AIDS Reviews.

2008;10
212
Role of Genetic Diversity amongst HIV-1 Non-B
Subtypes in Drug Resistance: A Systematic Review
of Virologic and Biochemical Evidence
Jorge L. Martnez-Cajas
1
, Nitika Pant-Pai
2
, Marina B. Klein
2
and Mark A. Wainberg
1
1
McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Canada;
2
Montreal Chest
Institute/Immunodeficiency Centre, McGill University Health Centre, Montreal, Quebec, Canada
Abstract
The genetic diversity of HIV-1 has required its classification into types and subtypes. There is contro-
versy as to how and to what extent genetic diversity may affect the emergence of antiretroviral drug
resistance in HIV-1 subtypes other than B. To better understand the impact of genetic diversity (rep-
resented by different HIV-1 subtypes) on resistance to reverse transcriptase and protease inhibitor
drugs, a systematic review was conducted on virologic and biochemical evidence obtained from work
with non-B HIV-1 subtypes. We searched 11 databases and retrieved 3,486 citations on all aspects of
non-B subtype-related resistance research. Twenty-seven studies with virologic and/or biochemical
data met the eligibility criteria for our systematic review. Nineteen studies were found that reported
phenotypes in non-B subtypes (304 from naive isolates and 242 from drug-exposed isolates) and 11 stud-
ies that used molecular biology techniques to study non-B resistance to antiretroviral drugs. Com-
pared to the NL4-3 laboratory strain, lower baseline susceptibilities of recombinant A/G subtype virus
to protease inhibitors were observed and a substantial proportion of subtype C isolates displayed
higher IC
50
at baseline for atazanavir. Some A/G isolates were found to have reduced susceptibility to
abacavir. Mutations not typical of B subtypes include the reverse transcriptase mutation V106M and
the protease mutations M89I/V and N83T. Virologic and biochemical data suggest that K65R is more
likely to emerge in subtype C HIV-1. There is evidence to suggest differential effects of other mutations
according to subtype, e.g. the protease inhibitor mutations I93L and M89I/V. Importantly, the most
widely used commercial phenotyping systems do not take into account gag variations among natural
isolates, which could limit the accuracy of measured susceptibility. Enzymatic and virologic data
support the concept that naturally occurring polymorphisms in different non-B subtypes can affect
the susceptibility of HIV-1 to different antiretroviral drugs, the magnitude of resistance conferred
by major mutations, and the propensity to acquire some resistance mutations. Tools may need to
be optimized to accurately measure drug susceptibility of non-B subtypes, especially for protease
inhibitors. (AIDS Rev. 2008;10:212-23)
Corresponding author: Mark A. Wainberg, mark.wainberg@mcgill.ca
Key words
Genetic diversity. HIV-1. Non-B subtypes. Drug resistance.
AIDS Rev. 2008;10:212-23
Correspondence to:
Mark A. Wainberg
McGill University AIDS Centre
Jewish General Hospital
3755, Cote-Ste-Catherine-Road
Montreal, Quebec
H3T 1E2, Canada
E-mail: mark.wainberg@mcgill.ca
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
Jorge L. Martinez-Cajas, et al.: HIV Resistance in Different Subtypes
213
Background
Almost all HIV genetic sequences can be classified into
subtypes or recombinant forms, even though problems in
classification can occasionally arise. Due to genetic relat-
edness, it is reasonable to think that viruses belonging to
a certain subtype will respond in a similar way to antiret-
roviral drug pressure. However, responsiveness of HIV-1
to antiretroviral drugs might vary among subtypes, par-
ticularly if major dissimilarities are present. It might also
be expected that responsiveness might be predictable for
each individual subtype.
Genotyping of HIV obtained from plasma samples has
been used for two important purposes. First, genotyping
has permitted the detection of mutations responsible for
drug resistance in patients failing therapy. Second, it al-
lows surveillance of epidemiological trends in various geo-
graphic areas and populations. Therefore, HIV-1 subtyp-
ing may become important for the prediction of resistance
patterns. Since the best opportunity for long-lasting anti-
retroviral treatment efficacy is the first therapeutic regi-
men, a clear knowledge of the potential for resistance and
cross-resistance in different subtypes is important.
Data used to define drug resistance mutations have
been generated almost entirely from subtype B HIV-1
1,2
.
However, non-B subtypes are dominant throughout the
world and are also increasingly prevalent in countries that
had traditionally been affected primarily by subtype B
3-7
.
This has resulted in a need to understand the following:
(i) how the frequency of resistance mutations among cer-
tain subtypes may be different than those described in
subtype B; (ii) whether the timing of the emergence of
resistance may differ among subtypes; (iii) how the effi-
cacy of second-line drugs might be affected to differential
extent by resistance mutations within each subtype; and
(iv) the influence of genetic characteristics among people
infected by different subtypes on clinical outcome (viro-
logic failure and/or CD4 responses).
The interpretation of degrees of resistance by popular
algorithms is widely accepted and used in clinical practice
in all countries in which B subtypes predominate. How-
ever, such algorithms may be less accurate in assessing
resistance in non-B subtypes, in part because of differ-
ences in the genetic backgrounds among subtypes. In
fact, several studies comparing different HIV resistance
algorithms revealed high degrees of discordance in the
interpretation of protease inhibitor resistance when ap-
plied to non-B subtypes
8-11
.
Our primary goal was to assemble the relevant virologic
and biochemical evidence on the role of polymorphisms of
non-B subtypes on resistance to reverse transcriptase in-
hibitors (RTI) and protease inhibitors (PI). Therefore, we
conducted a systematic review of all literature published in
English language on non-B subtypes. Our secondary ob-
jectives were to (i) summarize and evaluate available knowl-
edge in regard to antiretroviral resistance to non-B sub-
types, (ii) identify gaps in knowledge, and (iii) delineate
research that might be needed to fill such gaps. Herein we
review the literature on virologic and biochemical aspects
of HIV drug resistance in non-B HIV-1 subtypes.
Review strategy
This review strategy covers the period January 1996 to
December 2007. We limited our search to publications in
English. Eleven electronic databases for full text articles
and conference abstracts were searched: PUBMED (1996-
2007), Web of Science (1996-2007), EMBASE (1996-2005),
BIOSIS (1996-2007), AIDSLINE (1996-2007), OVID (1996-
2007), Psychinfor (1996-2007), Cochrane controlled
trials register (1996-2007), DARE (1996-2007), COCHRANE
(1996-2007), ILLUMINA (1996-2007). Bibliographies and
references from primary studies and review articles were
also searched. If full-text articles were not available, ab-
stracts or letters were used, provided they contained rele-
vant information and were of sufficient completeness. Con-
ference websites were also searched and abstracts with
pertinent data were reviewed. Abstracts which were later
found as full-text publications were excluded and the full-
text publication was examined instead. We gave primary
importance to sensitivity of the search. Our search string
included key words and search terms as follows:
Search #1: HIV[MeSH] OR HIV-1[MeSH] OR
HIV-1[TI]
Search #2: non-b[TIAB] OR subtype*[TI] OR clade[TI]
OR strain*[TI] OR variant*[TI] OR non-B subtype*[TIAB]
The same key words were used for searches in data-
bases in which MeSH terms are not applicable including
conference search engines. We also extracted additional
information from reference lists of reviews on non-B sub-
type HIV-1.
Study selection
The study selection methodology is shown in figure 1.
After pooling, a total of 5,288 citations were identified.
After excluding duplicates, 3,486 citations were selected
for further review. From these, 405 were related to resis-
tance to antiretrovirals in non-B subtype HIV-1. All cita-
tions had full reports and were reviewed. After applying
our inclusion and exclusion criteria, consistent with the
goals of our review, 30 articles were included and care-
fully examined.
Inclusion criteria
Based on our objectives, we included two types of stud-
ies: (i) those that identified in vitro drug susceptibility of
isolates (viral phenotype), and (ii) studies using molecular
biological techniques such as site-directed mutagenesis,
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
AIDS Reviews. 2008;10
214
enzymatic assays, and competition assays in order to
understand the effect of resistance mutations on viral and/
or enzymatic function.
Exclusion criteria
We excluded news reports, duplicates, animal experi-
ments, modeling studies, methods papers, comorbidity
studies, reviews, editorials, perspectives, and opinion
pieces. We also excluded (i) studies that dealt only with
genetic and epidemiologic diversity and that did not eval-
uate impact on antiretroviral resistance, (ii) vaccine stud-
ies related to non-B subtypes, and (iii) studies that com-
bined all non-B subtypes into a single analysis.
Results
The process of data selection is shown in figure 1. A
total of 30 studies were found to meet our inclusion crite-
ria. The studies of drug susceptibility testing of non-B
subtypes are summarized in table 1 and the studies that
used molecular biological methods and drug resistance
selection are summarized in table 2.
Studies of drug susceptibility testing
of non-B subtype HIV-1
Nineteen studies that measured HIV-1 susceptibility of
non-B subtype strains to antiretrovirals were included. The
total number of samples whose phenotypes have been
published and included in this review is 546, of which 242
were from patients who had been exposed to antiretrovi-
rals. Overall, the majority of non-B HIV-1 subtypes pos-
sessed wild-type susceptibilities similar to those of sub-
type B wild-type isolates (Table 1).
In untreated patients, decreased susceptibility to PI in
some non-B subtype isolates was found, though infre-
quently. Compared to subtype B, diminished susceptibili-
ties among wild-type isolates have been found for A/G
recombinant viruses in two different studies. In the first,
lower susceptibilities to nelfinavir and lopinavir were re-
ported, while in the second, four of 12 isolates had lower
susceptibility to atazanavir
12,13
. The first study did not test
atazanavir. Neither study was designed to test statistical
significance related to drug susceptibility levels based on
the presence of polymorphisms. The study by Kinomoto,
et al.
12
also performed molecular modeling and suggested
that distortions in the K26 pocket of A/G proteases appear
to be responsible for a lower binding energy of nelfinavir
and hence lower susceptibility of A/G viruses to this drug.
Vergne, et al.
14
reported isolates with susceptibilities
slightly above the biological test cutoffs for ritonavir as
follows: one of two CRF06 isolates (FC = 3), one of two
CRF13 isolates (FC = 4.7), and one of two F isolates (FC
= 2.4) (ritonavir biological cutoff = 2.4). For nelfinavir and
saquinavir, respectively, this was true for one of two B
isolates (FC = 2.6) (nelfinavir biological cutoff = 2.3) and
a single URF A/G/J isolate (FC = 2.1) (saquinavir biologi-
cal cutoff = 1.7).
With regard to RTI, the study by Vergne, et al.
14
reported
that one of three G isolates had low susceptibility to efa-
virenz but not nevirapine, one of 12 CRF02 had low sus-
ceptibility to efavirenz and nevirapine, one URFA/D/A isolate
Figure 1. Selection of studies in the systematic review.
Reviews
Editorials
Letters

Not relevant after title review
or these were epidemiologic
studies
Not relevant after abstract
review because of
absence of phenotypic
or enzymatic data Selected for review
5,288
Duplicates
405
30
3,486
1,910
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
Jorge L. Martinez-Cajas, et al.: HIV Resistance in Different Subtypes
215
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(
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No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
AIDS Reviews. 2008;10
216
T
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*
.
(
c
o
n
t
i
n
u
e
)
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
Jorge L. Martinez-Cajas, et al.: HIV Resistance in Different Subtypes
217
T
a
b
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1
.

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.
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
AIDS Reviews. 2008;10
218
T
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No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
Jorge L. Martinez-Cajas, et al.: HIV Resistance in Different Subtypes
219
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No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
AIDS Reviews. 2008;10
220
had reduced susceptibility to efavirenz and nevirapine,
and one CRF02 isolate had reduced susceptibility to zido-
vudine. Fleury, et al.
13
described that several viruses had
above average cutoff values in the absence of typical re-
sistance mutations. One of 12 A/E isolates, which had de-
creased susceptibility to nevirapine and efavirenz, pos-
sessed an I135T substitution. Of 12 A/G subtype isolates,
two had an IC
50
FC above the cutoff for abacavir. The
D123N + I135V mutations were associated with a decrease
in susceptibility to this drug. The I135T mutation was also
associated with lower susceptibility to nonnucleoside re-
verse transcriptase inhibitors (NNRTI) in subtype C.
Regarding isolates obtained from ART-treated patients,
a study by Weidle, et al.
15
reported that 96% (n = 94) of
isolates resistant to stavudine or zidovudine had at least
one commonly recognized mutation for zidovudine resis-
tance. Eleven of 17 had typical NNRTI resistance muta-
tions. Seventeen specimens had phenotypic resistance to
at least one PI. Two patients, who had never received PI,
had low-level PI resistance and had no major mutations.
All patients who received nelfinavir (n = 9) had resistance
to nelfinavir. Six of these possessed subtype D viruses, all
with the D30N mutation. Three were subtype A and did not
have mutations at position 30 (two had M46I and one had
N88S). Abecasis, et al.
16
studied the effect of the M89I
mutation on viral susceptibility to PI. Such a mutation, which
has been found to emerge in F, G, and C subtypes after
nelfinavir failure, was also associated with increased sus-
ceptibility to other PI, except for lopinavir. Also, the combi-
nation of mutations at position 89 together with the L90M
mutation had a differential effect on susceptibility to other
PI (Table 1). In addition, the same authors reported that
other polymorphisms were associated with either reduced
or increased susceptibility to PI in a differential fashion in
A/G, C, and F subtypes (Table 1)
17
.
Studies using molecular biological
and drug resistance selection techniques
Thermodynamic studies performed on target-inhibitor
interactions in proteases have specifically described a
lower affinity of some non-B subtype proteases for PI and
for selective amplification of primary resistance mutations
on the basis of background polymorphisms (Table 2).
Velasquez-Campoy, et al.
18,19
reported two important
findings: (i) the enzymatic efficiency or vitality of subtype
C proteases was higher than those of subtype B, and (ii)
natural polymorphisms in the subtype C and A protease
genes amplified the effect of resistance mutations on PI
binding. This suggests that subtype C proteases pro-
cessed their substrates faster that B proteases; for ex-
ample, the effect of the V82F and I84V resistance muta-
tions V82F and I84V in subtype A and C viruses was to
reduce binding to substrate by twofold to sevenfold more
than occurred in subtype B. Background polymorphisms
differentially affected the effect of the PI saquinavir, nel-
finavir, indinavir, and ritonavir, and the double mutation
V82F/I84V improved the relative vitality of the protease by
~ 100 in the presence of tested PI. This effect was more
pronounced in the protease of subtypes A and C, in
which relative vitality sometimes reached 1000-fold
19
.
Taken together, these results suggest that natural poly-
morphisms contribute to the HIV resistance phenotype by
both lowering drug discrimination and by restoring en-
zyme function.
Gonzlez, et al.
20
found that the IC
50
of a number of
subtype C isolates carrying the I93L mutation were
16.2 to 2.6 times lower than that of a reference strain
HXB2/NL4-3-PR (p < 0.0001; two-tailed t test). However,
when subtype B or C isolates lacking this specific substi-
tution were analyzed, the IC
50
were only 1.2 to 0.7 times
less than that of HXB2/NL4-3 (p = 0.77; two-tailed t test).
Nor was this phenotypic effect observed when I93L was
naturally present in subtype B (isolate Br-RS2172). The
authors suggested that the I93L substitution might cause
hypersusceptibility to lopinavir in subtype C HIV-1 rather
than resistance
20
.
Coman, et al.
21
performed important comparative enzy-
matic and structural analyses of proteases derived from
subtypes B and C that carried one or several nelfinavir-
selected resistance mutations (D30N, N88S, L90M). They
showed a differential effect of these resistance mutations
on the kinetics of B and C proteases as follows: (i) the
effect varied according to whether only a single mutation
or combinations of mutations were present, and (ii) the
effect differed based on each PI tested. Structural ana-
lyses of subtype C protease bound to nelfinavir and indina-
vir showed that these inhibitors form similar interactions
with residues in the active site of subtype B protease, and
that naturally occurring polymorphisms could alter the po-
sition of the outer loops of the subtype C protease, espe-
cially the 60 loop. Clemente, et al.
22
reported that high
levels of PI resistance in A/E and B proteases were due
to combinations of active site and non-active site muta-
tions that involve background polymorphisms.
Some mutations may also have important roles only in
certain subtypes. For example, N83T and M89L restored
replicative capacity only in subtype C. The M89I/V substi-
tutions that occur in subtypes G, F, and C had a differen-
tial effect, depending on which PI was tested and wheth-
er or not the L90M substitution was present
16
(Table 1).
In regard to reverse transcriptase, one drug resistance
selection study showed that subtype C viruses selected
the K65R mutation faster under tenofovir pressure com-
pared to subtype B
23
. A template-based mechanism for
this phenomenon has been proposed
24
. Certain NNRTI
mutations also appear to emerge preferentially in certain
subtypes, e.g. V106M in subtype C instead of V106A in
subtype B
25
. Furthermore, V106A in subtype B is mainly
associated with resistance to nevirapine but not efavirenz,
while V106M confers broad NNRTI cross-resistance.
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
Jorge L. Martinez-Cajas, et al.: HIV Resistance in Different Subtypes
221
Discussion
Biochemical and virologic data provide compelling evi-
dence as to the differential effect of viral genetic back-
ground on both the type and degree of antiretroviral drug
resistance in HIV-1. In regard to protease, genetic back-
ground can affect the degree of protein binding caused by
primary mutations and also help to restore enzymatic activ-
ity based on the presence of background polymorphisms
and subtype. This effect was not discernible in the absence
of typical major resistance mutations, but rather only when
particular backgrounds of combinations of major resistance
mutations and background polymorphisms were present.
In this regard, it seems that some background polymor-
phisms can act as secondary resistance mutations.
In addition, HIV phenotypic assays have failed to reveal
major differences in the susceptibilities of B versus non-B
subtypes, consistent with molecular data (Table 2). How-
ever, there are only few data on relative susceptibility levels
among subtypes carrying specific major resistance muta-
tions. More information is needed since many polymor-
phisms in non-B viruses are considered to be secondary
resistance mutations, based on the fact that they emerge
in B subtype viruses after drug exposure. However, the
effect of such polymorphisms within different genetic
backgrounds cannot always be extrapolated to B sub-
types. They might sometimes contribute to higher levels
of resistance in certain genetic backgrounds, but could
also have either a neutral effect or hypersensitize HIV to
certain antiretroviral drugs (e.g. I93L is a secondary resis-
tance mutation in subtype B but causes hypersusceptibil-
ity to PI in subtype C)
20
.
Novel NNRTI resistance mutations have been found in
subtype C that had not been recognized in subtype B. In
tissue culture, subtype C can acquire a different mutation
(i.e. V106M) under NNRTI drug pressure compared to what
it is seen in subtype B, and this mutation confers broad
NNRTI cross-resistance. Resistance surveillance of South
African patients has confirmed the importance of the V106M
mutation in subtype C HIV-1
26
. Similarly, rates of acquisition
of resistance could have important implications in regard
to durability of response to therapy. In vitro, the emergence
of the K65R mutation is faster in subtype C than in B. Bio-
chemical mechanisms have been proposed to explain this
observation based on subtype C template usage and pro-
cessing by reverse transcriptase
24
. In the clinic, K65R has
been seen in approximately 70% of patients failing di-
danosine- and stavudine-containing nucleoside backbones
in Botswana
27
, and this has now been confirmed in a larg-
er study performed in Malawi in patients with subtype C
viruses who received stavudine/lamivudine and nevirapine
as first-line therapy. Of these individuals, 19% developed
either the K70E or K65R mutations. However, K65R did not
appear to emerge frequently in subtype C patients who
participated in large clinical trials in which they received
either tenofovir or tenofovir/emtricitabine as part of a triple
therapy regimen
29
; this substantiates the view that suppres-
sion of viral replication will forestall the appearance of drug
resistance regardless of subtype. However, larger numbers
of patients and longer follow-up will be required to assess
the situation in patients receiving therapy in subtype C
endemic areas.
To date, most drug-susceptibility phenotypes in non-B
isolates have been determined by commercial and/or in-
house assays that were developed primarily to measure
B subtype drug susceptibilities and that are based on the
laboratory adapted strains HXB2 or NL4-3. These studies
employed modified molecular clones of laboratory strains
that lack both the terminal portion of Gag and most of Pol,
in order to generate a chimeric clone
12,30-33
. Only one
study used an assay that included a substantially more
complete Gag gene of a clinical isolate
13
and, interest-
ingly, is the only one to have reported higher frequencies
of resistance to atazanavir on the part of subtype C iso-
lates. The lack of a complete Gag gene in the constructs
to measure non-B drug susceptibility is a potential limita-
tion of studies in regard to the accuracy of PI susceptibil-
ity determinations in non-B isolates (Fig. 2).
Multiple clinical and in vitro studies have confirmed
that protease and gag are a functional unit and co-evolve
when HIV is subject to drug pressure. Both genes mutate
under PI pressure and Gag mutations can act as com-
pensatory substitutions that may increase levels of resis-
tance and viral replication capacity
34-38
. None of the re-
combinant phenotyping systems used to date for clinical
samples included the complete original Gag gene in the
chimeric virus used for determining phenotype. One sub-
type B study demonstrated that inclusion only of the p7/
p1 and p1/p6 regions (as is the case with current com-
mercial assays) instead of the complete Gag gene may
underestimate the reduction of drug susceptibility by at
least twofold in some viral isolates
39
. Although this differ-
ence appears small, non-B subtypes might develop com-
pensatory Gag mutations different from those seen in
subtype B, establishing a need to take the entire Gag
region into account in phenotyping assays used to as-
sess drug susceptibilities with non-B viruses.
A wide variety of mutations can impact on drug sensitiv-
ity to differential extent and only few data on the potential
for cross-resistance to PI among non-B subtypes have
been published. In regard to nelfinavir, there may be a
tendency to select for the L90M pathway in some subtype
C isolates, instead of D30N, which could reduce the po-
tential for salvage ability of PI therapy
47
. In addition, the
D30N mutation was not found in A/G recombinants from
Ivory Coast that instead possessed N88S after exposure
to nelfinavir
40
. N88S unlike D30N can cause cross-resis-
tance with other PI, including atazanavir. Therefore, ac-
curate cross-resistance information is not yet available for
most non-B subtypes due to a scarcity of paired geno-
typic and phenotypic data, yet this subject should now be
assumed to be of clinical significance. In regard to NNRTI,
No part of this publication may be
reproduced or photocopying
without the prior written permission
of the publisher
Permanyer Publications 2010
AIDS Reviews. 2008;10
222
only one study analyzed the genotypes and phenotypes
of non-B subtypes obtained from a clinical trial of use of
single-dose nevirapine for prevention of mother-to-child
transmission
41
.
In conclusion, the genetic diversity of HIV-1 can affect
the type of resistance mutations, degree of resistance,
and timing of emergence of antiretroviral resistance.
However, the accumulated published evidence is insuf-
ficient to adequately assess the contribution of the innate
genetic diversity of HIV-1 on resistance and cross-resis-
tance, particularly for PI in non-B HIV-1. The potential for
extensive cross-resistance acquires further importance
in settings with limited access to antiretroviral drugs in
which PI may be the only realistic option for salvage
therapy
42
. No in vitro selection study has ever been pub-
lished for PI in non-B subtypes, yet such data may be
crucial to understanding cross-resistance for specific
drugs. In addition, large numbers of paired samples
need to be systematically collected from naive and treat-
ed patients infected with subtypes C, A/E, A/G, A, and
G, in order to generate genotypic and phenotypic data
sets that will have applicability for both established drug
classes as well as for newer classes such as integrase
inhibitors.
Acknowledgements
JL Martnez-Cajas and N Pant-Pai, were supported by
fellowships from the Canadian HIV Trials Network. MB
Klein is a chercheur boursier of the FRSQ.
JL Martnez-Cajas and N Pant-Pai, contributed equally
to the manuscript.
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