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Background Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. BLV is closely related to human T cell leukemia virus. B cell epitopes are important for the use of antibodies as therapeutic agents, the epitope-driven vaccine design, and immunological assays. A common B cell epitope for BLV has not yet been found due to individual differences in disease susceptibility.ResultsWe used a peptide microarray with 156 synthetic 15-mer peptides covering the envelope glycoprotein gp51 and the Gag proteins p15, p24, and p12 to map B cell epitope and one B cell epitope, gp51p16, was recognized by all four cattle experimentally infected with BLV. A newly developed high-throughput peptide ELISA system revealed 590 (91.2 %) of 647 cattle naturally infected with BLV, carrying 25 different bovine leukocyte antigen class II DRB3 (BoLA-DRB3) alleles, responded to a 20-mer gp51p16-C peptide containing a C-terminal cysteine and gp51p16. Alanine mutation and comparison of the sequences at 17 amino acid positions within gp51p16-C revealed that R7, R9, F10, V16, and Y18 were the common binding sites to BLV antibodies, and two of these sites were found to be highly conserved. Transient expression in the cells of five infectious molecular clones of BLV with a single alanine mutation at five common antibody binding sites had no effect syncytia formation of the gp51 protein. In addition, the mutant proteins, R7A and R9A had no effect on the expression of gp51 protein; the gp51 protein expressions of F10A, V16A and Y18A were lower than that of the wild type protein.ConclusionsThis is the first report to identify a common B cell epitope in BLV by comprehensive screening of BLV-infected cattle with varied genetic backgrounds in BoLA-DRB3. Our results have important implications for disease control and diagnosis.

Several derivatives of human immunodeficiency virus type 1 (HIV-1) that evade macaque restriction factors and establish infection in pig-tailed macaques (PtMs) have been described. These monkey-tropic HIV-1s utilize CXCR4 as a co-receptor that differs from CCR5 used by most currently circulating HIV-1 strains. We generated a new monkey-tropic HIV-1 carrying env from a CCR5-tropic subtype C HIV-1 clinical isolate. Using intracellular homologous recombination, we generated an uncloned chimeric virus consisting of at least seven types of recombination breakpoints in the region between vpr and env. The virus increased its replication capacity while maintaining CCR5 tropism after in vitro passage in PtM primary lymphocytes. PtM infection with the adapted virus exhibited high peak viremia levels in plasma while the virus was undetectable at 12-16 weeks. This virus serves as starting point for generating a pathogenic monkey-tropic HIV-1 with CCR5-tropic subtype C env, perhaps through serial passage in macaques.

A new simian-human immunodeficiency virus (SHIV), carrying env from an uncloned HIV-1 subtype C clinical isolate (97ZA012), was generated through intracellular homologous recombination, a DNA repair mechanism of the host cell. PCR fragments amplified from an existing SHIV plasmid (a 7-kb fragment from the 5' end and a 1.5-kb fragment from the 3' end) and a 4-kb fragment amplified from 97ZA012 cDNA containing env were co-transfected to human lymphoid cells. The resulting recombinant was subjected to serial passage in rhesus peripheral blood mononuclear cells (RhPBMCs). The resulting SHIV 97ZA012 was replication competent in RhPBMCs and monkey alveolar macrophages, and possessed CCR5 preference as an entry co-receptor. Experimental infection of rhesus macaques with SHIV 97ZA012 caused high titers of plasma viremia and a transient but profound depletion of CD4(+) T lymphocytes in the lung. Animal-to-animal passage was shown to be a promising measure for further adaptation of the virus in monkeys.

Viral protein R (Vpr) is an accessory protein found in various primate lentiviruses, including human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2) as well as simian immunodeficiency viruses (SIVs). Vpr modulates many processes during viral lifecycle via interaction with several of cellular targets. Previous studies showed that HIV-1 Vpr strengthened degradation of Mini-chromosome Maintenance Protein10 (MCM10) by manipulating DCAF1-Cul4-E3 ligase in proteasome-dependent pathway. However, whether Vpr from other primate lentiviruses are also associated with MCM10 degradation and the ensuing impact remain unknown. Based on phylogenetic analyses, a panel of primate lentiviruses Vpr/x covering main virus lineages was prepared. Distinct MCM10 degradation profiles were mapped and HIV-1, SIVmus and SIVrcm Vprs induced MCM10 degradation in proteasome-dependent pathway. Colocalization and interaction between MCM10 with these Vprs were also observed. Moreover, MCM10 2-7 interaction region was identified as a determinant region susceptible to degradation. However, MCM10 degradation did not alleviate DNA damage response induced by these Vpr proteins. MCM10 degradation by HIV-1 Vpr proteins was correlated with G2/M arrest, while induction of apoptosis and oligomerization formation of Vpr failed to alter MCM10 proteolysis. The current study demonstrated a distinct interplay pattern between primate lentiviruses Vpr proteins and MCM10.

Simian-human immunodeficiency virus (SHIV) carrying the envelope from the clade B clinical human immunodeficiency virus type 1 (HIV-1) isolate MNA, designated SHIV MNA, was generated through intracellular homologous recombination. SHIV MNA inherited biological properties from the parental HIV-1, including CCR5 co-receptor preference, resistance to neutralization by the anti-V3 loop mAb KD-247 and loss of resistance in the presence of the CD4mimic small-molecule YYA-021. SHIV MNA showed productive replication in rhesus macaque PBMCs. Experimental infection of a rhesus macaque with SHIV MNA caused a transient but high titre of plasma viral RNA and a moderate antibody response. Immunoglobulin in the plasma at 24 weeks post-infection was capable of neutralizing SHIV MNA in the presence but not in the absence of YYA-021. SHIV MNA could serve a model for development of novel therapeutic interventions based on CD4-mimic-mediated conversion of envelope protein susceptible to antibody neutralization.

Bovine leukemia virus (BLV), which is closely related to human T-cell leukemia viruses, is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle. The transmembrane subunit of the BLV envelope glycoprotein, gp30, contains three completely conserved YXXL sequences that fit an endocytic sorting motif. The two N-terminal YXXL sequences are reportedly critical for viral infection. However, their actual function in the viral life cycle remains undetermined. Here, we identified the novel roles of each YXXL sequence. Syncytia formation ability was upregulated by a single mutation of the tyrosine (Tyr) residue in any of the three YXXL sequences, indicating that each YXXL sequence is independently able to regulate the fusion event. The alteration resulted from significantly high expression of gp51 on the cell surface, thereby decreasing the amount of gp51 in early endosomes and further revealing that the three YXXL sequences are independently required for internalization of the envelope (Env) protein, following transport to the cell surface. Moreover, the 2nd and 3rd YXXL sequences contributed to Env protein incorporation into the virion by functionally distinct mechanisms. Our findings provide new insights regarding the three YXXL sequences toward the BLV viral life cycle and for developing new anti-BLV drugs.