Molecular Architecture of the Retroviral Capsid.

Retroviral capsid cores are proteinaceous containers that self-assemble to encase the viral genome and a handful of proteins that promote infection. Their function is to protect and aid in the delivery of viral genes to the nucleus of the host, and, in many cases, infection pathways are influenced by capsid-cellular interactions. From a mathematical perspective, capsid cores are polyhedral cages and, as such, follow well-defined geometric rules. However, marked morphological differences in shapes exist, depending on virus type. Given the specific roles of capsid in the viral life cycle, the availability of detailed molecular structures, particularly at assembly interfaces, opens novel avenues for targeted drug development against these pathogens. Here, we summarize recent advances in the structure and understanding of retroviral capsid, with particular emphasis on assemblies and the capsid cores.

Analysis of bovine leukemia virus gag membrane targeting and late domain function.

Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain).

Cellular distribution of bovine leukemia virus proteins gp51SU, Pr72(env), and Pr66(gag-pro) in persistently infected cells.

Monoclonal antibodies (mAbs) against bovine leukemia virus (BLV) mature proteins and precursors were used to map the localization of these proteins in persistently infected non-lymphocytic cell lines using immunofluorescence assay (IFA) and immuno-electron microscopy. IFA staining was observed in the basolateral surface of live FLK-BLV cells. When using a mAb against Pr66(gag-pro), mottled pinpoint fluorescence was seen in the cell surface of polarized cells, but no reaction was observed in cells undergoing mitosis. However, a mAb against Pr72(env) stained only mitotic cells and cellular fragments. Additionally, in these dividing cells, this envelope (Env) precursor polyprotein was not evenly distributed but concentrated predominantly in only one daughter cell. To the best of our knowledge, this observation has not been reported previously, either for BLV or for other retroviruses. The results of immunogold electron microscopy confirmed the specificity of the mAbs in the intracellular level. In infected cells, Pr72(env) and gp51SU were seen in proximity at the plasma membrane in incipient budding sites. Additionally, the mAb against Pr72(env) also reacted with Env precursor polyproteins in the mitochondria of BLV-bat(2) ultrathin sections. These mAbs may be used as a tool for mapping virus excretion sites in the cell surface of naturally or in vitro infected cells in the different stages of the cell cycle.

In vitro infection of cells of the monocytic/macrophage lineage with bovine leukaemia virus.

The oncogenic retrovirus bovine leukaemia virus (BLV) primarily infects B cells. Most infected animals remain asymptomatic for long periods of time before an increase in circulating B cells or localized tumours can be observed. This long clinical latency period may be explained by cells of the monocyte/macrophage lineage (M/M) becoming infected and acting as a reservoir for the virus, as shown for other retroviruses (human immunodeficiency virus-1, feline immunodeficiency virus). M/M cells in different stages of differentiation (HL-60, THP-1, U-937, J774, BGM, PM2, primary macrophages of sheep and cows) were cultured with BLV produced by permanently infected donor cells (FLKBLV and BLV-bat(2)). Donor cells were inhibited from multiplying by either irradiation or treatment with mitomycin C. In other experiments, supernatant from donor cells containing virus was used. In co-culture with the donor cells, the less differentiated monocytic cells showed severe cellular changes such as differentiation, vacuolization, cell lysis and membrane blebbing; apoptosis was a frequent phenomenon. Budding and extracellular viruses were also observed. The more differentiated macrophage cells, although they showed less signs of infection by microscopy, had a complete BLV protein profile, as seen by Western blotting; bands corresponding to p24CA (Gag) and its precursors were clearly seen. In addition, gp51SU was identified by syncytia formation assays. It is concluded that M/M cells may be infected by BLV, the consequences of the infection differing according to the type of cell.

Bovine leukemia virus Gag particle assembly in insect cells: formation of chimeric particles by domain-switched leukemia/lentivirus Gag polyprotein.

A key stage in the life cycle of C-type retroviruses is the assembly of Gag precursor protein at the plasma membrane of infected cells. Here we report the assembly of bovine leukemia virus (BLV) gag gene product into virus-like particles (VLPs) using the baculovirus expression system. Expression of BLV Pr44(Gag) resulted in the assembly and release of VLPs, thereby confirming the ability of retroviral Gag polyprotein to assemble and bud from insect cells. Efficient particle formation required a myristoylation signal at the N-terminus of BLV Pr44(Gag). Recombinant baculoviruses expressing matrix (MA) or capsid-nucleocapsid (CA-NC) proteins of BLV were generated but neither of these domains was capable of assembling into particulate structures. To assess the compatibility of Gag domains between leukemia and lentivirus groups three different recombinant chimeras each expressing MA of one virus (e.g., simian immunodeficiency or BLV) and CA-NC of another (e.g., BLV or human T-cell leukemia virus type-I) were constructed. Each of the chimeric proteins assembled efficiently and budded as VLPs, suggesting that the MA and CA domains of these two evolutionary divergent retrovirus groups can be functionally exchanged without perturbation of Gag VLP formation. The lenti-leukemia chimeric Gag approach has potential for studying protein-protein interactions in other retroviruses.

Detection of bovine leukemia viruses (BLV) in mammary tissues of BLV antibody-positive cows affected by subclinical mastitis.

The mammary tissues of 6 cows with bovine leukemia virus (BLV) antibody and subclinical mastitis were investigated histopathologically, and their organ cultures were ultrastructurally observed. Numerous BLV particles, 110 to 120 nm in diameter, were seen around lymphocytes, which had infiltrated into mammary alveoli and showed blastogenesis under culture. Particles budding from the cell membrane were also found.

Detection of proviral DNA of bovine leukaemia virus in cattle by a combination of in-situ hybridization and the polymerase chain reaction.

Bovine leukaemia virus (BLV) proviral DNA was detected in lymphocytes isolated from cattle with persistent lymphocytosis (PL) by the polymerase chain reaction (PCR) and in-situ hybridization (ISH) with a biotinylated pX DNA probe. Many positive cells were observed when short-term culture and a combination of ISH with PCR were used. Immunohistochemical examination of lymphocytes isolated from the lymph node showed that BLV attached mainly to surface immunoglobulins (SIg) of positive B lymphocytes, and to a few tumour-associated antigen (TAA)-, PanT-, and CD8-positive cells and non-CD4 positive cells. Electron microscopical examination revealed colloidal gold particles within the nuclei and cytoplasm of lymphocytes. Lymphoid cells from neoplastic lymph node of enzootic bovine leukosis (EBL) cases gave particularly strong positive signals with the ISH-PCR method. The technique of combined ISH and PCR with a biotinylated pX probe may prove useful in future studies of EBL.

Retrovirus-like particles produced by vaccinia viruses expressing gag-pro-pol region genes of bovine leukaemia virus.

Processing and assembly of bovine leukaemia virus-like particles were studied in African green monkey kidney cells using recombinant vaccinia viruses (rVVs) expressing regions of the bovine leukaemia virus genome. Unprocessed gag precursor protein (Pr44) was detected in immunoblot analysis of lysed cells and particles sedimented from culture supernatants after infection with a rVV carrying the gag and truncated protease (pro) gene. Processing of Pr44 was observed after infection of cells with a rVV carrying the gag and pro gene or a rVV expressing the gag, pro and polymerase (pol) gene. Reverse transcriptase activity was detected only in association with particles produced by gag-, pro- and pol-expressing recombinants. Thin section electron microscopic analysis of infected cells and pelleted particles revealed that Pr44 and processed gag proteins assembled at the cell membrane. Pr44 was released into the cell culture media as immature virus-like particles, whereas processed gag proteins from rVVs expressing gag and pro or gag, pro and pol formed mature particles.

Dynamics of mitotic activity and expression of viral proteins gp51 and p24 of bovine leukemia virus producing cells.

The dynamics of expression of viral proteins gp51 and p24, virus particle production and mitotic activity of a highly productive bovine leukemia virus clone of the fetal lamb kidney (FLK) cell line were studied. The period of the highest protein production (20-44 h after incubation) was established by the indirect immunofluorescence method using specific monoclonal antibodies. It was followed by a complete formation of the cell monolayer and the most intensive period of the mitotic activity determined by the 3H-thymidine labeling method. After the active synthesis of viral protein, the formation of mature viral particles and their shedding in the intercellular spaces was established electron-microscopically and by the syncytia induction test. A similar comparative study was carried out with a BLV producing short-term lymphocyte culture (STLC).

[Electron microscopic, serological and hematological research on lambs infected with the bovine leukemia virus]. / Elektronno-mikroskopicheskoe, serologicheskoe i gematologicheskoe issledovanie iagniat, infitsirovannykh virusom leikoza krupnogo rogatogo skota.

A complex study on experimental oncornavirus infection in sheep was carried out. Fast development of infectious process, production of virus-specific precipitating antibodies, presence of BLV reproduction in cultivated leucocytes were found. Terms of antibody appearance ranged between 20-30 days after infection. Stable antibody carriage remained during the whole observation period (36 months). Moreover, no expressed specific changes were observed in the hemogram of tested animals. Use of electron microscopy in oncornavirus infection allows revealing cells with pathologic changes in organelles and nucleus which are characteristic of the leucosis.

[Structural organization and intravirion interactions of proteins of the bovine leukemia virus]. / Strukturnaia organizatsiia i vnutrivirionnye vzaimodeistviia belkov virusa leikoza krupnogo rogatogo skota.

Data on structural organization and interactions inside the virion of bovine leukemia virus (BLV) proteins are presented. Uniqueness of the structural organization of BLV proteins having no antigenic relationship between the corresponding animal retrovirus proteins is emphasized. Certain connection is also observed in the structural organization of surface glycoprotein gp69, major internal protein p24 and internal protein p12 with the corresponding polypeptides of human T-cell leukemia virus (HTLV). It is shown that BLV proteins form various complexes inside the virion which are not found in other known retroviruses. A structural model of BLV is presented.

The morphogenesis of BLV (bovine leukemia virus) cultivated on FLK (fetal lamb kidney): an ultrastructural study.

A new cell line, obtained by co-cultivation of fetal lamb kidney cells and lymphocytes collected from an adult calf affected by enzootic bovine leukemia, was studied for bovine leukemia virus (BLV) morphogenesis. In this new cell line, called FLK-BLV, persistently infected with BLV, we identified extracellular and intracellular BLV particles. We never observed "budding" particles along the cell surface, and therefore assumed it was a new BLV maturation process in the cell vacuoles. In fact we found mature and non-mature particles connected with the cell-membrane system or cellular debris within vacuoles. We suggest that the viral envelope could be supplied by vacuole membrane. In our samples we also observed a cytopathic effect with syncytia formations similar to those observed on other BLV-producing cell lines.

[Isolation of lymphocyte cultures from leukemic cattle and electron microscopic study of the leukemia virus]. / Poluchavane na limfotsitni kulturi ot bolni ot levkoza goveda na elektronnomikroskopsko prouchvane na virusa na levkozata.

Several methods were employed to obtain lymphocyte cultures from blood samples taken from normal cattle and from cattle affected with enzootic leukosis. Biologic and virologic experiments revealed that the cultures from diseased cattle contained an oncogenic leukosis virus, while those obtained from healthy animals were exempt from such virus. Electron microscopy was applied to study the morphologic aspects of the bovine leukosis virus with a total of 18 lymphocyte cultures. The viral particles noted were shown to have typical configuration and size of type C oncogenic viruses as described in the literature--possessing a central dense nucleotide and double membranes. Seen were also various stages in the development of the virus. Mature virus particles were likewise observed in a stable cell line FLK (percent of the virus) as well as in ultrathin cross sections of the spleen of leukosis-affected animals.

[Electron microscopy of bovine leukosis viruses (BLV) in a fetal lamb spleen cell line]. / Elektronová mikroskopie viru bovinní leukózy (BLV) v bunecné linii fetální jehnecí sleziny.

The ultra-thin section method, the method of negative staining, and immunoelectron microscopy were used for detecting BLV and for determining its morphological characteristics in the FLS continual cell line used as a virus antigen producer for the ELISA test. Particles of C type, about 110 nm in size, having a structure corresponding to BLV, were detected in the FLS cells on the ultra-thin sections. The viruses were located extracellularly, in cytoplasmic vacuoles, and in different stages of maturation by budding from cell plasma membrane. BLV presence was also demonstrated by immunoelectron microscopy.

Electron microscopic investigation on the fine structure of the virus of enzootic bovine leukosis (BLV).

Systematically electron microscopical control of BLV producing cells and stimulated lymphocytes of leukotic cattle in the last years revealed maturation processes of BLV in a different form from comparable type C viruses. Electron microscopic representation of bridge-like junctions between core and envelope within examined retroviruses were discussed and compared with new model concepts.

Ultrastructural comparison of Oncovirinae (type C), Spumavirinae, and Lentivirinae: three subfamilies of Retroviridae found in farm animals.

The successive steps of maturation of seven retroviruses from five species of farm animals and one retrovirus from a mouse were compared in cell cultures. The viruses included three type C oncoviruses, one spumavirus, and three lentiviruses. Although members of the 3 subfamilies shared some gross morphologic features such as budding on plasma membranes, core, and surface projections, differences were noted in the ultrastructural detail of these features. Type C oncoviruses did not show any structural differentiation in identifiable form in the cytoplasm as opposed to characteristic features observed in the spumavirus and lentivirus subfamilies, respectively. Budding viruses were distinct among the 3 subfamilies. The type C bovine leukemia virus budding on vacuole membranes differed from the two other type C viruses by lacking an electron-lucent intermediate layer as did the lentiviruses. Differentiation between type C oncoviruses and lentiviruses could be confusing because of the similarity of the fully mature virions appearing in the intercellular space. However, each subfamily of retroviruses can be readily differentiated from one another when each morphologic stage of virus replication is examined by electron microscopy.

Bovine leukemia virus (BLV)--a structural model based on chemical crosslinking studies.

Nearest neighbor relationships between lipid and protein as well as between high-molecular-weight viral RNA and protein were investigated in bovine leukemia virus (BLV) particles using chemical crosslinking reagents. Separation of dimethyl suberimidate (DMS) induced lipid-protein complexes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phosphoprotein pp 15 is linked to the lipid bilayer of the virus. By use of diepoxybutan (DEB) as crosslinking reagent p 12 and again pp 15 were found to be linked to the viral RNA. Based on these results and our previous data describing the spatial relationships of major structural proteins within BLV particles, a structural model of BLV is proposed.

[Molecular biology characteristics of cattle leukemia virus]. / Molekuliarno-biologicheskaia kharakteristika virusa leikoza krupnogo rogatogo skota.

The structure and morphogenesis of the cattle leukemia virus, the main biochemical and immunological characteristics and its antigenic relationship with other members of the family Retroviridae are reviewed.

[Electron microscopy study of an association of Mycoplasma and the bovine leukemia virus in tissue culture]. / Elektronno-mikroskopicheskoe izuchenie assotsiatsii mikoplazmy i bych'ego leikoznogo virusa v kul'ture tkani.

FLK cell culture chronically producing bovine leukemia virus (BLV) and spontaneously contaminated with mycoplasma (M. sp.) was studied by electron microscopy. Information on the features of the ultrastructural organization of M. sp. and its direct relationships with BLV was obtained. BLV virions are capable of adsorbing on mycoplasma surface and penetrate inside, apparently in the course of phagocytosis. In the cytosole of M. sp. free-lying BLV virions devoid of the limiting membrane were found. In FLK cells both agents were regularly produced in high amounts causing no signs of death of the cell culture itself. From the foregoing, the FLK cells--BLV-M. sp. system appears to be quite balanced.