Increased expression of CD4 molecules on Jurkat cells mediated by human immunodeficiency virus tat protein.

The tat gene of the human immunodeficiency virus, tat-III, when introduced into T-lymphoblastoid Jurkat cells by a Moloney retroviral recombinant DNA vector expressed high levels of the functional tat protein as measured by the chloramphenicol acetyltransferase assay. Immunofluorescence analysis with CD4-specific monoclonal antibodies demonstrated that the cell surface levels of the CD4 antigen were increased by 5- to 10-fold in the tat-III-infected Jurkat cells. Cellular cytoplasmic RNA analysis indicated that the enhanced CD4 expression was mediated at the mRNA level. Our findings suggest that the single expression of the human immunodeficiency virus tat protein in the absence of the other viral proteins causes an upregulation of CD4 gene expression on helper T cells, although infection of these cells by the virus, thus expressing all the viral gene products including tat, is known to downregulate CD4 antigen expression.     

Synthesis of functional human immunodeficiency virus tat protein in baculovirus as determined by a cell-cell fusion assay.

The human immunodeficiency virus tat protein is a strong trans-activator of the expression of mRNAs originating from the viral long terminal repeat. We have expressed the first 72 amino acids (coding exon 1) of this protein in eucaryotic Spodoptera frugiperda SF9 cells by using a baculovirus vector, Autographa californica nuclear polyhedrosis virus. We show that the baculovirus vector stably produced the 72-amino-acid form of the tat protein but was unable to stably synthesize a larger 101-amino-acid full-length version of the same polypeptide. The 72-amino-acid tat protein, when introduced into mammalian fibroblasts by using a cell-cell fusion technique, functionally trans-activated the expression of the human immunodeficiency virus long terminal repeat.     

Recognition of viral antigens by human influenza A virus-specific T lymphocyte clones

The nature of the viral antigens recognized by influenza A virus-immune cytotoxic T lymphocytes (CTL) is still a matter of debate. We have used four human influenza A virus-specific T lymphocyte clones with antigen-specific cytotoxic and proliferative activity to investigate the requirements for recognition of viral antigens on infected cells. One clone recognized a cross-reactive determinant on the viral hemagglutinin, and two clones were specific for different epitopes on the viral nucleoprotein (NP). A fourth clone seemed to be specific for the viral M protein. Target cell recognition was abrogated by the addition, during infection, of the lysosomotropic drug chloroquine, known to inhibit antigen processing. Furthermore, target cells that had been pulsed with soluble purified NP were recognized and were lysed by the NP-specific clone. This reaction could also be abrogated by the addition of chloroquine during pulsing. These results were obtained irrespective of whether EBV-transformed B lymphoblastoid cells or Ia antigen-expressing T cell blasts were used as target cells. It is concluded that CTL can recognize internal viral proteins that are actively presented at the surface of the target cell. These data indicate that probably every viral protein can function as a target molecule for virus-immune CTL.     

DNA immunization with HIV-1 tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type Tat

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.     

Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression

The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.     

Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.

In this study, we examined the mechanism of translation of the human immunodeficiency virus type 1 tat mRNA in eucaryotic cells. This mRNA contains the tat open reading frame (ORF), followed by rev and nef ORFs, but only the first ORF, encoding tat, is efficiently translated. Introduction of premature stop codons in the tat ORF resulted in efficient translation of the downstream rev ORF. We show that the degree of inhibition of translation of rev is proportional to the length of the upstream tat ORF. An upstream ORF spanning 84 nucleotides was predicted to inhibit 50% of the ribosomes from initiating translation at downstream AUGs. Interestingly, the distance between the upstream ORF and the start codon of the second ORF also played a role in efficiency of downstream translation initiation. It remains to be investigated if these conclusions relate to translation of mRNAs other than human immunodeficiency virus type 1 mRNAs. The strong inhibition of rev translation exerted by the presence of the tat ORF may reflect the different roles of Tat and Rev in the viral life cycle. Tat acts early to induce high production of all viral mRNAs. Rev induces a switch from the early to the late phase of the viral life cycle, resulting in production of viral structural proteins and virions. Premature Rev production may result in entrance into the late phase in the presence of suboptimal levels of viral mRNAs coding for structural proteins, resulting in inefficient virus production.     

Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat.

Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.     

河南HIV感染者的HIV-1B型株tat基因的克隆及序列分析

克隆河南人免疫缺陷病毒(HIV)感染HIV-1B型株tat基因完整编码框序列,并分析比较其编码产物的序列结构特点。使用重叠PCR技术,从河南省1名HIV-1感染外周血标本中扩增出to／基因第一和第二外显子并重组为完整的tat基因序列。获得的HIV-1B病毒株tat基因,第一外显子为263bp,第二外显子为214bp。将该基因编码产物与其他HW-1株Tat蛋白经DNA软件编辑并翻译成蛋白质,使用Clustal X1．81进行多序列对比分析发现,第一外显子编码产物的3个保守区域的氨基酸组成大致相同,只有少数氨基酸存在差异。由于Tat蛋白不同病毒株间有高度保守的Cys富集区、核心区和碱性氨基酸富集区,tat基因的克隆为研究其功能并以其为靶点设计和筛选抗艾滋病的药物奠定了基础。     

The expression of HIV-1 tat and nef genes induces cell-specific changes in growth properties and morphology of different types of rat cells

Among the viral regulatory genes the tat and nef genes of HIV-1 encode the proteins playing a central role in viral replication and exerting pleiotropic effects on the survival and growth of the cells. These effects differ in various cell types, possibly due to the use of genes from different HIV-1 isolates. In this work, we studied the effects of the tat and nef genes on three types of cultured rat cells: primary embryo fibroblasts, pseudonormal Rat-2, and pheochromocytoma PC12. Both genes affected growth properties and morphology of cells, the effects being cell-specific. The proliferative activity of both Rat-2 and PC12 cells was considerably increased after transfection with the tat gene. In primary rat embryo fibroblasts the tat gene induced multilayered foci. More importantly, it was shown that the efficiency of transformation was higher in cells coexpressing tat and nef. The nef gene caused considerable suppression of Rat-2 cell proliferation, but no changes in their morphology. The nef gene transfection of PC12 cells also led to suppression of their proliferative activity. In addition, cellular agglomerates which were morphologically similar to multinuclear syncytial cells were detected in these cells for the first time.     

Human immunodeficiency virus type 1 escape from RNA interference

Sequence-specific degradation of mRNA by short interfering RNA (siRNA) allows the selective inhibition of viral proteins that are critical for human immunodeficiency virus type 1 (HIV-1) replication. The aim of this study was to characterize the potency and durability of virus-specific RNA interference (RNAi) in cell lines that stably express short hairpin RNA (shRNA) targeting the HIV-1 transactivator protein gene tat. We found that the antiviral activity of tat shRNA was abolished due to the emergence of viral quasispecies harboring a point mutation in the shRNA target region. Our results suggest that, in order for RNAi to durably suppress HIV-1 replication, it may be necessary to target highly conserved regions of the viral genome. Alternatively, similar to present antiviral drug therapy paradigms, DNA constructs expressing multiple siRNAs need to be developed that target different regions of the viral genome, thereby reducing the probability of generating escape mutants.