Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor - PubMed (original) (raw)

Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor

R Salvi et al. J Virol. 1998 May.

Abstract

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4+ T-cell counts (>1,000 CD4 cells/microl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5' long terminal repeat (5'LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5'LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5'LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.

PubMed Disclaimer

Figures

FIG. 1

(A) Kinetics of p24 antigen accumulation in the supernatants of undepleted (closed symbols) and CD8-depleted (open symbols) PBMC cultures from patient SG1 with (squares) and without (circles) exogenous infection with HIV-1. (B) Kinetics of p24 antigen accumulation in the supernatants of HIV-infected undepleted (▪) and CD8-depleted (□) PBMC cultures from an HIV-seronegative individual. Error bars indicate standard deviations.

FIG. 2

Analysis of length polymorphism of 5′LTR/gag leader (bp 128 to 784) and nef gene regions in PBMC obtained from patient SG1 in May 1994 (lanes 2 and 6), October 1995 (lanes 3 and 7), and June 1996 (lanes 4 and 8). DNA fragments from the second PCR amplification were separated by electrophoresis through a 2% agarose gel and visualized by ethidium bromide staining. The positive control (lanes 1 and 5) consists of plasmid pNL4-3, which yields two full-length bands of 657 bp (5′LTR/gag leader) and 705 bp (nef gene).

FIG. 3

Alignment of 11 HIV-1 5′LTR/gag leader sequences in PBMC derived from patient SG1 in June 1996, along with the alignment of the consensus for these sequences and the alignment of the NL4-3 sequences with respect to the consensus. Asterisks indicate identity to the consensus sequence. A number symbol indicates the absence of a base pair. The positions of the motifs for the NF-AT, USF, TCF-1 alpha, NF-κB, and Sp1 sites, TATAA box, bulge and loop elements of the TAR, primer binding site (PBS), and SD 1 (major 5′ splice donor) are boxed. A schematic drawing of the LTR/gag leader region is shown above the alignment, and the positions of the nested-PCR primers are indicated by arrows at the top.

FIG. 3

FIG. 4

Locations of the deletions present within the nef gene in PBMC derived from patient SG1 in June 1996. A schematic drawing of the genomic structure of the NL4-3 HIV-1 clone (1) for the same region is shown at the top. The positions of the nested-PCR primers are indicated by arrows. Black boxes represent normal sequence, while blank spaces represent deletions. Nucleotide numbering refers to NL4-3 sequence positions. The numbers above the blank regions represent the sizes of the deletions. The deletions shown in the LD row are referred to the sequencing of the LD-PCR product.

FIG. 5

Identification of proviruses with partially deleted 5′LTR termini: Southern blotting and sequence analysis of the 5′LTRs of proviruses in SG1 PBMC. Hybridization with L2-F (A) and L3-R (B) 32P-labelled probes is shown. Lanes −, negative controls (DNA extracted from 105 PBMC of a healthy blood donor). Lanes 1 and 2, positive controls (1 pg [lane 1] and 1 fg [lane 2] of pNL4-3 DNA mixed with DNA extracted from 105 PBMC of a healthy donor). The other lanes show PCR products of DNA extracted from 105 PBMC collected from patient SG1 at the May 1995 and June 1996 time points. (C) Alignment of partial sequences relative to the 5′LTR regions of clones obtained from the 680-bp PCR product.

FIG. 6

Phylogenetic analysis of HIV-1 nef/U3 quasispecies in patient SG1, showing the relationship between the sequences derived from different sets of experiments. The tree was constructed by the neighbor-joining method, using Clustal (16). One thousand bootstrap replications were performed. NL4-3 was chosen as the prototype sequence.

FIG. 7

LD-PCR analysis of nearly full-length HIV-1 provirus (about 9 kb) and restriction enzyme analysis of the 3′ half of the viral genome (about 4.5 kb). (Top) Schematic illustration of HIV-1 proviral DNA showing the positions and the orientations of the nested PCR primers used to generate either the 9- or 4.5-kb fragment. The magnified 3′ half of the provirus shows the locations of the regulatory and accessory genes, the restriction sites (K, _Kpn_I; X, _Xho_I), and two nef deletions (white boxes). The reference is the HIV-1 NL4-3 map. (Bottom left) Agarose gel electrophoresis (0.8%) of the second-round PCR products obtained from the 9-kb amplification (nested primer pairs, 626s-9680a for first round and 691s-9614a for second round). Lane 1, pNL4-3 (10 fg of plasmid diluted in DNA extracted from PBMC of a seronegative subject). Lane 2, SG1 (DNA extracted from 2 × 105 PBMC). Lane L, molecular size ladder (MBI, Fermentas). (Bottom right) Restriction site analysis of the 3′ half of the viral genome amplified by the nested primer pairs 626s-9680a (first round) and 5048s-9614a (second round). Lane 1, undigested pNL4-3; lane 2, undigested SG1; lanes 3 and 5, pNL4-3 digested with _Kpn_I and _Xho_I, respectively; lanes 4 and 6, SG1 digested with _Kpn_I and _Xho_I, respectively.

FIG. 8

DNA sequence at the deletion junction. Sequences of nef clones, 5′LTR deletion clones (consensus) and the LD-PCR product (LD) are compared with the corresponding sequence of HIV-1 NL4-3. The direct repeats CCAA are underlined. The numbers underneath the nef and 5′LTR deletion clones refer to the frequency of the repeats among them. nt, nucleotides.

References

1. Adachi A, Gendelman H E, Koenig S, Folks T, Willey R, Rabson A, Martin M. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986;59:284–291. - PMC - PubMed
1. Aiken C, Trono D. Nef stimulates human immunodeficiency virus type 1 proviral DNA synthesis. J Virol. 1995;69:5048–5056. - PMC - PubMed
1. Aiken C, Konner J, Landau N R, Lenburg M E, Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell. 1994;76:853–864. - PubMed
1. Benedetto A, Garbuglia A R, Di Caro A, Lo Presti E, Alfani E, Delfini C. Virus free survival and down-regulation of CD4 in C8166 cells infected with human immunodeficiency virus type 1 at low density. J Gen Virol. 1993;74:2595–2601. - PubMed
1. Blumberg B M, Epstein L G, Saito Y, Chen D, Sharer L R, Anand R. Human immunodeficiency virus type 1 Nef quasispecies in pathological tissue. J Virol. 1992;66:5256–5264. - PMC - PubMed

Grossly defective nef gene sequences in a human immunodeficiency virus type 1-seropositive long-term nonprogressor - PubMed (original) (raw)