Longitudinal analysis of human immunodeficiency virus type 1 nef/long terminal repeat sequences in a cohort of long-term survivors infected from a single source

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) in a cohort of long-term survivors infected with an attenuated strain of HIV-1 acquired from a single source. Although the cohort members experienced differing clinical courses, we demonstrate similar evolution of HIV-1 nef/long-terminal repeat (LTR) sequences, characterized by progressive sequence deletions tending toward a minimal nef/LTR structure that retains only sequence elements required for viral replication. The in vivo pathogenicity of attenuated HIV-1 is therefore dictated by viral and/or host factors other than those that impose a unidirectional selection pressure on the nef/LTR region of the HIV-1 genome.     

Characterization of nef sequences in long-term survivors of human immunodeficiency virus type 1 infection.

Studies with the simian immunodeficiency virus have shown that nef deletion results in a low level of viremia and a lack of disease progression in monkeys. Given the similarity of this clinical profile to that observed in long-term survivors of human immunodeficiency virus type 1 (HIV-1) infection, we sought to examine the nef gene in 10 patients who are clinically healthy and immunologically normal despite 12 to 15 years of infection. PCR and DNA sequencing were used to determine nef sequences in peripheral blood mononuclear cells obtained from long-term survivors. We found that there is no gross deletion within nef in the cases studied; most nef sequences (91.1%) obtained from 10 subjects contained a full-length and intact open reading frame. In addition, at the protein level, there were no discernible differences between the Nef consensus sequences derived from long-term survivors and those from patients with AIDS. We therefore conclude that deletion of or gross sequence abnormality within nef is not likely to be a common explanation for the well-being of long-term survivors of HIV-1 infection. Moreover, phylogenetic analysis of nef sequences suggests that HIV-1 strains found in our study subjects do not have a common origin.     

Biological characterization of nef in long-term survivors of human immunodeficiency virus type 1 infection.

We have previously shown that there were no gross deletions or obvious sequence abnormalities within nef of human immunodeficiency virus type 1 (HIV-1) in the 10 long-term survivors studied (Y. Huang, L. Zhang, and D. D. Ho, J. Virol. 69:93-100, 1995). Here we extend our study to examine these nef alleles in a functional context. Using a new technique, termed site-directed gene replacement, we have precisely replaced the nef of an infectious molecular clone, HIV-1HXB2, with nef alleles derived from 10 long-term survivors as well as from a patient with AIDS. The replication properties of these chimeric viruses demonstrated that the nef alleles derived from long-term survivors neither significantly increased nor decreased viral replication, compared with the nef allele of Nef+ HIV-1HXB2 and that derived from a patient with AIDS. However, Nef+ viruses always replicated faster than virus lacking nef. Moreover, single-cell infection analysis by the MAGI assay showed that these chimeric viruses, as well as Nef+ HIV-1HXB2, were more infectious than Nef- HIV-1HXB2 was. Therefore, we conclude that the genotypic and phenotypic features of nef are not likely to account for the nonprogression of HIV-1 infection in the 10 cases studied, unless the function of the nef gene in vivo is not accurately reflected by the in vitro assays we used.     

Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus.

A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted. To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed. CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers. Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells. CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA. These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.     

Analysis of long terminal repeat circle junctions of human immunodeficiency virus type 1.

Circle junctions of unintegrated human immunodeficiency virus type 1 strain IIIB were analyzed after polymerase chain reaction amplification. Among the 28 colonies sequenced, eight unique circle junction species were detected. Five of the eight species resulted in circle junctions with larger inserts than predicted. A majority of these could result from heterogeneity in generating the U5' long terminal repeat terminus.     

Effects of long terminal repeat mutations on human immunodeficiency virus type 1 replication.

The effects of deletions within three functional regions of the long terminal repeat of human immunodeficiency virus type 1 upon the ability of the long terminal repeat to direct production of the chloramphenicol acetyltransferase gene product and upon the ability of viruses that carry the mutations to replicate in human cell lines was investigated. The results show that the enhancer and TATAA sequences were required for efficient virus replication. Deletion of the negative regulatory element (NRE) yielded a virus that replicated more rapidly than did an otherwise isogeneic NRE-positive virus. The suppressive effect of the NRE did not depend upon the negative regulatory gene (nef), as both NRE-positive and NRE-negative viruses were defective for nef. We conclude that factors specified by the cell interact with the NRE sequences to retard human immunodeficiency virus type 1 replication.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.     

Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by cis-platin

We constructed a recombinant plasmid, pBHIV1 carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1), linked to the chloramphenicol acetyl transferase (CAT) gene plasmid. Plasmid pBHIV1 also contains the aminoglycoside phosphotransferase gene as a selectable marker. We introduced pBHIV1 in rat 208F fibroblasts and obtained stable geneticin resistant RFBHIV1-1 transfectant cells. A further control used was plasmid p202A, which carries the mutant T24 H-ras1 promoter linked to the promotorless cat gene. Plasmid p202A also carries the aph gene as a selectable marker and was transfected into 208F cells to obtain stable transfectant RF202A-1 cells. Both RFBHIV1-1 and RF202A-1 cells expressed CAT activity from the HIV LTR and T24 H-ras1 promoters. The response to cis-platin, a platin derivative and hexadecyl-phosphocholine was studied on the HIV LTR and H-ras1 regulated CAT activity in RFBHIV1-1 and RF202A-1 cells. It was found that at 5 x 10(-5) M concentrations cis-platin stimulates by 22-fold the expression of CAT from the HIV LTR, whereas only a 4-fold stimulation was observed on the T24 H-ras1 promoter. Our results suggest caution against therapy including this compound at cytotoxic concentrations in the treatment of AIDS patients.     

Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in cocultured lymphocytes.

One of the unexplained aspects of the progression of AIDS is that immunological abnormalities are detectable before CD4+ T-helper cell depletion occurs (A.R. Gruters, F.G. Terpstra, R. De Jong, C.J.M. Van Noesel, R.A.W. Van Lier, and F. Miedema, Eur. J. Immunol. 20:1039-1044, 1990; F. Miedema, A.J. Chantal-Petit, F.G. Terpstra, J.K.M.E. Schattenkerk, F. de Wolf, B.J.M. Al, M. Roos, J.M.A. Lang, S.A. Danner, J. Goudsmit, and P.T.A. Schellekens, J. Clin. Invest. 82:1908-1914, 1988; G.M. Shearer, D.C. Bernstein, K.S. Tung, C.S. Via, R. Redfield, S.Z. Salahuddin, and R.C. Gallo, J. Immunol. 137:2514-2521, 1986). In this report, we describe a mechanism by which human immunodeficiency virus type 1 (HIV-1)-infected cells can influence neighboring HIV-1-infected T lymphocytes and uninfected T cells as well. We have examined the interaction of T-cell and macrophage cell lines that are transfected with HIV-1 DNA by using cocultured lymphocytes. The HIV-1 constructs we used lack a functional pol gene and therefore do not produce infectious virus. Cocultivation results in the transcellular activation of the HIV long terminal repeat in the cocultured T cells. This transcellular activation is evident in as little as 3 h of cocultivation, at ratios of HIV-expressing cells to target cells as low as 1:1,000, and is dependent on the Tat-responsive element. The demonstration that a small number of HIV-expressing cells can affect a large number of uninfected bystander cells in a short period of time suggests a mechanism by which global immune dysfunction can precede the high prevalence of infected cells.     

Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro.

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A. Meyerhans, R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asj?, and S. Wain-Hobson, Cell 58:901-910, 1989). Once again in vitro culture resulted in the selection of minor forms. Over a 4-year period in vivo, there was no obvious selection for, or outgrowth of, any particular nef or U3/R sequence. Few defective nef protein sequences were observed, which argues against nef acting as a negative regulatory factor. Although no functionally defective promoter/trans-activation-responsive elements were identified, the transactivation efficiencies varied between 0.2 and 2 times that of the control. The sequence encoding the most efficient trans-activation-responsive region did not outgrow others. The extreme genetic heterogeneity of the different samples of the locus, either in vivo or in vitro, indicates that there is no such thing as a single, distinct HIV sequence. It is suggested that different HIV-1 loci evolve independently, recombination being responsible for their uncoupling.     

An integration-defective U5 deletion mutant of human immunodeficiency virus type 1 reverts by eliminating additional long terminal repeat sequences.

Nonoverlapping deletions that eliminated the 5' (HIV-1US/603del), middle (HIV-1U5/206del), and 3' (HIV-1U5/604del) thirds of the U5 region of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) were studied for their effects on virus replication (transient transfection of HeLa cells) and infectivity (T-cell lines and peripheral blood mononuclear cells). All three mutants exhibited a wild-type phenotype in directing the production and release of virus particles from transfected HeLa cells. In infectivity assays, HIV-1U5/206del was usually indistinguishable from wild-type virus whereas HIV-1U%/603del was unable to infect human peripheral blood mononuclear cells or MT4 and CEM cells. Investigations of HIV-1U5/603del particles revealed a packaging defect resulting in a 10-fold reduction of encapsidated genomic RNA. The HIV-1U5/604del mutant either was noninfectious or exhibited delayed infection kinetics, depending on the cell type and multiplicity of infection. Quantitative competitive PCR indicated that HIV-1U5/604del synthesized normal amounts of viral DNA in newly infected cells. During the course of a long-term infectivity assay, a revertant of the HIV-1U5/604del mutant that displayed rapid infection kinetics emerged. Nucleotide sequence analysis indicated that the original 26-nucleotide deletion present in HIV-1U5/604del had been extended an additional 19 nucleotides in the revertant virus. Characterization of the HIV-1U5/604del mutant LTR in in vitro integration reactions revealed defective 3' processing and strand transfer activities that were partially restored when the revertant LTR substrate was used, suggesting that the reversion corrected a similar defect in the mutant virus.     

Targeted derepression of the human immunodeficiency virus type 1 long terminal repeat by pyrrole-imidazole polyamides

The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.     

Viral phenotypes and antibody responses in long-term survivors infected with attenuated human immunodeficiency virus type 1 containing deletions in the nef and long terminal repeat regions

The Sydney Blood Bank Cohort (SBBC) consists of eight blood transfusion recipients infected with nef-attenuated human immunodeficiency virus type 1 (HIV-1) acquired from a single donor. Here, we show that viral phenotypes and antibody responses differ considerably between individual cohort members, despite the single source of infection. Replication of isolated virus varied from barely detectable to similar to that of the wild-type virus, and virus isolated from five SBBC members showed coreceptor usage signatures unique to each individual. Higher viral loads and stronger neutralizing antibody responses were associated with better-replicating viral strains, and detectable viral replication was essential for the development of strong and sustained humoral immune responses. Despite the presence of strong neutralizing antibodies in a number of SBBC members, disease progression was not prevented, and each cohort member studied displayed a unique outcome of infection with nef-attenuated HIV-1.     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.