Bovine Immunodeficiency VirustatGene: Cloning of Two Distinct cDNAs and Identification, Characterization, and Immunolocalization of thetatGene Products

Abstract

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.

Functional Differences between Human and Bovine Immunodeficiency Virus Tat Transcription Factors

“…Therefore, if bTat binding to bTAR is indeed cofactor independent, one might predict that bTat would not show a comparable species tropism. Earlier reports have not resolved this issue, as bTat has been reported to be active in bovine, human, and murine cells but poorly active in lapine cells (16). Also, while the bTat protein has been reported to activate the HIV-1 LTR in certain tissue culture cell lines (16,25), in vitro data indicate that bTat binding to bTAR is far more efficient than binding to hTAR (7).…”

“…The conserved cysteine-rich domain of hTat is critical for both CycT1 binding and hTat function, and mutation of cysteine 22 in hTat to serine (C22S) therefore blocks both of these activities (2, 23, 26). Because hTat cysteine 22 is conserved in bTat (16,23,25), we examined whether mutation of the equivalent residue, i.e., mutation of bTat cysteine 38 to serine (C38S), would also affect bTat function and CycT1 binding. As we do not have access to a bTat-specific antiserum, we performed this mutational analysis in the context of an HIV-1 Rev-bTat fusion protein that can be readily detected using an anti-Rev antiserum.…”

“…All primate lentiviruses, as well as equine infectious anemia virus (EIAV) and bovine immunodeficiency virus (BIV), encode an HIV-1 Tat homolog (4-7, 13,16,25,37). In contrast, feline immunodeficiency virus and the ovine and caprine lentiviruses lack an equivalent RNA sequence-dependent transcriptional activator (11,29).…”

“…Thus, the 17-amino-acid basic domain of bTat was shown to bind to bTAR with high affinity and specificity in vitro and could also efficiently recruit a fused heterologous effector domain to bTAR when expressed in bacteria (7,20,31). While these earlier experiments did not address a possible role for CycT1 in facilitating bTAR binding by bTat, the similarity in domain organization of bTat and hTat, and in particular the conservation of the cysteine-rich and core domains that mediate CycT1 binding to hTat (2, 16,23,25,26,38), suggests that CycT1 is likely to play a role in mediating bTat function.…”

Binding of Tat to TAR and Recruitment of Positive Transcription Elongation Factor b Occur Independently in Bovine Immunodeficiency Virus

“…Therefore, these Tat proteins increase transcription in a TAR-independent manner. In sharp contrast, HIV-1, HIV-2, SIV (22), BIV (12,24,25), the Jembrana disease virus (JDV) (5), and equine infectious anemia virus (EIAV) (10) depend completely on promoter proximal TAR elements. That Tat proteins from this group affect gene expression via RNA makes them unique among other eukaryotic transcriptional activators, which act via DNA.…”