Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins.

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.     

Biological activity and intracellular location of the Tat protein of equine infectious anemia virus

The Tat protein of equine infectious anemia virus (EIAV) was synthesized in Escherichia coli using the inducible expression plasmid, pET16b, which contains a His.Tag leader, thus allowing for rapid and efficient enrichment of the histidine-tagged protein by metal affinity chromatography. Yields of up to 20 mg of Tat were obtained from 10¹¹ bacterial cells. The recombinant Tat protein was shown to potently trans-activate the EIAV long terminal repeat (LTR) following its introduction into canine cells by ‘scrape loading’. The EIAV Tat protein was found to localize predominantly within the cytoplasm, in contrast to HIV-1 Tat. The availability of large amounts of purified functional EIAV Tat protein should greatly facilitate detailed structure-function analyses.     

Analysis of the EIAV Rev-responsive element (RRE) reveals a conserved RNA motif required for high affinity Rev binding in both HIV-1 and EIAV

A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.     

人免疫缺陷病毒1型TAT蛋白点突变对其反式激活作用的影响

不同免疫缺陷病毒（ＨＩＶ－１,ＨＩＶ－２和ＳＩＶ）的Ｔａｔ均有３个高度保守的结构域：Ｃｙｓ富集域、核心域和碱性氨基酸富集域,用ＰＣＲ定点突变法在ＨＩＶ－１Ｔａｔ蛋白的这些区域引入单氨基酸或多氨基酸突变;构建了以ＨＩＶ－１ＬＴＲ－１５８到＋８Ｏ区域为启动子,含有不同突变点的突变Ｔａｔ基因表达质粒;以荧光酶基因为报告基因,瞬时共转染Ｊｕｒｋａｔ细胞：分析不同氨基酸突变对Ｔａｔ的反式激活作用的影响。结果发现,突变后的Ｔａｔ蛋白的活性均极大地降低或丧失,表明这些序列内的氨基酸的确定性对Ｔａｔ的活性至关重要。     

Late domain-dependent inhibition of equine infectious anemia virus budding

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.     

Solution structure of the capsid protein from the human T-cell leukemia virus type-I.

The solution structure of the capsid protein (CA) from the human T-cell leukemia virus type one (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, has been determined by NMR methods. The protein consists of independent N and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (HIV-1) and equine infectious anemia virus (EIAV) capsid proteins, although several important differences exist. In particular, hydrophobic residues near the major homology region are partially buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in HIV-1 and EIAV CA project from the C-terminal domain and promote dimerization. These differences in the structure and oligomerization state of the proteins appear to be related to, and possibly controlled by, the oxidation state of conserved cysteine residues, which are reduced in HTLV-I CA but form a disulfide bond in the HIV-1 and EIAV CA crystal structures. The results are consistent with an oxidative capsid assembly mechanism, in which CA oligomerization or maturation is triggered by disulfide bo nd formation as the budding virus enters the oxidizing environment of the bloodstream.     

Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization

DNA vaccines have been successful in eliciting potent immune responses in mice. Their efficiency, however, is restricted in larger animals. One reason for the limited performance of the DNA vaccines is the lack of molecular strategies to enhance immune responses. Additionally, genes directly cloned from pathogenic organisms may not be efficiently translated in a heterologous host expression system as a consequence of codon bias. To evaluate the influence of codon optimization on the immune response, we elected to use the Tat antigens of human immunodeficiency virus type 1 (HIV-1) (subtype C) and HIV-2, as these viral antigens are poorly immunogenic in natural infection and in experimental immunization and they are functionally important in viral infectivity and pathogenesis. Substituting codons that are optimally used in the mammalian system, we synthetically assembled Tat genes and compared them with the wild-type counterparts in two different mouse strains. Codon-optimized Tat genes induced qualitatively and quantitatively superior immune responses as measured in a T-cell proliferation assay, enzyme-linked immunospot assay, and chromium release assay. Importantly, while the wild-type genes promoted a mixed Th1-Th2-type cytokine profile, the codon-optimized genes induced a predominantly Th1 profile. Using a pepscan strategy, we mapped an immunodominant T-helper epitope to the core and basic domains of HIV-1 Tat. We also identified cross-clade immune responses between HIV-1 subtype B and C Tat proteins mapped to this T-helper epitope. Developing molecular strategies to optimize the immunogenicity of DNA vaccines is critical for inducing strong immune responses, especially to antigens like Tat. Our identification of a highly conserved T-helper epitope in the first exon of HIV-1 Tat of subtype C and the demonstration of a cross-clade immune response between subtypes B and C are important for a more rational design of an HIV vaccine.     

Novel Tat-encoding bicistronic human immunodeficiency virus type 1-based gene transfer vectors for high-level transgene expression

We describe bicistronic single-exon Tat (72-amino-acid Tat [Tat72])- and full-length Tat (Tat86)-encoding gene transfer vectors based on human immunodeficiency virus type 1 (HIV-1). We created versions of these vectors that were rendered Rev independent by using the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV). Tat72-encoding vectors performed better than Tat86-expressing vectors in gene transfer experiments. CTE-containing vectors, produced in a Rev-independent packaging system, had gene transfer efficiencies nearly equivalent to those produced using a combination RNA transport (CTE and Rev-Rev response element)-based packaging system. The Tat72-encoding vectors could be efficiently transduced into a variety of cell types, showed higher levels of transgene expression than vectors with the simian cytomegalovirus immediate-early or the simian virus 40 early promoter, and provide an alternative to HIV-1 vectors with internal promoters.     

Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.