Entire genome of a strain of human immunodeficiency virus type 1 with a deletion of nef that was recovered 20 years after primary infection: large pool of proviruses with deletions of env

Abstract

We report the complete sequence analysis of the provirus harbored in a long-term nonprogressor (patient SG1) 20 years after the first infection with a human immunodeficiency virus type 1 strain lacking nef. The sequencing showed large deletions in the nef-nef and nef-U3 regions. Except for vpu, all of the other accessory genes were intact. The gag and pol genes did not show significant alterations. We found large deletions in env, spanning the V1, V2, V3, V4, and V5 regions. We believe that, when down-regulation of the class 1 major histocompatibility complex molecules is inhibited by the lack of nef function, the cells containing Env-defective molecules evade cytotoxic T lymphocyte killing and accumulate progressively.

FIG. 1.

Analysis of the entire HIV-1 genome in PBMCs of SG1. (A) Schematic representation of HIV-1 genome. Numbers represent nucleotide positions. (B) Second-round-PCR products encompassing the entire HIV-1 genome of SG1. These PCR fragments were subjected to sequence analysis.

FIG. 2.

Schematic representation of nef-LTR deletions of HIV-1 detected from patient SG1 in 2005. (A) Deletions in SG1 provirus. The positions of the PCR primers are indicated by arrows: primers NEF1 and NEF2 were used for the first round, and primers NEF2 and NEF3 were used for the second round. Black boxes represent normal sequences; blank spaces represent deletions. Nucleotide numbering refers to HXB2 sequence positions. The numbers in the blank regions represent the sizes (in base pairs) of the deletions. (B) Deletions detected in viral RNA present in plasma by a single-round PCR using primers NEF1/NEF2.

FIG. 3.

Characterization of env gene in the provirus harbored in patient SG1. The upper part of the figure shows a schematic representation of the first and second rounds of the PCR protocols designed for the amplification of the proximal (A upper), middle (B upper), and distal (C upper) parts of the env gene. Nucleotide numbering refers to HXB2 sequence positions. The lower part of the figure shows the gel patterns of the nested-PCR products for the proximal (A lower) and distal (C lower) tracts of env. The lengths of the polymorphisms of the clones derived from the PCR product designed for the middle part of env are shown in panel B lower. cl-1, clone no. 1; cl-3, clone no. 3; etc.

FIG. 4.

Alignment of the entire Env protein. Shown are sequences of proximal and distal parts of Env and of the subclones (cl-1, cl-3, cl-4, cl-5, cl-6, and cl-7) derived from a PCR performed for the middle part of Env. The changed amino acids are shown in bold characters. Dashes indicate the deleted amino acids. The start and end of the V1, V2, V3, V4, and V5 domains are indicated by vertical lines. Two oblique lines indicate the gp120-gp41 proteolytic domain.