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Background: Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate lentiviruses since SRLVs encode only three auxiliary proteins, namely, Tat, Rev, and Vif, in addition to the products of gag, pol, and env genes common to all retroviruses. Here, we investigated the central part of the SRLV genome to identify new splice elements and their relevance in viral mRNA and protein expression., Results: We demonstrated the existence of a new 5' splice (SD) site located within the central part of CAEV genome, 17 nucleotides downstream from the SD site used for the rev mRNA synthesis, and perfectly conserved among SRLV strains. This new SD site was found to be functional in both transfected and infected cells, leading to the production of a transcript containing an open reading frame generated by the splice junction with the 3' splice site used for the rev mRNA synthesis. This open reading frame encodes two major protein isoforms of 18- and 17-kDa, named Rtm, in which the N-terminal domain shared by the Env precursor and Rev proteins is fused to the entire cytoplasmic tail of the transmembrane glycoprotein. Immunoprecipitations using monospecific antibodies provided evidence for the expression of the Rtm isoforms in infected cells. The Rtm protein interacts specifically with the cytoplasmic domain of the transmembrane glycoprotein in vitro, and its expression impairs the fusion activity of the Env protein., Conclusion: The characterization of a novel CAEV protein, named Rtm, which is produced by an additional multiply-spliced mRNA, indicated that the splicing pattern of CAEV genome is more complex than previously reported, generating greater protein diversity. The high conservation of the SD site used for the rtm mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation in vivo, likely by competing with Env protein functions.

Small ruminant lentiviruses (SRLV) are widely spread in many countries, including Spain. However, little is known about the genetic characteristics of Spanish goat and sheep SRLV. In this study, segments from three genomic regions (pol, gag-p25 and LTR) were amplified using DNA isolated from three Spanish autochthonous sheep (one) and goats (two). Animals (one per flock) belonged to distantly located, single-species flocks (goat or sheep). Sequence analysis showed conservation of regions that are putatively relevant to viral survival. Sequences of Spanish goat and sheep SRLV were allocated into phylogenetic trees (phylograms) with known SRLV groups. The phylograms corresponding to the pol, gag-p25 and LTR regions analyzed presented a compatible topology. This showed that Spanish caprine and ovine SRLV sequences belonged to the A or D phylogenetic groups and were closer to sheep SRLV prototypes (A1 group) than to goat SRLV prototypes (B or C groups), according to the current classification [Shah, C., Boni, J., Huder, J.B., Vogt, H.R., Muhlherr, J., Zanoni, R., Miserez, R., Lutz, H., Schupbach, J., 2004a. Phylogenetic analysis and reclassification of caprine and ovine lentiviruses based on 104 new isolates: evidence for regular sheep-to-goat transmission and worldwide propagation through livestock trade. Virology 319 (1), 12-26]. It was not possible to amplify in the three genetic regions the expected fragment in additional Spanish caprine and ovine SRLV proviral DNA sequences with the PCR primers used. This suggests that there is heterogeneity at the primer binding site among Spanish SRLV sequences. It also illustrates the need to develop diagnostic tests that are sensitive in local breeds.

During retroviral particle formation, the capsid precursors (Gag) associate with the cell membrane via their matrix (MA) domain to form viral assembling particles. After budding, Gag and its proteolytically matured MA, form a shell in the released immature and mature particles, respectively. Although the arrangement of Gag domains in vitro and their radial organisation in retroviral particles have been extensively studied, little is known concerning Gag inter-subunit interactions in authentic retroviruses. We report that human T-cell leukemia virus type 1 Gag homodimerises in the cell via a disulphide bonding at cysteine 61 in the MA domain. Most Gags are homodimeric after budding and MAs are also dimeric in mature authentic virions. Molecular modelling of the MA domain indicates that non-covalent interactions at the MA dimer interface may also be important for Gag (and MA) dimerisation. In addition, all amino acids previously reported to be involved in MA-transmembrane (TM) interactions are located on the MA face opposite to the dimer interface. The model reveals that homodimerisation is compatible with a hexameric network of Gag and MA dimers that look like the hexameric networks observed for other retroviruses. These data, together with previous studies, lead us to propose a supra-molecular arrangement model in which the transmembrane glycoproteins of the virion envelope are anchored in a hexameric cage hole formed by the MA.

Primary goat synovial membrane (GSM) cells are widely used to study small ruminant lentiviruses (SRLV), i.e. maedi visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV), but their limited life-span of 15-20 passages in vitro is problematic. Here, we report that ectopic expression of the catalytic subunit of human telomerase (hTERT) was sufficient to immortalize primary GSM cells. Cultures of hTERT-transfected GSM cells have been passaged for 2 years without showing any phenotypic difference from the original primary GSM cells. The hTERT-transfected cells continued to grow beyond a population doubling number of 250, while no net telomere lengthening was observed for these cells. Moreover, the immortalized GSM cells were susceptible to infection by both CAEV and MVV and were able to propagate theses viruses. Such cell line provides a useful source of standard and robust cells for both research and veterinary purposes.

Small ruminant lentiviruses (SRLV), i.e. caprine arthritis-encephalitis virus (CAEV) (which infects goats) and maedi-visna virus (MVV) (which infects sheep) are two closely related lentiviruses but the relationship between goat and sheep lentiviruses has not been clearly established. To better understand their genetic relationship, we reinvestigated the phylogeny of SRLV using new sequences from an Irish and a Norwegian strain together with sequences available from databases. The phylogenetic analyses were carried out on the gag, pol and env fragments using four methods: neighbor-joining (NJ), Fitch and Margoliash (Fitch), Fitch and Wagner parsimony (Pars) and maximum likelihood (ML). The tree topologies were consistent whether derived from any of the four methods or any of the gene fragments, but the phylogenetic analyses in the pol and env regions were more informative than in the gag region. The Tamura-Nei model with variable rates across sites (described by a gamma distribution) provides a more accurate description of SRLV evolution than simple methods. The newly described Irish lentivirus strain, which was isolated from a goat, was closely related to the lentivirus that infects sheep: MVV. The novel Norwegian CAEV strain belonged to a cluster specific to the CAEV strains from Norway. Together, both data confirm the previously reported subdivision of the different SRLV strains into six clades. The caprine and ovine lentivirus sequences are interspersed in phylogenetic trees, supporting the existence of cross-species transmission. Nevertheless, the transmission of an ovine lentivirus to a goat could trigger the emergence of some goat-adapted phylums. Our new sequences confirm the complex situation in SRLV phylogeny but more sequences are needed to elucidate more precisely the relationship between SRLV.

The human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) capsid proteins (CA) display similar structures formed by two independently folded N-terminal (NTD) and C-terminal (CTD) domains. To characterize the functions harbored by the HTLV-1 CA domains in particle formation, 12 sites scattered throughout the protein were mutated. The effects of the mutations on Gag membrane binding, proteolytic processing, and virus-like particle secretion were analyzed. It appears that the NTD is the major partner of indirect or direct Gag-Gag interactions. In particular, most of the NTD mutations impaired virion morphogenesis, and no mutation located in the NTD could be fully rescued by coexpression of wild-type Gag. In contrast, the CTD seems not to be involved in Gag-Gag interactions. Nevertheless, an unknown function required for particle formation is located in the CTD. Thus, despite an overall structural similarity between the HIV-1 and HTLV-1 CA proteins, their NTDs and CTDs exhibit different functions.

In this study, we examined the ability of human T-cell leukemia virus type I (HTLV-I) Gag and Gag-Pro to assemble immature virus-like particles (VLPs) and bud from insect and mammalian cells. Transmission electron microscopy of insect cells infected with a recombinant baculovirus carrying the entire gag gene revealed that Pr53(Gag) is targeted to the plasma membrane, where it extensively accumulates and forms electron-dense evaginations. However, no particles could be detected either inside the cells or in the culture supernatants. With the Gag-Pro-expressing construct, we observed HTLV-I-specific cytoplasmic proteolysis of the Gag precursor, but again no particle released in the culture supernatants. Transmission electron microscopic analysis of insect cells expressing Gag-Pro polyprotein revealed large vacuoles in the cytoplasm and no budding particles at the plasma membrane. In contrast, human immunodeficiency virus type 1 Gag polyprotein expressed in insect cells is able to release VLPs. These data showed that unlike other retroviruses, Pr53(Gag) is unable to be released as immature VLPs from insect cells. To determine whether the block in particle budding and release is due to an intrinsic property of Pr53(Gag) or the absence of essential cellular factors in insect cells, we expressed Gag and Gag-Pro polyproteins in human 293 cells. The results indicate that Pr53(Gag) and p24 capsid are released within particles into the culture supernatants of human 293 cells. We found that the myristylation of the N-terminal glycine residue is essential for Gag release. Altogether, these results strongly suggest that the proper assembly of HTLV-I particles is dependent on mammalian host cell factors., (Copyright 2000 Academic Press.)

The complete surface glycoprotein (SU) nucleotide sequences of three French isolates of caprine arthritis-encephalitis virus (CAEV) were determined and compared with those of previously described isolates: three American isolates and one French isolate. Phylogenetic analyses revealed the existence of four distinct and roughly equidistant evolutionary CAEV subtypes. Four conserved and five variable domains were identified in the SU. The fine specificities of antibodies produced against these domains during natural infection were examined using a pepscan analysis. Nine immunogenic segments were delineated throughout the conserved and variable domains of SU, two of them corresponding to conserved immunodominant epitopes. Antigenic determinants which may be involved in the immunopathogenic process induced by CAEV were identified. These results also provide sensitive and specific antigen peptides for the serological detection and differentiation of CAEV and visna/maedi virus infections.

The first description of an active form of a recombinant human T-cell leukemia virus type 1 (HTLV-1) reverse transcriptase (RT) and subsequent predictions of its amino acid sequence and quaternary structure are reported here. By using amino acid alignment methods, the NH2 and COOH termini of the RT, RNase H (RH), and integrase (IN) domains of the Pol polyprotein were determined. The HTLV-1 RT seems to be unique since its NH2 terminus is probably encoded by the pro open reading frame (ORF) fused downstream, via a transframe peptide, to the polypeptide encoded by the pol ORF. The HTLV-1 Pol amino acid sequence was revealed to be highly similar to that of Rous sarcoma virus (RSV), particularly at the RT-RH hinge region. These two domains remain linked for RSV; this may also be the case for HTLV-1. In light of these results, RT, RT-RH, and RT-RH-IN genes were constructed and introduced into His-tagged protein expression vectors. The corresponding proteins were synthesized in vitro, and the DNA polymerase activities of different protein combinations were tested. Solely the RT-RH-RT-RH-IN combination was found to have a significant activity level. Velocity sedimentation analysis suggested that the HTLV-1 RT-RH and RT-RH-IN monomers are likely associated in an oligomeric structure, probably of the alpha3/beta type.

The full extent of genetic diversity among small ruminant lentiviruses (SRLVs), i.e., caprine arthritis encephalitis viruses (CAEVs) and maedi-visna viruses (MVVs), remains unknown. This is due in part to the fact that few sequences of CAEV are available. To contribute to this knowledge, gag, pol, and env nucleotide sequences from an SRLV named CA680 originating from a goat from western France were determined. This analysis revealed that this virus is closely related to the Cork and 63 CAEV American isolates. Mismatched amino acids between the CA680 virus and prototype CAEVs ranged from 6.7, 0. 7, and 17.5% for gag, pol, and SU sequences, respectively. The differences between the CA680 virus and MVV prototypes ranged from 16.5, 12.5, and 32.3% for the protein sequences, respectively. A screening using a heteroduplex mobility assay (HMA) adapted to SRLVs revealed that 6 of 10 caprine virus field isolates were closely related to CA680, indicating that this latter isolate was a prototype of CAEVs common in the west of France. Phylogenetic trees drawn using CA, RT, or SU sequences of numerous SRLVs and rooted with EIAV sequences revealed that CA680 and CAEV prototypes, all infectious for goat, clustered in one group. From these HMA and phylogenetic analyses, it appears that U.S. and French caprine SRLVs form a clade that had emerged from a much more diverse group containing all SRLVs infectious for sheep. These ovine SRLVs form a more ancient group in which the EIAV is rooted., (Copyright 1997 Academic Press.)

The proteases (PR) of retroviruses are expressed as gag-PR fused polyprotein. The active PR is a dimer obtained after the aggregation of the gag and gag-pro precursors, which leads to the formation and the release of the viral particle. Subsequently, in the cell, the PR is present essentially as a monomeric polyprotein. To mimic the antigenic properties of such an intracellular form of the PR, we produced a monomeric form of the HTLV-I (human T-cell leukemia virus, type-I) PR fused to the maltose binding protein (MBP-PR). Monoclonal antibodies (mabs) directed against MBP-PR were developed. Three mabs were obtained that recognized different epitopes. Two were directed against the NH2-terminus, a region that contributes to the dimerization interface. The other was specific to a peptide that lines the substrate binding pocket. This latter epitope is located just downstream of the D-T-G peptide of the catalytic site. The two identified regions contained the amino acids Asp6, Arg10 and Asp36, which were previously shown to be important in the stabilization of the dimer. In view of the localization of the recognized epitopes, these mabs will be useful for inhibition studies of the HTLV-I PR by intracellular immunization.

The cytoplasmic domains of the transducing subunits associated with B and T cell antigen receptors contain a common amino acid motif consisting of two precisely spaced Tyr-X-X-Leu/Ile sequences (where X corresponds to a variable residue). Expression of a single copy of this motif suffices to initiate B or T cell activation. The bovine leukaemia virus (BLV) is a B cell lymphotropic retrovirus which causes a non-neoplasic proliferation of B cells. The cytoplasmic domain of the BLV transmembrane envelope glycoprotein, gp30, possesses two overlapping copies of the Tyr-X-X-Leu/Ile-containing motif which could participate in the induction of B cell activation. Similarly, the N-terminal cytoplasmic domain of the latent membrane protein 2A (LMP2A) of the Epstein-Barr virus (EBV) contains a single copy of the Tyr-X-X-Leu/Ile-containing motif which could play a critical role in B cell transformation. To determine whether these two virus-encoded cytoplasmic domains are endowed with signalling functions, we constructed chimeric proteins by replacing the cytoplasmic tail of CD8-alpha with that of either BLV gp30 or EBV LMP2A. We show here that, once separately expressed in B or T cell lines, these chimeras are capable of triggering both calcium responses and cytokine production when cross-linked with an antibody to CD8-alpha. Furthermore, using site-directed mutagenesis, we demonstrated unequivocally that this signalling function may be accounted for by the Tyr-X-X-Leu/Ile motifs they contain.(ABSTRACT TRUNCATED AT 250 WORDS)

To develop efficient bovine leukemia virus (BLV) protease (PR) inhibitors, pure enzyme is required. For this, we have developed a two-step chromatographic nondenaturing purification protocol of PR from virions. As expected, the purified protein presents a molecular weight (14 kDa) and a NH2 terminal end fitting with previously reported data. The enzymatic activity of BLV PR was characterized using a synthetic peptide containing a potential cleavage site of the BLV gag-pro polypeptide precursor as substrate. The protease was most active at pH 6, 40 degrees, and high salt concentration (1-2 M NaCl or ammonium sulfate). In contrast, using a natural substrate such as a human T-cell leukemia virus recombinant gag precursor, BLV PR activity was higher at a low salt concentration (0.5 M NaCl). Besides, the use of different potentially cleavable molecules revealed that PR activity may be influenced by the substrate conformational structure around the cleavage site. Replacement of the two amino acids of a synthetic substrate cleavable site by a statin residue completely inhibited the enzymatic activity of the BLV PR.

The nucleotide sequences of the env genes of seven bovine leukemia viruses and the encoded peptide sequence were compared, with the objective of (i) determining the genetic distance separating bovine leukemia virus isolates from different geographical regions, (ii) identifying particular amino acids that contribute to the sequential and conformational epitopes, and (iii) relating such epitopes to their projected position in a three-dimensional model of the structure of the gp51 surface glycoprotein. Two bovine leukemia virus subgroups were clearly identified, a Japanese-American subgroup represented by strains lambda BLV-1, VdM, and FLK-BLV and a European subgroup by strains T15-2, LB285, and LB59. It was possible to identify amino acids that were important in determining three of the epitopes (F, G, and H) recognized by neutralizing monoclonal and polyclonal antibodies. On the model, these epitopes were adjacent and located on the exposed region of the molecule. Amino acid sequences contributing to a fourth cryptic epitope were identified; as predicted by the model, they lay on the opposite side to the neutralizable epitopes in a region involved in glycoprotein subunit association. The fact that this region is not normally exposed on the virion surface provides further evidence for the validity of the model.

Bovine leukaemia virus (BLV) is known to be the aetiological agent of enzootic bovine lymphosarcoma. As the mechanism of tumour induction is unknown, we analysed the viral proteins expressed in cultured bovine cells of different origin, i.e. from enzootic or sporadic tumourous tissues, normal cells infected or not with BLV, and the reference FLK-BLV cells. We also investigated BLVs of different origins. As well as the previously described BLV polyproteins in FLK-BLV cells (pr70 and pr45) we have also found two additional polyproteins, p52 and p27. In these four proteins we observed the combined antigenicities of p24, p15 and p12. We observed an additional polyprotein in p42, with the antigenicities of the p24 and the p15 in cells derived from tumour tissue or infected in vitro with naturally occurring BLV isolates. None of these BLV-coded proteins, nor others with cross-reacting antigenicities, were encountered in non-BLV-producing cultured cells from enzootic or sporadic tumours. The use of autologous sera did not permit the detection of any proteins in addition to the above gag-coded and env-coded proteins. Thus, there is no evidence for the existence of a gag-x fusion protein which could play a role in BLV-induced tumourigenicity.

This report describes pulse-chase experiments performed with cells infected with tumour-derived bovine leukaemia virus (BLV) and using purified gamma-globulins directed against BLV structural proteins, namely gp51, p24, p15 and p12. A gpr72 was found to be the precursor of the gp51 with a gpr70 intermediate. The p12 was shown to be derived from a pr40 with numerous intermediates (pr35, pr22, pr16 and pr14). The p24 and p15 originate from a pr42. Both the pr42 and pr40 are derived from a common pr70. A P45, a P52 and a P27 were also detected. Because these three proteins were found to accumulate progressively in cells and because they were not observed to be processed, they might play a physiological role in infected cells.

A cell line (LB59Ly) derived from the leukocytes of a leukaemic cow was established as a monolayer, which spontaneously released large amounts of a retrovirus. This virus was found to be indistinguishable from the bovine leukaemia virus (BLV) produced by the commonly used high-producing heterologous cell line (FLK-BLV). Like the latter, its reverse transcriptase activity was greater in the presence of Mg2+ cations than in the presence of Mn2+ cations; its polyacrylamide gel electrophoresis pattern showed the presence of gp51, p24, p15, p12 and p10, and the antigenicity of the two major proteins completely cross-reacted with those of BLV from FLK-BLV cells. The virus was infectious and induced early and late polykaryocytosis, the specificity of which was demonstrated by use of specific anti-BLV sera.

The bovine leukemia virus mRNAs expressed in cultured bovine cells of various origins are a 9.0-kilobase genomic RNA, a 5.1-kilobase env RNA, and a newly detected 2.1-kilobase RNA corresponding to the transcription of pX sequences located in between the env gene and the 3' end of the provirus.