Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by a two-amino-acid insertion in HIV-1 Vif from a nonprogressing mother and child

Abstract

We studied a 15-year-old girl, patient X, who has maintained consistently low plasma loads of human immunodeficiency virus type 1 (HIV-1) RNA, as well as normal and stable CD4(+) T-cell concentrations. She has presented no clinical manifestations of AIDS, despite having only received zidovudine monotherapy for a part of her life. Patient X's HIV-positive mother (patient Y) has also not progressed to AIDS and has never been treated with antiretroviral agents. HIV-1 isolated from patient X replicated poorly in human peripheral blood mononuclear cells (PBMC). In order to map the determinant of the poor growth of patient X's isolate, viral sequences from patient X were determined and examined for insertion or deletion mutations. These sequences contained a two-amino-acid insertion mutation in the Vif gene, which was also observed in uncultured PBMC acquired at different times. Furthermore, Vif sequences harbored by patient Y contained the identical mutation. These observations suggest that polymorphic HIV-1 was transmitted to patient X perinatally 15 years previously and has been maintained since that time. Recombinant HIV-1, engineered with Vif sequences from patient X, replicated in PBMC to levels approximately 20-fold lower than that of wild type. Removal of the insertion mutation from this recombinant restored replication efficiency to wild-type levels, while introduction of the insertion mutation into wild-type Vif sequences resulted in greatly decreased replication. Furthermore, Vif protein from patient X's HIV-1 was aberrantly cleaved, suggesting a mechanism for loss of Vif function. Since HIV-1 containing these sequences replicates poorly, the implication is that the two-amino-acid insertion mutation in Vif contributes significantly to the nonprogressor status of this mother and child. Further studies of these sequences might provide information regarding contributions of Vif structure and/or function to HIV-1 virulence.

FIG. 1.

Plasma viremia and CD4⁺ T-cell counts for patient X. The black diamonds indicate the concentrations of HIV RNA in plasma and the white circles indicate the numbers of CD4⁺ T-cells/ml in PBMC from patient X. The vertical arrow indicates the time when zidovudine monotherapy was stopped.

FIG. 2.

(A) Nucleotide alignment of LTR sequences from strains from patients X and Y and clade B consensus LTR sequences. (B) Amino-acid alignment of HIV-1 Vif sequences from strains from patients X and Y and clade B consensus HIV-1 Vif sequences. (C) Amino-acid alignment of the sequence from the strain from patient X and Clade B consensus, NL4-3, and engineered HIV-1 Vif sequences derived from the strain from patient X or from NL4-3. The stars indicate conservation between the Clade B consensus sequence and the sequences from the strains from patient X. Dashes denote nucleotides or amino acids that are not contained in a particular sequence. The areas shaded in grey indicate the locations of the mutant sequences.

FIG. 3.

(A) Replication in CEMx174 cells of HIV-1 strain NL4-3 and an NL4-3 recombinant (XL) that contains the 5-base insertion mutation in the LTR observed in HIV-1 isolated from patient X. (B) Replication in CEMx174 cells that express Vif (CEMx174Vif) of HIV-1 strain NL4-3, an NL4-3-based recombinant that contains a large deletion in Vif (ΔVif), and an NL4-3 recombinant that contains Vif sequences from the strain from patient X (XV). (C) Replication of the viral strains described in panel B in PBMC isolated from HIV-1-seronegative donors. (D) Replication in H9 cells of the viral strains described for panel B.

FIG. 4.

Replication in PBMC isolated from HIV-1-seronegative donors of HIV-1 strain NL4-3, an NL4-3-based recombinant that contains the insertion mutation (DS) observed in Vif from the strain from patient X (NL4-3+DS), and an XV-based recombinant with the insertion mutation removed (XVΔDS).

FIG. 5.

(A) Western immunoblot of strains from patient X and strains with NL4-3 or derivative Vif sequences. Cells transfected with vector sequences without Vif sequences (Vector) served as the control. The migration of molecular size markers is indicated to the left. (B) RT-PCR of strains from patient X and strains with NL4-3 or derivative Vif sequences. A plus sign indicates that the isolated RNA was reverse transcribed prior to PCR amplification, and a minus sign indicates that the isolated RNA was not reverse transcribed prior to PCR amplification. A 100-nucleotide ladder is shown on the left, and the identity of the 600-nucleotide band is indicated.