virus contains, in addition to its RNA genome, a nucleocapsid protein (C) and two membrane proteins (E2 and E1). We have studied the association of these proteins during viral assembly and when expressed from cDNA constructs. The C protein was found to dimerize very shortly after synthesis; this dimer became disulfide-linked in the virion. Formation of the dimer was independent of the presence of other RV proteins. The membrane glycoproteins formed an E2E1 heterodimer, a minor fraction of which was also found to be disulfide-linked in the virion. This heterodimer also formed when the two proteins were coexpressed from cloned cDNA. Formation of the heterodimer preceded the transport of E2 to the Golgi, as judged by modification of the protein by Golgi-located enzymes. In the absence of E2, the E1 protein was slowly converted to high molecular weight aggregates.

Capsid↗