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M A Gonda

Publications and source records attributed to M A Gonda.

At least 19 recordsLinked to original sources
PubMed1992

Seroprevalence of bovine immunodeficiency-like virus and bovine leukemia virus in a dairy cattle herd.

To determine the prevalence of single vs. dual infection with bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV), sera (n = 95) from a dairy cattle herd were analyzed for anti-BIV and anti-BLV antibodies by an enzyme linked immunosorbent assay. Twenty-one percent (20/95) of samples were BIV-seropositive, while 52% (49/95) of the same samples were BLV-seropositive. A significantly greater percentage of BIV-seronegative samples were BLV-seropositive, 57% (43/75), than were BIV-seropositive samples, 30% (6/20). There was no significant correlation between data ranked from least to greatest amount of anti-viral antibody. Five cattle had persistent lymphocytosis (PL); all five were BLV-seropositive and two were BIV-positive. The mean anti-BLV titer was significantly greater in PL cattle, as compared at non-PL cattle, whereas there was no significant difference between the mean anti-BIV titer in PL cattle, as compared with non-PL cattle. These results provide additional information on the seroprevalence of naturally occurring BIV infection, and indicate that BIV can exist independent of other common infectious agents, such as BLV. Further, the results suggest that infection with BIV is not associated with an increased rate of infection with other infectious agents such as BLV.

Animals↗
PubMed1992

Conservation of amino-acid sequence motifs in lentivirus Vif proteins.

The nonstructural/regulatory genes of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are believed to play an important role in the replication and pathogenesis of these viruses. In HIV-1 and other lentiviruses, the vif (viral infectivity factor) open reading frame (ORF) (also termed sor or Q in some lentivirus genomes) is located in the central region, overlapping the 3' end of the pol ORF, but in a different reading frame. Among the lentiviruses, only equine infectious anemia virus lacks a vif ORF. The predicted Vif protein sequences from 38 lentiviruses were analyzed for the presence of global and local sequence similarity. The Vif proteins of closely related lentiviruses are highly conserved (HIV-1HXB2:HIV-1mn = 91% identity), while those of more distantly related lentirviruses have diverged significantly (HIV-1HXB2:simian immunodeficiency virusmax = 30% identity). A search for local sequence similarity revealed that a unifying feature of predicted lentivirus Vif proteins is the presence of at least one of two short, highly conserved sequence motifs, SL(I/V)X4YX9Y and SLQXLA. SLQXLA was present in 34 of 38 lentiviruses examined, while the remaining four lentiviruses had one (three viruses) or two (one virus) substitutions in this motif (of five total substitutions, three were conservative changes). The SL(I/V)X4YX9Y motif was found only in primate lentiviruses and in bovine immunodeficiency-like virus. Based on these findings, we suggest that the locus designation vif be used to denote all lentivirus ORFs previously called vif, Q, or sor.

Amino Acid Sequence↗
PubMed1992

Expression of bovine immunodeficiency-like virus envelope glycoproteins by a recombinant baculovirus in insect cells.

The bovine immunodeficiency-like virus (BIV) env open reading frame (ORF) contains both sequences encoding env and sequences for exon 1 of the putative rev gene. Recombinant baculoviruses incorporating BIV env ORF sequences were constructed to characterize the expression, processing, and immunogenicity of products of the BIV env ORF in insect cells and to develop reagents to study native BIV Env glycoproteins. A recombinant baculovirus containing the entire env ORF synthesized a nonglycosylated, 20-kDa, BIV-specific protein, apparently unrelated to native BIV Env proteins. In contrast, a recombinant baculovirus containing a truncated env ORF in which the coding sequences for rev exon 1 were deleted synthesized three size classes of glycosylated proteins in insect cells related to the BIV Env precursor (gp145), surface (gp100), and transmembrane (gp45) glycoproteins observed in BIV-infected mammalian cells. Oligomers of recombinant BIV Env proteins also formed in these baculovirus-infected insect cells. Immunofluorescence staining of intact insect cells infected by the baculovirus expressing BIV Env with BIV-specific serum demonstrated that the recombinant Env glycoproteins were expressed on the cell surface. Antisera raised to recombinant Env glycoproteins immunoprecipitated native gp145, gp100, and gp45 in BIV-infected bovine cells similar to sera from animals naturally or experimentally infected with BIV.

Animals↗
PubMed1992

Immunological characterization of the gag gene products of bovine immunodeficiency virus.

The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.

Amino Acid Sequence↗
PubMed1992

Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat.

Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.

Animals↗
PubMed1992

Persistent infection of rabbits with bovine immunodeficiency-like virus.

Chronic infection of rabbits was induced by a single intraperitoneal injection of bovine immunodeficiency-like virus (BIV)-infected cells. Ten BIV-infected animals were monitored serologically for up to 2 years. Results of serologic and virus rescue assays indicated that all animals became infected and demonstrated a rapid and sustained BIV-specific humoral response. BIV was rescued by cocultivation from spleen, lymph nodes, and peripheral blood leukocytes of infected animals. Viral DNA in immune tissues was confirmed by polymerase chain reaction amplification of BIV sequences. These data and specific immunohistochemical staining of mononuclear cells of the spleen for BIV antigen suggest that the infection is targeted to immune system cells.

Animals↗
PubMed1992

The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif.

We show that the open reading frame transcribed from the unr gene (immediately upstream of N-ras) in mammals consists of multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif found in prokaryotic cold-shock proteins, and eukaryotic DNA-binding proteins. Alignment of the CSD sequences of unr with those from other proteins reveals a core of similarity for which a consistent secondary structure prediction can be derived. This prediction suggests that the CSD consists primarily of beta-sheet, in contrast to most known eukaryotic DNA-binding proteins. Sequence analysis of the 3' end of the guinea pig unr gene shows that the core of one CSD repeat is encoded in a single exon, consistent with the modular assembly of the gene from ancestral CSD-coding units.

Amino Acid Sequence↗
PubMed1991

Structure and transcriptional status of bovine syncytial virus in cytopathic infections.

The genomic structure of bovine syncytial virus (BSV), a virus commonly infecting cattle, was examined in order to gain insights into the nature of viral DNA (vDNA) intermediates and the transcriptional status of the virus in cytopathic infections. In dog Cf2Th cells, the DNA intermediate of BSV was found to exist predominantly as linear unintegrated vDNA (uvD) molecules. The uvD molecules were cloned directly from total cellular DNA by addition of EcoRI linkers and subsequent ligation into the phage lambda EMBL4 vector. Of the eleven clones characterized, seven were full length as judged by restriction fragment analysis. The remaining four clones showed varying degrees of heterogeneity in the form of internal deletions or terminal truncations. Heat denaturation and S1 nuclease analyses were used to show that vDNA isolated from Cf2Th cells contains a single-stranded (ss) gap structure located in the central region of the genome. In addition, a double-stranded (ds) 1.3-kb fragment is observed in this vDNA population. Northern-blot analysis revealed the presence of virus-specific transcripts of 11.0, 6.4, 2.8, and 2.4 kb. This suggests that BSV is similar in complexity to the lentiviruses in terms of linear intermediates containing ss gap structures, and the presence of several RNA transcripts which may direct complex regulatory functions.

Animals↗
PubMed1991

Analysis of the transcription pattern and mapping of the putative rev and env splice junctions of bovine immunodeficiency-like virus.

The bovine immunodeficiency-like virus (BIV) genome contains the obligatory structural genes of all retroviruses and, in addition, the complex central region of lentiviruses; this novel region may code for at least five nonstructural/regulatory genes in BIV (K.J.Garvey, M.S. Oberste, J.E. Elser, M.J. Braun, and M.A. Gonda, Virology 175:391-409, 1990). As a prelude to determining the function of these novel open reading frames, the transcriptional pattern of BIV was studied by Northern analysis of RNA from BIV-infected cells. Five size classes of BIV-specific RNAs of 8.5, 4.1, 3.8, 1.7, and 1.4 kb were detected. The 8.5-kb RNA contains sequences from all regions of the genome; it is the virion RNA and probably serves as the gag-pol transcript as well. By using gene-specific probes, subgenomic viral RNAs of 3.8, 1.7, and 1.4 kb were tentatively identified as the env, tat, and rev spliced messages, respectively. The 4.1-kb RNA could not be unambiguously identified but may encode vif. The hybridization patterns of the putative tat and rev mRNAs suggest that they are the products of multiple splicing events. Discrete transcripts for the BIV W and Y central region open reading frames were not defined. The characterization of partial cDNA clones has permitted the mapping of the env and putative rev splice junctions.

Amino Acid Sequence↗
PubMed1991

Isolation of bovine leukemia virus infected endothelial cells from cattle with persistent lymphocytosis.

Incubation of adherent cells derived from peripheral blood mononuclear cells of cattle naturally infected with bovine leukemia virus (BLV) led to the establishment of three, persistently infected, primary cell cultures. These cultures were obtained exclusively from animals exhibiting persistent lymphocytosis, and not from uninfected or infected, hematologically normal cattle. The cells contained monoclonally integrated, full length BLV provirus, indicating that each culture resulted from clonal expansion of a single cell. They expressed high levels of all BLV specific mRNAs and showed intracellular reactivity to antibodies directed to viral gag and env proteins. Viral particle morphogenesis was highly restricted as determined by low levels of reverse transcriptase activity in cell supernatants and the paucity of viral particles on the cell surface. Analysis of cellular antigenic determinants, using monoclonal antibodies to bovine leukocyte differentiation and major histocompatibility complex antigens, was inconclusive. Cytochemical, morphologic, and ultrastructural analyses were consistent with endothelial cells and they exhibited the distinctive functional capacity of endothelial cells derived from specialized postcapillary venules, which constitute sites of lymphocyte extravasation. These data suggest that infection of these endothelial cells may be involved in the development of persistent lymphocytosis in BLV-infected animals.

Animals↗
PubMed1990

Characterization of virus-like particles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus.

The entire gag gene of the bovine immunodeficiency-like virus (BIV) was inserted behind the strong polyhedron promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). The resultant recombinant baculovirus (AcNPV-BIVgag) was used to infect insect cells in order to overexpress and characterize BIV gag gene products. The infection resulted in the high-level expression of a protein similar in size to the predicted BIV gag precursor (Pr53gag). BIV Pr53gag was detected in AcNPV-BIVgag-infected insect cells and in culture supernatants. Electron microscopy of these cells revealed an abundance of virus-like particles (VLPs) in the cytoplasm, budding from the cell membrane, and free in the culture medium. The size and morphology of the VLPs were similar to those of the immature forms of BIV observed in infected mammalian cells. The VLPs sedimented at a density of 1.16 g of sucrose per milliliter in linear gradients and were shown to contain the majority of the supernatant Pr53gag. Antigenic determinants on Pr53gag from VLPs were recognized by BIV and HIV-1 antiserum, and serum from rats immunized with VLPs reacted with recombinant and viral BIV Pr53gag and processed products. The protease (PR) activity in BIV virions was capable of processing recombinant Pr53gag; this activity was blocked by pepstatin A, a potent aspartyl PR inhibitor. Baculovirus-expressed BIV Pr53gag appears to be an excellent source of gag precursor; it may prove useful for structural studies and enable the development of assays to detect retroviral PR inhibitors. The data further suggest that unprocessed BIV Pr53gag plays a major role in the assembly of BIV particles. The expression of other BIV structural genes in insect cells may prove instructive in the study of molecular events involved in the assembly and processing of these BIV proteins.

Animals↗
PubMed1990

Nucleotide sequence and genome organization of biologically active proviruses of the bovine immunodeficiency-like virus.

The complete nucleotide sequences and translations of major open reading frames (ORF) of two distinct, infectious, proviral molecular clones (106 and 127) of the bovine immunodeficiency-like virus (BIV), obtained from a single virus isolation, were determined and compared. The genomes of BIV 127 and 106 are 8482 and 8391 nucleotides (nt), respectively, in the form predicted for the viral RNA. The structural organization of the genomes of BIV 127 and 106 are identical to one another and most similar to that of the lentivirus subfamily of retroviruses. In addition to gag, pol, and env genes, the BIV genome contains five short ORFs between and overlapping pol and env in the "central region," a hallmark of the lentiviruses which is believed to play an important role in their pathogenesis. Three of the short ORFs in the central region of BIV have been identified by location and structural similarity to the nonstructural/regulatory genes (vif, tat, and rev) of other lentiviruses; we also discovered two unique ORFs, termed W and Y, which may serve as exons for novel genes. BIV does not have the nef gene found in primate lentivirus genomes. The proviral LTR of BIV 127 is 589 nt, contains regulatory signals for initiation, enhancement, and termination of viral transcription, and has sequences related to the Sp1 and NF-kappa B binding sites. A major deletion (87 nt) in the env gene and 2 minor deletions (2 nt each) in the R regions of the LTRs account for the smaller size of clone 106. Numerous point mutations were also present; some caused coding substitutions that were most prevalent in the env encoding ORF. These data suggest that, within a single virus isolate, BIV displays extensive genomic variation. These infectious clones of BIV represent well-defined tools with which to analyze the function of the various ORFs and to dissect the molecular mechanisms of replication and pathogenesis.

Amino Acid Sequence↗
PubMed1990

Development of the bovine immunodeficiency-like virus as a model of lentivirus disease.

The bovine immunodeficiency-like virus (BIV) is morphologically, serologically, and genetically related to the lentivirus subfamily of retroviruses which includes human and simian immunodeficiency viruses and other lentiviruses causally associated with debilitating diseases of domestic animals. There are many parallels in the biology and pathologic characteristics of BIV infections with those of HIV that make its development as a model of HIV-like infection and disease potentially attractive. In order to obtain a better understanding of the molecular basis of BIV-induced disease, two biologically active proviruses of BIV were molecularly cloned and sequenced. The BIV genome is 9.0 kilobases in the form of the proviral DNA. It contains the obligate retroviral structural genes, gag, pol, and env. In addition, in the BIV central region, between and overlapping pol and env, there are five potential coding regions for non-structural/regulatory genes; three are analogous to vif, tat, and rev in HIV and two, called W and Y, are unique to BIV. There is no coding region analogous to nef in BIV. Sequence comparisons of two functional proviruses obtained from the DNA of cells carrying an infection from a single virus isolation indicate that the genome of BIV is highly variable within a single biological isolate. Moreover, the greatest number of substitutions occur in the env gene. The results suggest the presence of multiple genotypes which may be of significance in defining the disease potential of a BIV isolate. These clones will be useful in dissecting the replicative cycle and mechanisms of pathogenesis of BIV in various animal models.

Amino Acid Sequence↗
PubMed1990

Characterization of clones of HIV-1 infected HuT 78 cells defective in gag gene processing and of SIV clones producing large amounts of envelope glycoprotein.

Single-cell clones of HIV-1 (FRE-3) or SIV/Mne infected HuT 78 cells were obtained by plating dilutions of virally infected HuT 78 cells on a monolayer of sheep choroid plexus cells in 96-well microtiter plates. Several of these clones produce HIV-1 virus mutants that accumulate the gag precursor polyprotein and lack a functional protease. These protease-deficient viruses are non-infectious and consist of aberrant "immature" virus particles as determined by electron microscopy. Several SIV mutants are also described that produce large amounts of either the envelope glycoprotein gp120 or the nucleic acid binding gag protein. These mutants are useful for the purification of these retroviral proteins, in developing assays of protease inhibitors, and in preparing SIV envelope protein vaccines.

Animals↗
PubMed1989

Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution.

The gene for bovine interphotoreceptor retinoid-binding protein (IRBP) has been cloned, and its nucleotide sequence has been determined. The IRBP gene is about 11.6 kilobase pairs (kb) and contains four exons and three introns. It transcribed into a large mRNA of approximately 6.4 kb and translated into a large protein of 145,000 daltons. To prove the identity of the genomic clone, we determined the protein sequence of several tryptic and cyanogen bromide fragments of purified bovine IRBP protein and localized them in the protein predicted from its nucleotide sequence. There is a 4-fold repeat structure in the protein sequence with 30-40% sequence identity and many conservative substitutions between any two of the four protein repeats. The third and fourth repeats are the most similar pair. All three of the introns in the IRBP gene fall in the fourth protein repeat. Two of the exons, the first and the fourth, are large, 3173 and 2447 bases, respectively. The introns are each about 1.5-2.2 kb long. The human IRBP gene has a sequence that is similar to one of the introns from the bovine gene. The unexpected gene structure and protein repeat structure in the bovine gene lead us to propose a model for the evolution of the IRBP gene.

Amino Acid Sequence↗
PubMed1989

Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector.

Infectious retrovirus particles consist of a core structure containing RNA and gag-pol polypeptides surrounded by a lipid membrane studded with env proteins. A recombinant vaccinia virus was designed to express the entire gag-pol precursor protein of the human immunodeficiency virus type 1. Synthesis and processing of gag proteins occurred in mammalian cells infected with this live recombinant virus, and reverse transcriptase was detected largely in the medium. Electron micrographs revealed immature retrovirus-like particles budding from the plasma membrane and extracellular particles with morphological characteristics of immature and mature human immunodeficiency virus. The latter contained functional reverse transcriptase as well as processed p24 and p17 gag polypeptides. Thus, assembly and maturation of human immunodeficiency virus-like particles can occur in the absence of either infectious RNA molecules or env proteins. The production of noninfectious virus-like particles by expression vectors should be useful for biochemical studies and could provide a safe source of material for the development of vaccines.

Animals↗
PubMed1988

Structure of the gene encoding the beta-subunit of chicken prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) converts peptidyl proline to peptidyl hydroxyproline in procollagen polypeptides during collagen biosynthesis. The active enzyme is a tetramer which is composed of two pairs of non-identical subunits in a molecular form of alpha 2 beta 2. In addition to the tetrameric prolyl 4-hydroxylase (alpha 2 beta 2), the free beta-subunit is also found inside cells. Recently it was shown that the beta-subunit of prolyl 4-hydroxylase is identical to the protein disulfide isomerase and cellular thyroid hormone-binding protein. We previously isolated and characterized cDNAs of the beta-subunit of chicken prolyl 4-hydroxylase. The cDNA of beta-subunit was used to screen a chicken genomic DNA library constructed with the lambda EMBL-3 vector. Two clones, lambda gCPH beta-22 and beta-50, were isolated and characterized by restriction enzyme analysis, heteroduplex analysis, and nucleotide sequencing. The results showed that the 2.5-kb mRNA of the beta-subunit is divided into eleven exons and that the gene is 9.0 kb long. The gene contains consensus sequence for TATA at -24 bp and four CAAT at -57, -157, -194 and -223 bp in the 5' end flanking sequence of the transcription start point. In addition, there are three GC boxes upstream from the TATA box and four GC boxes in the first intron. This is similar to the structure of the alpha 1(I) collagen coding gene (COL1A1). These elements may interact with nuclear factors and play important roles in expression regulation of the beta-subunit gene as has been described in COL1A1.

Amino Acid Sequence↗