Mutational analysis of the HTLV-I trans-activator, Tax

The gene coding for the trans-activating factor (Tax) of the human T-cell leukemia virus, type I (HTLV-I) was mutagenized in vitro using oligonucleotide-directed mutagenesis and recombinant DNA techniques. All except one of the mutagenized tax constructs failed to trans-activate the HTLV-I LTR in a eukaryotic test system. Moreover, negative Tax mutant Arg-39----Gly was found to be trans-dominant. This observation suggests that Tax contains distinct functional domains mediating different interactions of the protein in the process of trans-activation.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

Identification of functional regions in the human T-cell leukemia virus type I SU glycoprotein.

Single conservative and nonconservative amino acid substitutions were introduced into the gp45 external envelope protein (SU) of human T-cell leukemia virus type I (HTLV-I). The mutated amino acids were those identified as being conserved in HTLV-I, HTLV-II, and simian T-cell leukemia virus type I (but not in bovine leukemia virus). The mutated envelopes were tested for intracellular maturation and for function. Mutants with three major phenotypes could be defined: (i) 9 mutants with a wild-type phenotype, which included most of the conservative amino acid changes (five of seven) distributed throughout the SU protein; (ii) 8 mutants with affected intracellular maturation, 6 of which define a region in the central part of the SU protein essential for correct folding of the protein; and (iii) 13 mutants with normal intracellular maturation but impaired syncytium formation. These mutations likely affect the receptor binding step or postbinding events required for fusion. Five of these mutations are located between amino acids 75 and 101 of the SU protein, in the amino-terminal third of the molecule. The other mutations involve positions 170, 181, 195, 197, 208, 233, and 286, suggesting that two other domains, one central and one carboxy terminal, are involved in HTLV-I envelope functions.     

Genetic stability of human T lymphotropic virus type I despite antiviral pressures by CTLs

Human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is an inflammatory neurological disease. Patients with HAM/TSP show high proviral load despite increased HTLV-I Tax-specific CTL. It is still unknown whether the CTL efficiently eliminate the virus in vivo and/or whether a naturally occurring variant virus becomes predominant by escaping from the CTL. To address these issues, we sequenced a large number of HTLV-I tax genes from HLA-A*02 HAM/TSP patients and estimated synonymous and nonsynonymous changes of the genes to detect positive selection pressure on the virus. We found the pressures in three of six CTL epitopes in HTLV-I Tax, where amino acid substitutions preferentially occurred. Although some of variant viruses were not recognized by the CTL, no variant viruses accumulated within 3-8 years, indicating genetic stability of HTLV-I tax gene. These results suggest that CTL eliminate the infected cells in vivo and naturally occurring variant viruses do not predominate. As Tax is a regulatory protein which controls viral replication, the amino acid substitutions in Tax may reduce viral fitness for replication. Viral fitness and host immune response may contribute to the viral evolution within the infected individuals. Furthermore, the genetic stability in the epitopes despite the antiviral pressures suggests that the three epitopes can be the candidate targets for HTLV-I vaccine development.     

Naturally occurring variants of human T-cell leukemia virus type I Tax protein impair its recognition by cytotoxic T lymphocytes and the transactivation function of Tax.

There is a high degree of intraisolate sequence heterogeneity in the tax gene of human T-cell leukemia virus type I (HTLV-I), although the sequence variation between patients is small compared with that of human immunodeficiency virus type 1. In the present study, we investigated whether naturally occurring amino acid substitutions changed the properties of the Tax protein in two respects: first, recognition of the protein by cytotoxic T lymphocytes (CTL), and second, the ability of the Tax protein to transactivate various promoters. We found that (i) all of the observed amino acid substitutions that occur in known CTL epitopes abolished the recognition of the synthetic peptide representing the respective epitope; (ii) these substitutions occurred significantly more frequently in subjects carrying HLA-A2; and (iii) most of the amino acid substitutions severely reduced the ability of Tax protein to transactivate three promoters: the HTLV-I long terminal repeat, the c-fos promoter, and the interleukin-2 receptor alpha chain promoter.     

Mutational analysis of the human immunodeficiency virus type 1 Vif protein

Lentivirus Vif proteins are potent regulators of virus infectivity. However, relatively little is known about the functional domains, peptide motifs, or residues of any Vif protein. In this report, we present the first extensive mutagenesis analysis of the 192-amino-acid human immunodeficiency virus type 1 (HIV-1) Vif protein. A large number of scanning missense (mostly alanine substitution) and deletion mutations were introduced into the HIV-1HXB3 vif gene, and the resulting proteins were evaluated for the induction of virus infectivity as well as subcellular localization. The results show that amino acids dispersed throughout Vif's linear sequence are important for function. However, because many of the inactive proteins also appear to be mislocalized, we suggest that many of them may actually be misfolded rather lacking an intracellular targeting signal. Interestingly, disruptions within an internal region spanning residues 114 to 146 give rise to mutant proteins that either retain function or are inactive but are not substantially mislocalized. We therefore speculate that this region, which harbors two essential cysteine residues and one essential serine residue, may contain aspects of a putative Vif effector domain.     

Target epitope in the Tax protein of human T-cell leukemia virus type I recognized by class I major histocompatibility complex-restricted cytotoxic T cells.

A trans-acting regulatory gene product p40tax (Tax) of human T-cell leukemia virus type I (HTLV-I) is one of the main target antigens recognized by cytotoxic T lymphocytes (CTL) specific for HTLV-I. A CTL epitope within the Tax protein was identified in this report. HTLV-I-specific CD8+ CTL lines established from two HTLV-I carriers with HTLV-I-associated myelopathy or Sj?gren syndrome were previously demonstrated to kill predominantly the target cells expressing HTLV-I Tax. The CTL from two patients showed significant levels of cytotoxicity to autologous target cells pulsed with a synthetic peptide of 24 amino acids corresponding to the amino-terminal sequences of the Tax protein. Allogeneic target cells were also sensitized for CTL by this peptide when the target cells have HLA-A2. Tax-specific cytotoxicity, detected as cytolysis of the target cells infected with vaccinia virus-HTLV-I recombinant expressing Tax protein, was almost completely inhibited by competitor cells pulsed with the synthetic peptide. This indicates that a major CTL epitope is present in this peptide. Further analysis using shorter peptides revealed that the core sequence of the CTL epitope was LLFGYPVYV at positions 11 through 19. This sequence can be aligned with the HLA-A2-specific motifs reported recently.