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. 1999 Jul;73(7):5497-508.
doi: 10.1128/JVI.73.7.5497-5508.1999.

Sequence variations in human immunodeficiency virus type 1 Nef are associated with different stages of disease

F Kirchhoff  1 , P J Easterbrook, N Douglas, M Troop, T C Greenough, J Weber, S Carl, J L Sullivan, R S Daniels
Affiliations

Sequence variations in human immunodeficiency virus type 1 Nef are associated with different stages of disease

F Kirchhoff et al. J Virol. 1999 Jul.

Abstract

nef alleles derived from a large number of individuals infected with human immunodeficiency virus type 1 (HIV-1) were analyzed to investigate the frequency of disrupted nef genes and to elucidate whether specific amino acid substitutions in Nef are associated with different stages of disease. We confirm that deletions or gross abnormalities in nef are rarely present. However, a comparison of Nef consensus sequences derived from 41 long-term nonprogressors and from 50 individuals with progressive HIV-1 infection revealed that specific variations are associated with different stages of infection. Five amino acid variations in Nef (T15, N51, H102, L170, and E182) were more frequently observed among nonprogressors, while nine features (an additional N-terminal PxxP motif, A15, R39, T51, T157, C163, N169, Q170, and M182) were more frequently found in progressors. Strong correlations between the frequency of these variations in Nef and both the CD4(+)-cell count and the viral load were observed. Moreover, analysis of sequential samples obtained from two progressors revealed that several variations in Nef, which were more commonly observed in patients with low CD4(+)-T-cell counts, were detected only during or after progression to immunodeficiency. Our results indicate that sequence variations in Nef are associated with different stages of HIV-1 infection and suggest a link between nef gene function and the immune status of the infected individual.

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Figures

FIG. 1
FIG. 1
Alignment of Nef protein sequences derived from HIV-1-infected individuals with different rates of disease progression. Representative consensus Nef protein sequences from 41 NPs, 18 SPs, and 32 RPs were aligned. Nef protein sequences were derived from the present study (NPs 1 to 28, SPs 1 to 15, and RPs 19 to 38) and from studies by Huang et al. (15) (NPs 29 to 38), Michael et al. (27) (NPs 39 to 41, SPs 16 to 18, and RPs 39 to 41), and Shugars et al. (38) (patients 42 to 50). The consensus amino acid sequence is shown at the bottom. The NP and progressor consensus sequences and the HIV-1 NL4-3 sequence are also indicated. The first column indicates the index number for all NPs and progressors (SPs and RPs combined) in the study. In the second column, the first two to four numbers or letters specify the individual patient, and the last letter(s) specifies the progression grouping. Some conserved sequence elements are indicated schematically, and additional PxxP motifs close to the N terminus are in boldface. The position of the polypurine tract (PPT) and the start of the 3′ LTR (➩) are also indicated. Stars above the alignment indicate positions where amino acid variations between the different progressor groups are observed; the number gives the corresponding amino acid position in the NL4-3 Nef. Dashes indicate identity with the consensus sequence, dots indicate gaps introduced to optimize the alignment, and asterisks indicate stop codons.
FIG. 1
FIG. 1
Alignment of Nef protein sequences derived from HIV-1-infected individuals with different rates of disease progression. Representative consensus Nef protein sequences from 41 NPs, 18 SPs, and 32 RPs were aligned. Nef protein sequences were derived from the present study (NPs 1 to 28, SPs 1 to 15, and RPs 19 to 38) and from studies by Huang et al. (15) (NPs 29 to 38), Michael et al. (27) (NPs 39 to 41, SPs 16 to 18, and RPs 39 to 41), and Shugars et al. (38) (patients 42 to 50). The consensus amino acid sequence is shown at the bottom. The NP and progressor consensus sequences and the HIV-1 NL4-3 sequence are also indicated. The first column indicates the index number for all NPs and progressors (SPs and RPs combined) in the study. In the second column, the first two to four numbers or letters specify the individual patient, and the last letter(s) specifies the progression grouping. Some conserved sequence elements are indicated schematically, and additional PxxP motifs close to the N terminus are in boldface. The position of the polypurine tract (PPT) and the start of the 3′ LTR (➩) are also indicated. Stars above the alignment indicate positions where amino acid variations between the different progressor groups are observed; the number gives the corresponding amino acid position in the NL4-3 Nef. Dashes indicate identity with the consensus sequence, dots indicate gaps introduced to optimize the alignment, and asterisks indicate stop codons.
FIG. 2
FIG. 2
Nef sequence variation within and between NPs and progressors (P). The middle lines depict the NP (upper) and P (lower) consensus sequences with the predominant residue at each position. The sequence variation is represented above (NP) and below (P) the consensus sequences. Subscripted numbers show the number of patient-specific consensus Nef protein sequences in which that amino acid was observed. Several conserved motifs and the locations of five Nef features more frequently observed in NPs (NP-1 to NP-5) and nine features more frequently observed in progressors (P-1 to P-9) are indicated by shaded ovals. Differences between the NP and P consensus sequences are indicated by vertical bars. Amino acids that differ markedly between the two groups of patients are in white with black background. For symbols, see the legend to Fig. 1.
FIG. 3
FIG. 3
Correlation between the NefProg score and the CD4+-cell count and the viral load in patients in the Worcester cohort. The circles represent the absolute CD4+-cell counts (A), the plasma viral RNA levels (B), and the proviral copy numbers (C) in relation to the NefProg score given on the x axis. Pearson correlation coefficients obtained with the Fisher transformation test were used to assess the statistical significance.
FIG. 4
FIG. 4
CD4+-cell counts in relation to NefProg score. The dots represent the CD4+-cell count and NefProg score for each of the 100 patients analyzed. In addition to those from the patients described in the legend to Fig. 1, data from nine additional HIV-1-infected persons were included (33). The linear regression line is indicated.
FIG. 5
FIG. 5
Profile of CD4+-T-cell counts in patients FA and MB. The month and year of blood sampling are given on the x axis. The vertical arrows indicate the time points at which PBMCs were collected for genomic DNA extraction and PCR analysis. ■, percentage of CD4+ T lymphocytes; □, absolute number of CD4+ T lymphocytes.
FIG. 6
FIG. 6
Alignment of the predicted Nef amino acid sequences derived from patients FA and MB. The two-digit number in the left column gives the year of PBMC collection, and the last number specifies the individual clone. The Nef consensus sequences derived from NPs (Non-Con) and progressors (Pro-Con) are shown at the bottom for comparison. The Non-ex and Pro-ex sequences also contain those variations that were more frequently observed in NPs or RPs but did not alter the NP and RP consensus Nef sequences. The nine amino acid positions corresponding to those at which differences between Nef sequences derived from NPs and RPs were observed are indicated by stars above the alignment. The positions of additional N-terminal PxxP motifs in deduced FA and MB Nef sequences are shaded. Abbreviations and symbols are described in the legend to Fig. 1.
FIG. 7
FIG. 7
Localization of amino acid positions at which variations between NPs and RPs were observed on the tertiary structure of Nef. Residues H102, N157, and E182 were frequently found at these positions in NPs. The four α-helices (αa to αd), the four β-strands (βa to βd), and the approximate position of the (PxxP)3 motif are also indicated. Regions encompassing the remaining six amino acid positions used to define the NefProg score (residues 15, 39, 51, 63, 169, and 170) were deleted from the molecule used to determine the nuclear magnetic resonance solution structure of Nef (14).
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