Matthew A. Gonda

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, thevif (viral infectivity factor) open reading frame (ORF) (also termedsor orQ in some lentivirus genomes) is located in the central region, overlapping the 3′ end of thepol ORF, but in a different reading frame. Among the lentiviruses, only equine infectious anemia virus lacks avif 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 virusmac=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 designationvif be used to denote all lentivirus ORFs previously calledvif, Q, orsor.

Production of recombinant MART-1 proteins and specific antiMART-1 polyclonal and monoclonal antibodies: use in the characterization of the human melanoma antigen MART-1

Recombinant human MART-1 protein was produced by bacterial and baculoviral-insect cell expression systems. By immunization with bacterial MBP-MART-1 fusion protein or MBP cleaved MART-1 protein, a rabbit polyclonal and two murine monoclonal antibodies specific for MART-1 were produced. These antibodies specifically detected MART-1 in immuno-precipitation, Western blotting, flow cytometric assays and in immunohistochemical analysis of tissue sections. They also stained cytoplasmic components in melanocytes and most melanoma cells in frozen or paraffin embedded tissue sections, indicating that these antibodies may be useful for the diagnosis of melanoma. One of the monoclonal antibodies M2-7 C10 recognized only human MART-1, but the other monoclonal antibody M2-9 E3 recognized both human and murine MART-1. The size of the human MART-1 molecule detected by SDS-PAGE with these antibodies was approximately 18 kDa, suggesting possible posttranslational modifications in the MART-1 protein. Subcellular fractionation studies suggested that MART-1 was present in melanosomes and endoplasmic reticulum, although known melanogenic enzymatic activities were not detected in the MART-1 protein. These reagents may be useful for biological studies on melanocytes and melanoma cells as well as for the development and monitoring of immunotherapy for patients with melanoma.

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.

Molecular cloning of biologically active proviruses of bovine immunodeficiency-like virus

A series of independent proviral molecular clones of bovine immunodeficiency-like virus (BIV) obtained from a genomic library of BIV-infected bovine cell DNA were physically and biologically characterized. Heteroduplex mapping shows that two of these BIV clones (106 and 127) contain uninterrupted proviral sequences approximately 9.0 kb in length, flanked by nonhomologous bovine cellular sequences. Microinjection of purified DNA from BIV clone 106 or 127 into susceptible bovine cells produces virus-specific cytopathic effects, including syncytium induction, supernatant reverse transcriptase activity, and infectious virus particle formation, similar to the effects produced by parental virus stock. Using restriction enzyme mapping, it was determined that the two infectious clones share 13 of 14 sites mapped within the provirus; thus, based on this criterion, the two clones are nearly identical, with the exception of a single polymorphic site recognized in the 3 half of the genome. BIV appears to be an exogenous pathogenic virus, because Southern hybridization analyses detected no endogenous sequences related to BIV in DNA from a variety of uninfected bovine cells and tissues. Most of the BIV-related DNA found in cells 96 hr after infection is present as linear unintegrated viral DNA, although the presence of host flanking sequences in our proviral clones indicates that integration takes place. These biologically active clones of BIV will be of use in defining further the mechanisms of BIV pathogenesis and in engineering specific diagnostic reagents to determine the prevalence of BIV in cattle populations.

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.

Encephalitis, lymphoid tissue depletion and secondary diseases associated with bovine immunodeficiency virus in a dairy herd

Encephalitis, lymphoid tissue depletion and secondary infections occurred over a 5-yr-period in Holstein cows infected with bovine immunodeficiency virus (BIV). There were 59 cattle studied, the majority during 1991, when a severe environmental stress occurred, each with one or more primary causes of death, natural or by euthanasia, and most with several secondary diseases. The encephalitis was characterized by meningeal, perivascular and parenchymal infiltration with lymphocytes, occasional plasma cells and macrophages with perivascular edema in some cows. Affected areas included the cerebrum, cerebellum, and spinal cord with no particular distribution pattern recognized. The lymphoid depletion was primarily an absence of follicular development in nodes draining regions with secondary infections such as chronic mastitis and chronic suppurative pododermatitis. Paucity of lymphocytes in thymic-dependent regions of lymph nodes and the spleen suggested a primary depletion of T cells. Secondary infections were often multiple with each cow having several minor conditions, usually considered short-term and treatable. These included mastitis and pododermatitis, with many cows having non-responding abscesses, cellulitis and myositis attributed to injection site infections. A large number of the cattle had parturition difficulties such as dystocia, obturator paralysis, and metritis. Pulmonary, cardiovascular, and intestinal disease were recognized as both primary and secondary disease conditions. There was a high level of infection with bovine leukemia virus with 4 of the 59 cattle having lymphosarcoma. Under practical conditions, the infection with BIV has a different effect on the host than has been observed under experimental conditions. The presence of BIV combined with the stresses associated with parturition and a modern dairy production system were considered causal for the development of untreatable secondary diseases in immunocompromised cattle. The peak incidence in 1991 was attributed to increased environmental stress during renovation of the barn facility. During this time the cattle were kept on open pasture, exposed to an extremely wet winter, and spring weather conditions. The effect of co-infection with bovine leukemia virus, the influence of immunocompromise on the chronicity of mastitis, the relationship with laminitis and pododermatitis, and several questions related to viral transmission, complementarism with bovine leukemia virus, viral reactivation and immunoprophylaxis all remain as viable avenues for future investigations.

Serologic evidence for bovine immunodeficiency virus infection in France

We report herein on the first serologic detection of antibodies to bovine immunodeficiency virus (BIV) in France. Serum samples from dairy and beef cattle from southwestern and western France (Landes and Vendée) were tested using a western blot assay with a recombinant 53 kDa gag precursor derived from the Louisiana BIV R29 isolate. We performed our study on the oldest animals from 37 different herds that were under serologic follow up for previous bovine leukemia virus infection. Overall, 398 selected bovine sera were assayed and 15 serum samples from 8 herds reacted with the recombinant 53 kDa BIV R29 gag. Interestingly, reactions obtained with French sera were weaker than with positive Louisiana sera, a finding that may indicate the occurrence of distinct French and Louisiana BIV variants.

Natural and Experimental Bovine Immunodeficiency Virus Infection in Cattle

Since 1989, the LSU dairy herd, with its high seroprevalence of BIV, was recognized to have a high incidence of common diseases that reduced the economic viability of the dairy. The herd had a high percentage of cows with encephalitis associated with depression and stupor, alteration of the immune system associated with secondary bacterial infections, and chronic inflammatory lesions of the feet and legs. The occurrence of disease problems was associated with the stresses of parturition and early lactation and/or with unusual environmental stress cofactors.

Feline oncornavirus-associated cell membrane antigen: A viral and not a cellularly coded transformation-specific antigen of cat lymphomas

The feline oncornavirus-associated cell membrane antigen (FOCMA) was defined as a tumor antigen common to cat lymphomas and fibrosarcomas induced by feline leukemia virus (FeLV) and feline sarcoma virus (FeSV), respectively. The antigen was recognized by sera from cats thought to be resistant to leukemogenesis. We report here that a common denominator in the activity of naturally occurring viremic cat antisera to FOCMA is, in fact, their reactivity to FeLV C antigenic determinants. The cat antisera, monoclonal antibodies to FOCMA, and monoclonal antibodies to FeLV C, all reacted in immunofluorescence assays with FeLV C-infected cells and immunoprecipitated a molecule electrophoretically indistinguishable from envelope glycoprotein of FeLV. Viremic cat antisera to FOCMA bound to budding virus particles of FeLV C-infected cells, even though some of them could not be absorbed by mature virion proteins. Thus, the unusual feature of cat antibodies to FOCMA is their binding to nascent but not to mature virus particles. FOCMA-positive cat lymphomas expressed antigenic determinants of FeLV-C gp70, with or without productive infection. FeLV-negative tumors not expressing FeLV C gp70 were also FOCMA negative. Furthermore, most of the viremic cat sera and the monoclonal antibodies to FOCMA did not react with FeSV-transformed nonproducer cells. The absence of FOCMA from these cells and from FeLV-negative lymphoid tumors and its presence in FeLV-C infected fibroblasts indicated that this antigen is virus encoded and not a cellular tumor-specific antigen.

Heteroduplex analysis of cloned rat endogenous replication-defective (30 S) retrovirus and Harvey murine sarcoma virus

Abstract DNA representing endogenous rat replication-defective (30 S) retrovirus has been cloned at the SacI sites of the EK2 vector λgtWes.λB. Heteroduplex analysis of this cloned 30 S DNA to cloned Harvey murine sarcoma virus (Ha-MuSV) DNA suggests that the region of Ha-MuSV which codes for the p21 “src” protein is not found in the rat endogenous replication-defective (30 S) virus and that the p21 gene represents a 1.1-kb (kilobase) substitution of genetic material in the 30 S genome prior to or during the formation of Ha-MuSV. Additionally, a small region (0.56 kb) of nonhomology, representing either a deletion or an insertion, has been found near the middle of the 30 S viral genome and a large region of nonhomology (1.02 kb), representing that portion of Ha-MuSV which is mouse in origin, has been observed at the 3′ end of the respective genomes. These results are in good agreement with the localization of the p21 gene as determined by restriction enzyme analysis and transfection studies of both 30 S and Harvey murine and sarcoma virus DNAs (Ellis et al., submitted for publication).