JOURNAL OF VIROLOGY, Apr. 2010, p. 3644–3653
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 7
Meta-Analysis To Test the Association of HIV-1 nef Amino Acid
Differences and Deletions with Disease Progression?†
Ravindra Pushker,1,2,3Jean-Marc Jacque ´,2,3,4and Denis C. Shields1,2,3*
UCD Complex and Adaptive Systems Laboratory,1UCD Conway Institute of Biomolecular and Biomedical Research,2
School of Medicine and Medical Science,3and Centre for Research in Infectious Diseases,4
University College Dublin, Belfield, Dublin 4, Ireland
Received 15 September 2009/Accepted 30 December 2009
Previous relatively small studies have associated particular amino acid replacements and deletions in the
HIV-1 nef gene with differences in the rate of HIV disease progression. We tested more rigorously whether
particular nef amino acid differences and deletions are associated with HIV disease progression. Amino acid
replacements and deletions in patients’ consensus sequences were investigated for 153 progressor (P), 615
long-term nonprogressor (LTNP), and 2,311 unknown progressor sequences from 582 subtype B HIV-infected
patients. LTNPs had more defective nefs (interrupted by frameshifts or stop codons), but on a per-patient basis
there was no excess of LTNP patients with one or more defective nef sequences compared to the Ps (P ? 0.47).
The high frequency of amino acid replacement at residues S8, V10, I11, A15, V85, V133, N157, S163, V168, D174, R178,
E182, and R188in LTNPs was also seen in permuted datasets, implying that these are simply rapidly evolving
residues. Permutation testing revealed that residues showing the greatest excess over expectation (A15, V85,
N157, S163, V168, D174, R178, and R188) were not significant (P ? 0.77). Exploratory analysis suggested a
hypothetical excess of frameshifting in the regions9SVIG and118QGYF among LTNPs. The regions V10and
152KVEEA of nef were commonly deleted in LTNPs. However, permutation testing indicated that none of the
regions displayed significantly excessive deletion in LTNPs. In conclusion, meta-analysis of HIV-1 nef se-
quences provides no clear evidence of whether defective nef sequences or particular regions of the protein play
a significant role in disease progression.
HIV-infected people can be categorized according to the
number of years in which they progress to AIDS. Long-term
nonprogressors (LTNPs) do not progress to AIDS even after
more than 10 years of infection, and they maintain stable CD4
lymphocyte counts (5, 8). Nonprogression status may reflect
differences in either in the host, in viral genetics, or in envi-
ronmental factors. Within the virus, R77Q, a mutation in the
HIV-1 vpr gene, was associated with both LTNP infection and
impaired induction of apoptosis (38). However, this mutation
was not statistically significant, and no other clearly attenuating
mutations or deletions were detected (20). Most attention,
however, has focused on role of the viral nef protein.
In rhesus monkeys infected with simian immunodeficiency
virus (SIV), a model for studying AIDS pathogenesis (37),
animals infected with nef-deficient SIV showed an attenuated
course of infection (17, 30, 31, 51). nef was also a major de-
terminant of pathogenicity in transgenic mice with AIDS-like
symptoms induced by HIV-1 (27). Some patients with LTNP
strains of HIV were found to have gross deletions in the nef
gene (16, 33, 49), suggesting the importance of nef for HIV-1
progression in humans.
Previous studies related to phylogenetic analysis have re-
ported that nef sequences from patients with different rates of
progression do not form distinct clusters (28, 29, 40, 43). Each
patient had sequences that clustered together and could be
differentiated from those of the other patients, supporting the
monophyletic origin of the infections. The absence of intra-
group clustering suggested that no correlation existed between
the phylogenetic relationship of the nef sequences and the
progression rate in the patients (10). The differences in genetic
distance between LTNP and progressors (Ps) were not statis-
tically significant, suggesting that the degree of sequence vari-
ation in nef is unlikely to reflect the stage of HIV-1 disease (4).
Amino acids 25 to 36 in HIV-1 nef are important both for
several well-defined in vitro functions of nef and for the patho-
genicity of HIV-1 in humans, and nef’s ability to enhance virion
infectivity was fully restored when the deletion was repaired by
the insertion of that region (8). Nef proteins derived from
LTNPs and slow progressors (SPs) were found to be defective
or far less capable of enhancing viral replication and/or viral
infectivity in herpesvirus saimiri-transformed human T cells
and peripheral blood mononuclear cells (PBMC) (24). The
sizes of the deletions in the nef/LTR (long terminal repeat)
region increased progressively from 84 to 1,400 bp during the
5-year follow-up period in one case of a SP (35). Gross defects
were also present in the RNA-derived sequences of an LTNP
individual because of a frameshift and the premature termina-
tion of the protein (4). HIV-1 sequences from the isolates or
patient PBMC had similar deletions in the nef gene and in the
region of overlap of nef and the U3 region of the LTR (16).
There was a 36-bp deletion close to the 5? end of nef that
impaired nef function in an LTNP (8).
Many studies not only have described nef as carrying large
deletions in LTNPs (16, 33) but also found a higher proportion
* Corresponding author. Mailing address: 2M, UCD-CASL (Com-
plex and Adaptive Systems Laboratory), University College Dublin,
Belfield, Dublin 4, Ireland. Phone: 353-1-7165344. Fax: 353-1-7165396.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 13 January 2010.
of disrupted nef gene sequences in LTNPs. A study of six HIV
patients who reached at least 11 years of age without or with
mild symptoms revealed that LTNPs had higher proportions of
disrupted nef sequences (10). Seven LTNPs, all belonging to
the same cohort of infected hemophiliacs, had more defective
nef sequences than in progressors; the number of disrupted nef
sequences within each individual was significantly higher in
LTNPs than in progressors (4).
The nef amino acid sequence has been reported to be highly
polymorphic even within a particular subtype (4, 22, 28, 29, 40,
42, 53). Single amino acid deletions have been found predom-
inantly at three locations that are structurally less defined loop
regions: positions 8 to 11, 49 to 51, and 155 to 162 (25). Five
variants (T15, N51, H102, L170, and E182) have been noted
among LTNPs, whereas nine variants (N-terminal PxxP motif;
A15, R39, T51, T157, C163, N169, Q170, and M182) have been
noted among progressors (32). nef has been often changed at
residues localized in the folded core domain at cytotoxic-T-
lymphocyte epitopes (E105, K106, E110, Y132, K164, and R200);
moreover, LTNP-associated variations occur in the core do-
main of nef. Recently, nef sequence variations have been found
in the WL motif of the CD4 binding site, as well as a premature
stop codon in infected LTNPs that could potentially contribute
to the attenuation of the virus; however, these deletions were
found to be insignificant (13).
There has been a broad agreement that grossly defective nefs
are associated with an attenuated course of infection (17, 30,
31, 51) but rare in HIV-1 infection (32). Grossly defective nef
genes or significant changes from relevant clade reference se-
quences were not identified in a study of 32 LTNP children
(13). One study noted that the proportion of disrupted nef se-
quences within each patient was significantly higher in LTNPs
compared to Ps; however, the proportions of individuals with
nef defects (in LTNPs, 5 of 7, and in Ps, 6 of 8) were similar (4).
No major defects have been reported in a few other studies
(28, 39, 40). Another study of a small number of patients does
not indicate that gross deletions play any major role in delaying
or halting disease progression in infected drug abusers in Italy
(11), and premature stop codons were observed at equivalent,
yet low, frequencies among the different clinical groups (41). In
addition, disease progression has been reported in a HIV pa-
tient with a virus grossly deleted of nef (26).
Thus, overall, most of these studies were based on observa-
tion or case study rather than systematic scientific evaluation
(11). The objective of the present study was to determine in a
substantially larger sample than investigated to date whether
there is any association between disease progression and par-
ticular nef amino acid differences or deletions.
MATERIALS AND METHODS
Data collection. HIV-1 subtype B nef nucleotide and protein sequences from
Pa, LTNPs, and UPs (those for whom the “progression” field was null) were
TABLE 1. Previously published studies related to LTNPs and Ps
No. of patients
(no. of sequences)a
CountryYr Isolation sourceObservations
Age, 16-49 yrs
ItalyIntravenous drug users
Age, 43 yrs; homosexual
Age, 35 yrs
A cohort of six blood or blood
Perinatally infected children
Homosexual and intravenous
10 (89) PBMC; plasma
Age, 25-54 yrs
10 1995Age, 38-47 yrs
aThe total represents the total number of patients (or the total sequences studied ?in parentheses?) as reported by the authors of that reference and could include
sequences from other progressions in a few cases.
bUS, United States.
VOL. 84, 2010HIV-1 PROTEINS AND DISEASE PROGRESSION 3645
collected from the Los Alamos National Laboratory (LANL) HIV Sequence
Compendium 2009 (36) and the LANL website (http://www.hiv.lanl.gov) in No-
vember 2009. Details of all of the patients such as patient identification (ID) data
and sampling details were also retrieved from the LANL website. Some of the
information was retrieved from NCBI GenBank files and, in a few cases, from the
LANL publications. A few of these sequences belong to previous studies (de-
scribed in Table 1). We used the LTNP and P definitions of the LANL database,
which records the rate of progression of the patient as recorded by the study.
Table S1 in the supplemental material provides details such as the patient IDs,
country, number of sequences (defective and nondefective), mean lengths of
genes and proteins, PubMed IDs, risk factor(s), year of infection, number of
sampling time points, name of the isolate or clone, sampling year, patient age,
health, viral load, CD4 count, number of days from infection, etc. (if available),
for all LTNP and P patients considered in these analyses. The numbers of
sequences and the numbers of patients in the present study are summarized in
Table 2. The nef sequence from the HIV-1 NL4.3 strain (206 amino acids) was
used as a reference sequence for the whole analysis since nef is defective in the
standard reference strain HXB2, containing a mutation at position 124, which
produces a stop codon. NL4.3 has been used as a reference strain in previous
studies (see, for example, references 15, 34, and 56). All amino acid positions are
given relative to the reference sequence.
Defective nefs. To detect frameshifts, DNA sequences from all patients were
compared to the nef reference sequence. All nefs which had deletions due to
frameshifts or premature stop codons (pseudo genes) were considered to be
defective sequences and excluded from the main analysis. Only nondefective
nefs, i.e., 153 P sequences from 22 patients, 615 LTNP sequences from 155
patients, and 2,311 UP sequences from 405 patients were considered for these
main analyses (Table 2). The numbers of patients from different isolation sources
are summarized in Table 2. The primary comparison was done between LTNPs
and Ps, and a secondary comparison, for reason of patient numbers, was made
between LTNPs and UPs.
Amino acid replacement. To find consensus sequence corresponding to a
patient, all sequences of that patient were aligned by using MUSCLE (18, 19).
The aligned sequences were used to generate consensus sequence using the
“consensus” program from EMBOSS (46) that uses the sequence weights and a
scoring matrix to calculate a score for each amino acid residue or nucleotide in
the alignment; the highest-scoring residue goes into the consensus sequence if
the score is higher than half the total weight of all of the sequences. For primary
comparison, all consensus sequences from LTNPs and Ps were aligned by using
the MUSCLE alignment program (18, 19). CLUSTAL X (54) was used to create
phylogenetic trees using the neighbor-joining method (48). A total of 1,000
bootstrap replicates were performed in order to determine the confidence of the
We then matched pairs of most closely related LTNP versus P patients,
choosing the P patient whose consensus sequence was most closely related to the
LTNP consensus from the phylogenetic tree of all LTNP and P consensus
sequences. This was done as an attempt to reduce the amount of noise created
by sequence changes during evolution that are not associated with the potential
evolution of a switch in progression status. This matching of pairs was defined
manually, based on inspection of the nef consensus sequence phylogenetic tree.
A total of 20 pairs of LTNPs which were closely related to the Ps were obtained
(Fig. 1). The details of the above 20 pairs of patients are provided in Table S2 in
the supplemental material. For each pair, differences in residues were calculated
and an observed score (SOi) was determined for each residue as defined by
j ? 1
j ? NC?
k ? 1
k ? AL
Dji??1; if amino acids at ith position in jth pair are diferent
ALand NCrepresent alignment length and the number of pairs, respectively.
We wanted to determine whether the residue changes that defined the differ-
ences between a matched pair of LTNP and P consensuses were distributed
differently compared to the differences seen with any pairwise combination of the
20 patients. Since we had matched the pairs to be closely related, then if there
were subsequent amino acid changes occurring at a particular residue position
altering the phenotype, such changes should be enriched among the matched
pairs compared to the randomly matched pairs, which would then be expected to
be in practically all cases more evolutionarily divergent. Thus, to calculate ex-
pected values of differences in residues, all combinations of 20 pairs of LTNP
versus P pairs were generated, giving rise to 400 combinations. Subsequently, for
each pair, similar to SOi, an expected score (SEi) was determined for each residue
as defined by equation 3.
j ? 1
j ? NC
k ? 1
k ? AL
We then defined a score (Si) for each residue i as the ratio of observed and
expected scores for that residue, as follows:
TABLE 2. Number of HIV-1 subtype B patients and nef sequences studied
Data type and source
No. of patients (no. of sequences)No. of sequences (%)a
Avg no. of
LTNPs Ps UPsTotal LTNPs PsUPs TotalTotal NDD
No. of patients
Downloaded data/frameshift study
No. of sequences
Avg no. of sequences sampled
aBased on isolation sources.
bND, nondefective; D, defective.
cThe number of patients is the number with at least one nondefective nef.
dThe number of patients is the number with at least one defective nef.
3646 PUSHKER ET AL. J. VIROL.
To visualize these findings along the nef sequence, we plotted histograms of
SOiand SEi(Fig. 2). Scores for the most extreme residue (R1) and for the fifth
most extreme residue (R5) were recorded as SR1and SR5, respectively. In order
to assess whether R1andR5were higher than expected by chance, we carried out
permutation tests. A total of 10,000 permuted datasets were generated in which
all 20 pairs of sequences were randomly assigned as being either Ps or LTNPs
(i.e., typically ca. 50% of the pairs had their phenotypic status switched around),
giving arise to 400 pairs. For all iterations, Siwas recorded for each residue. For
the 10,000 permutations, we then recorded in how many of them one or more
residues with values greater than SR1were observed and in how many permu-
tations five or more residues with values greater than SR5were observed.
The secondary comparison was done in between LTNP and UP and the whole
process was repeated by replacing P by UP. We matched pairs of the most closely
related LTNP versus UP patients, choosing the UP patient whose consensus
sequence was most closely related to that of LTNP. A total of 47 closely related
FIG. 1. Phylogenetic relationship of HIV-1 subtype B nef consensus sequences from LTNP and P patients. Each LTNP patient paired with the
closest P patient has been marked. There were 20 such pairs. Bootstrap values greater than 70% are indicated in the figure. Groupings are made
in the figure purely for ease of viewing.
VOL. 84, 2010HIV-1 PROTEINS AND DISEASE PROGRESSION 3647
pairs were obtained. For each pair, differences in residues were calculated by
using SOi. To calculate the SEi, all combinations of 47 pairs of LTNP versus P/UP
pairs were generated, giving rise to 2,209 combinations. Score for the most
extreme residue (SR1) and the fifth most extreme residue (SR5) were calculated,
and a permutation test was carried out as explained above.
Deletion. To study deletion patterns in nef sequences from all LTNP, P, and
UP nucleotide sequences, consensus sequences corresponding to each patient
were generated by using the consensus program EMBOSS (46). All consensus
sequences, as reported in Table 2, were aligned by using the MUSCLE alignment
program (18, 19). The number of deletions for each codon was calculated. Then,
for each codon, we performed the Fisher exact test to assess the level of asso-
ciation between the deleted/nondeleted genotype and the LTNP-versus-P or
LTNP-versus-UP phenotype. All statistical analyses were performed by using the
statistical package R (http://www.R-project.org). The Spearman’s correlation
statistics was used to find correlations between LTNP versus P or UP. Probabil-
ities for comparison between LTNP and P or between LTNP and UP are
denoted by PNPor PNU, respectively.
Since the Fisher exact test was calculated over every codon/residue of nef, we
carried out a permutation test to determine the significance of the P value
obtained from each residue’s test. Similar to the replacement analysis, all con-
sensus sequences were randomly assigned a P/UP or LTNP status, and the P
values for each residue based on the Fisher exact test were calculated for each of
the 1,000 simulations. The whole deletion study was repeated for the longest
sequences corresponding to each patient. To find the longest sequence for a
patient, we calculated the length of all of the sequences for that patient. The
sequence with the maximum length out of the total sequences for a given patient
was selected as the longest sequence.
Defective nef genes are common among LTNPs. LTNPs have
a higher number of sequences containing frame shifts. The
frameshifts appear to be distributed generally across both
LTNPs and UPs (Fig. 3A). There are two peaks of frameshift-
ing in LTNPs occurring at
9SVIG (24 sequences) and
118QGYF (15 sequences) and one peak in UP patients at
8SSVI (10 sequences). In Ps, only one sequence had frame-
shifting at N52. We repeated the same for consensus (Fig. 3B)
and longest sequences (Fig. 3C) per patient. In the case of
LTNPs, there was one peak (S9) for both consensus sequences
and longest sequences (two patients). Similarly, in UPs, there
was one peak at9SVI in both types of sequences, whereas in P
patients no frameshifting was observed in case of consensus or
longest sequences. Although it is possible that such frameshift-
ing increases reflect selection maintaining an N-terminal activ-
ity while deleting a C-terminal one (e.g., see reference 9), it is
difficult to assess significance, given that assessment of posi-
tional biases of frameshifts was not a predefined primary end-
point of our study. It is worth noticing that the length varia-
tions near the N terminus, as well as in some other parts of nef,
are normal and usually do not have disruptive effects on the
function of nef (11, 52).
Most of the LTNP patients sequenced were from the United
States, Italy, and Australia, whereas the P patients were from
the United States, Italy, and Japan (see Fig. S1 in the supple-
mental material). When we compare the total number of se-
quences obtained, there are many more defective nef genes
among the LTNPs. LTNPs have a total of 149 defective nef
sequences (20%) out of a total of 764 sequences, whereas Ps
have 17 (10%) out of 170 and UPs have 315 (12%) out of a
total 2,626 sequences. Ps and UPs combined have 332 defec-
tive nef genes (12%) out of a total 2,796 sequences, suggesting
that defective nef genes are common among LTNPs. However,
this may simply be an artifact of sampling, with a few LTNPs
overinvestigated that have many defective nefs. The average
FIG. 2. Amino acid differences between HIV-1 subtype B nef consensus sequences of 20 LTNP patients, each paired with the nearest related
P patient. SOand SErepresent observed and expected scores, respectively (as defined in Materials and Methods).
3648 PUSHKER ET AL.J. VIROL.
lengths of nondefective nef genes in LTNPs, Ps, and UPs were
206.8, 207.1, and 207.7, respectively. A box plot of the lengths
in all three categories is shown in Fig. 4A. We also studied
percent defective proteins per patient in all three categories
(see Table S1 in the supplemental material). The average num-
ber of patients sequenced in Ps is higher than for LTNPs and
UPs (Table 2). However, when we calculated the average num-
ber of percent defective nef genes, we found that LTNPs had
higher average number of percent defective nef genes
(15.25%) than Ps (8.09%) and UPs (13.92%). The distribution
of the percent defective nef genes in LTNPs and others is
shown in Fig. 4B.
However, the results presented above may be biased by the
number of sequences investigated. When the analysis is per-
FIG. 3. Frameshifts in the sequences of LTNPs compared to those of UPs. The sequence of nef from the HIV-1 NL4.3 strain is shown on the
x axis. The y axis represents the cumulative percentage of frameshifts. (A) All HIV-1 subtype B nef sequences; (B) consensus sequences per patient;
(C) longest sequences per patient.
FIG. 4. Box plots of protein lengths and percent defective sequences of HIV-1 subtype B nef. (A) Distribution of protein lengths in LTNPs,
Ps, and UPs; (B) distribution of percent defective nef sequences per patient in LTNPs, Ps, and UPs.
VOL. 84, 2010 HIV-1 PROTEINS AND DISEASE PROGRESSION3649
formed on a patient-by-patient basis, there is no evidence that
LTNPs have more defective nef genes (Table 3). Thus, there is
no association between LTNP status and whether the patient
has one or more nondefective nef genes (PNP? 0.23 and
PNU? 0.39 [Fisher exact test]; Table 3, footnote a). Similarly,
there was no association between LTNP status and whether a
patient has one or more defective nef genes (PNP? 0.47 and
PNU? 0.052; Table 3, footnote b). Thus, we conclude that
there is no significant association between defective nef genes
and disease progression in this data set.
Intragroup clustering. Phylogenetic trees of all nondefective
sequences from LTNPs and Ps or from LTNPs and UPs were
generated. Each patient has sequences clustered together and
clearly differentiable from those of other patients as described
earlier (10). Consistent with previous studies (28, 29, 40, 43),
visual inspection of this tree does not provide any strong evi-
dence of phylogenetic grouping of LTNPs within the tree. We
investigated more closely a set of LTNPs paired with closely
related sequences from either Ps (Fig. 1) or UPs (see Fig. S2 in
the supplemental material) to investigate amino acid replace-
ments. The rationale behind this pairing was to try and focus
on evolutionary changes that are associated with differences
related to progression. This matching of pairs is intended to
reduce some of the noise caused by other evolutionary changes
that are not relevant to progression status, in the same way that
a matched case-control study reduces the noise due to other
Amino acid replacements. We examined the frequency of
changes between the consensus sequence of LTNPs and the P
consensus that they are paired with (as their nearest phyloge-
netically related sequence). We were interested in residues
that show more differences between the consensus sequence of
LTNPs and Ps. We found that certain residues show a much
higher frequency of changes (Fig. 2). In particular, 13 resi-
dues—S8, V10, I11, A15, V85, V133, N157, S163, V168, D174, R178,
E182, and R188—had high scores (SO? 0.015). The scores are
normalized so that the evidence from all 20 pairs is treated
equally, regardless of their phylogenetic distance. However,
the scores obtained from inspection of the permuted pairs are
also elevated for these residues (Fig. 2), implying that these
residues are simply rapidly evolving residues. The correlation
between SOand SEwas significantly very high for all residues
(? ? 0.96; P ? 2.2 ? 10?16).
Inspection of Fig. 2 reveals that one residue, N157, shows a
relatively marked excess of observed change over expectation.
To our knowledge, this residue has not been identified in
previous studies as contributing to nonprogression. Other res-
idues with a more modest excess included A15, V85, S163, V168,
D174, R178, and R188. To test whether the excess of SOover SE
seen for N157or any other residues was significant, we did a
more rigorous permutation test by looking at the number of
permutations in which we observed a score as high as SR1or
SR5. Although some of these residues did show an excess
of observed score over the expectation, the test revealed that a
score at least as high as the highest score (SR1? 2.28) occurred
in 76.7% of the permuted datasets and that a score at least as
high as the fifth highest score (SR5? 1.48) occurred in 99.9%
of the permuted datasets (for ranked residues, see Table S3 in
the supplemental material). Thus, there is no evidence that any
individual residue shows a significant excess of amino acid
replacement between LTNP patients and their nearest related
We also investigated amino acid replacements between the
consensus sequence of LTNP and the closest related UP con-
sensus sequence (see Fig. S3 in the supplemental material). In
all 47 pairs, we found that nine residues—S8, V10, I11, D28, K39,
V133, S163, R178, and E182—had high scores (SO? 0.015).
However, the scores obtained from the permuted pairs were
also elevated, as observed in the case of previous 20 LTNP and
P pairs. Similarly, the correlation between SOand SEwas
significantly very high for all residues (? ? 0.98; P ? 2.2 ?
10?16). A more rigorous permutation test was done by looking
at the number of permutations in which we observed a score as
high as SR1? 5.53 or SR5? 1.84, and this revealed that scores
as high as SR1or SR5were observed in all permutations.
Overall deletions. We tested whether there was an excess of
codon deletion overall among LTNPs. Table 4 shows the num-
ber of patients with one or more deletions in all three catego-
ries of sequences. There is a slight but not significant (P ?
0.83) excess of deletions among the LTNPs (26.45%) com-
pared to UPs (26%) in case of consensus sequences. However,
when only LTNPs and Ps are compared, the LTNPs seem to
have fewer proteins with codon deletions compared to Ps
(36%), but again these differences are not significant (P ?
When the analysis was repeated, replacing the consensus
sequence for a patient with the longest sequence per patient
(Table 4B), there was still a slight excess of deletions among
the LTNPs (27%) compared to UPs (26%), but this was not
significant (P ? 0.75). However, there was a lack of deletions
among LTNPs compared to Ps (41%) only, but the Fisher
exact test revealed that this finding was not significant (P ?
0.21). Thus, LTNPs do not appear to have a markedly in-
creased or decreased number of codon deletions overall.
Deletions of specific regions of the Nef protein in LTNPs.
We looked for deletions of specific regions of the Nef protein
in all consensus sequences from LTNPs and others. Each
codon present in the Nef NL4.3 sequence was compared to the
codon present at that position in the alignment of all consensus
sequences of LTNPs and other patients (either Ps or Ps and
UPs). We calculated the probability (using the Fisher exact
test) to determine whether there was a significant association
between LTNP status and codon deletion for each amino acid
TABLE 3. Association between defective nefs (containing
frameshifts or stop codons) and progression
No. of patients (%) witha:
No. of patients (%) withb:
One or more
One or more
Total582 67168 481
aThe probability, determined by using the Fisher exact test for comparison
between LTNPs and Ps (PNP), is 0.23; for comparison between LTNPs and UPs
(PNU), it was is 0.39.
bThe respective P values for these comparisons (see footnote a) were PNP?
0.47 and PNU? 0.052.
3650 PUSHKER ET AL. J. VIROL.
along the nef sequence. The codons that had in-frame dele-
tions in LTNPs are listed in Table 5. Comparison of LTNPs
and Ps revealed that four codons AGT (S9), GTG (V10), ATT
(I11), and GCA (A49) were deleted in both Ps and LTNPs;
however, only one codon GTG (V10) was found to be signifi-
cantly deleted in LTNPs (P ? 0.04). We also looked for dele-
tions of specific regions of the Nef protein in all consensus
sequences from LTNP and UP. The codons that had significant
in-frame deletions are marked in boldface in Table 5. These
are located in the152KVEEA region.
Since we performed multiple tests across each codon of nef,
we considered whether such low P values are likely to arise by
chance by carrying out permutation tests (see Materials and
Methods). The appropriate cutoffs for significance (equivalent
to P ? 0.05) were 8.5 ? 10?4(LTNPs and Ps) and 8.1 ? 10?5
(LTNPs and UPs), values lower than were seen for any of the
observed residues (Table 5). We repeated the analysis de-
scribed above by taking the longest sequence per patient in-
stead of the consensus. The codons whose deletion is most
significantly associated with LTNP (P ? 1) are listed in Table
S4 in the supplemental material. However, none of these
codons were found to have P values lower than for those which
occur by chance.
We found more defective/disrupted nef genes among
LTNPs, as suggested by earlier studies (4, 10, 16, 33), appar-
ently favoring the hypothesis that LTNPs have higher propor-
tion of disruptive nef genes than Ps or UPs. However, this
association was only seen when we looked at all sequences.
When the analysis was carried out on a per-patient basis, the
excess was not seen. The initial observation may reflect biased
sampling in numbers of sequences per patient. Alternatively,
later ascertainment of sequences from LTNPs postinfection
may impact in some way on the intrapatient variability of the
sequences observed. Therefore, we consider the evidence that
there is an excess of defective sequences among LTNPs to be
inconclusive. A prospective study would be required to address
this question more fully. At the very least, this would require
careful adjustment and control of the length of time postinfec-
tion among the comparison groups. Ideally, it would involve
long-term follow-up with sequences from different disease
stages to help avoid other potential confounding effects of the
disease process on the mutation rate. Clearer and more con-
sistent criteria for defining LTNP would also be beneficial,
since the study presented here relied on definitions of nonpro-
gression that are not identical across all studies. The study we
present here can also be confounded by technical artifacts of
the various study designs investigated, including bias in the
number of sequences observed per patient, differing degrees of
sequence variability per patient (which may partly relate to
number of years postinfection), and variability of the sequenc-
ing techniques (Table 2). Such biases are easier to investigate
in a positive association (to determine whether the positive
association arises through such confounding of causative fac-
tors) than in a negative study such as ours. It is clear that such
confounding factors in our study serve to somewhat reduce the
power of the study to detect a true effect.
Other studies have suggested that amino acid replacements
at particular residues are increased significantly among LTNPs
(8, 10). However, in reviewing the evidence among the largest
group of patients analyzed to date, we failed to confirm any of
these findings or to detect any residues whose replacement is
increased among LTNPs. Analysis of codon deletion revealed
that a few regions of nef were more commonly deleted among
LTNPs, in particular residues 152 to 156 (KVEEA) among
nine patients (see Fig. S4 and Table S5 in the supplemental
material). The region152KVEEA is involved in ?-COP recruit-
ment (3, 21) and also predicted to be highly disordered (see
TABLE 4. Number of HIV-1 subtype B patients with codon deletions in the nef sequences
No. (%) of HIV-1 subtype B patients with a codon
deletion in the nef sequencea
No. (%) of HIV-1 subtype B patients with a codon
deletion in the nef sequenceb
LTNPsPs UPsTotalLTNPs PsUPs Total
One or more codon deletions
Total 155 22405 582155 22 405582
aComparison of the codon deletions between consensus sequences of the LTNPs and Ps (PNP? 0.32) or between those of LTNPs and UP (PNU? 0.83) was
determined to be insignificant.
bComparison of the codon deletions between the longest sequences of LTNPs and Ps (PNP? 0.21) or between those of LTNPs and UPs (PNU? 0.75) were
determined to be insignificant.
TABLE 5. Codon deletions in HIV-1 subtype B nef consensus
sequences from all patientsa
No. of patients with
1.1 ? 10?4
aOnly deletions that have P ? 1 for at least one category are shown. aa, amino
acid. P values of ?0.05 are indicated in boldface. P values based on the Fisher
exact test were not significant after correction for multiple testing (appropriate
level PNP? 8.5 ? 10?4and PNU? 8.1 ? 10?5from permutation analysis).
VOL. 84, 2010 HIV-1 PROTEINS AND DISEASE PROGRESSION3651
Fig. S5 in the supplemental material), a feature shared by
many regions that encode short signaling motifs (23). How-
ever, after a permutation test, we determined that this deleted
region was not significant.
Although our study was focused on residue deletion and
replacement, we did note an excess of frameshifting in the
LTNPs occurring at specific regions of nef, which was not seen
in the non-LTNP sequences (9SVIG and118QGYF). This was
not a primary endpoint in our study, and it is unclear exactly
what statistical methods are most appropriate to address
whether the excess in these regions represents a statistical
departure from expectation. We can hypothesize that such
frameshifting may be selected for in generating nef sequences
that maintain the N-terminal activities while removing the C-
terminal activities of the protein. However, analysis of an in-
dependent set of sequences would be required in order to
formally test whether there is enrichment of frameshifting in
Thus, we can conclude that a significant proportion of non-
progression is unlikely to be attributable to amino acid replace-
ment or deletion in specific regions of HIV-1 nef. Although we
could not find any direct correlation between LTNP and P or
UP based on the sequences studied, it is possible that, in
addition to the fact that there are more deletions in nef from
LTNPs, other parameters, such as the immune system re-
sponse and virus fitness (other HIV gene products), could in
combination with nef variability explain the outcome of other
studies. Finally, it is also possible that rarer variants may con-
tribute to the disease progression in individual patients.
This study was funded by Science Foundation Ireland and University
College Dublin, Dublin, Ireland.
We thank Cathal Seoighe (NUI, Galway, Ireland) for helpful com-
ments. We also thank two anonymous reviewers for their comments
and suggestions, which helped us in improving the manuscript.
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