Advances in Infectious Diseases, 2011, 1, 1-13
doi:10.4236/aid.2011.11001 Published Online September 2011 (http://www.SciRP.org/journal/aid)
1
HIV-1 Env gp120 C2V5 Potential N-Linked
Glycosylation Site(s) (PNGs) Variations and
Amino Acid Length Polymorphisms among
Infected Family Members
Duri Kerina1*, Felicity Zvanyadza Gumbo2, Knut Ivans Kristiansen3, Munyaradzi Paul Mapingure4,
Simba Rusakaniko5, Mike Zvavahera Chirenje6, Babill Stray-Pedersen4, Fredrik Müller3
1Department of Immunology, University of Zimbabwe, Harare, Zimbabwe; 2Department of Pediatrics and Child Health, University
of Zimbabwe, Harare, Zimbabwe; 3 Department of Microbiology, University of Oslo and Oslo University Hospital, Rikshospitalet,
Oslo, Norway; 4Letten Foundation Research Centre, Harare, Zimbabwe; 5Department of Community Medicine, University of Zim-
babwe, Harare, Zimbabwe; 6Department of Obstetrics and Gynecology, University of Zimbabwe, Harare, Zimbabwe; 7 Division of
Women and Children, Oslo University Hospital, Rikshospitalet and Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
Received August 2nd, 2011; revised September 6th, 2011; accepted September 13th, 2011.
ABSTRACT
Objective: To ascertain the role of HIV-1 gp120 env PNGs variations and sequence length polymorphism following
transmission events as possible suppo rting forensic evidence to determine directionality of HIV tran smission. Method:
An observational study of HIV-1 infected family members, where median and range values of the amino acid lengths
and PNGs for the genotyped C2V5 region were calculated. Wilcoxon rank-sum test was used to determine differences in
these parameters between different family members. Results: For heterosexual transmission, two mothers had longer
C3 sequences relative to tha t of their spou ses; p = 0.006 and p = 0.025 whilst the opposite was observed for one mother,
p = 0.028. No clear trends were observed for PNGs. In three families, index children had longer C 2V5 amino acid se-
quences compared to their mothers; p= 0.013, 0.040 and 0.043. Second siblings’ V4 and V5 sequences were generally
shorter relative to the maternal ones; p = 0.039 and 0.028, respectively. Generally adults had longer V3 amino acid
sequences compared to the children; p = 0.018. Similar trends were also observed regarding PNGs within the entire
C2V5 region, C3 and V4 sub-regions; p= 0.0025, 0.005 and 0.008, respectively. First siblings’ C2V5 and C3 sequence
lengths were significantly longer relative to tho se of the second sibling s; p = 0.005 and 0.007, respectively. Conclusion:
Our results are suggestive that HIV-1 env C2V5 amino acid length polymorph ism and PNGs tend to increase with age
and HIV disease progression. Though sensitive and should be cautiously handled, it is tempting to propose the direc-
tionality of the HIV transmission events with respect to C3 sequence length polymorphisms. Correlating HIV-1 env
C2V5 amino acid length polymorphism and age of infection may be the first step towards a possible valuable piece of
forensic evidence which may be useful in criminalisation of willful HIV infections. However, bigger studies are war-
ranted to substantiate the authenticity of this potentially useful application.
Keywords: HIV-1 Env120 C2V5, Glycosyl a tion, Amino Acid Length Polymorphism, Parent to Child Transmission
1. Introduction
Human immunodeficiency virus type 1 envelope glyco-
protein 120 (HIV-1 env gp120) is composed of relatively
conserved constant (C) C1 to C5 and variable (V) V1 to
V5 regions [1]. It is ranked one of the most heavily gly-
cosylated proteins known in nature with N-linked glycans
constituting over 55% of its molecular weight [2,3].
This extensive glycosylation is known to play an impor-
tant role in evasion of the host immune response by
masking key neutralization epitopes and presentation of
the glycosylated env (glycan shield) to the immune sys-
tem as “self” [4-9]. Under host immune response or anti-
retroviral therapy selection pressures, it is postulated that
HIV-1 evolves towards a denser glycan shield [10], an
observation also supported by others [11-13]. According

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and
2 Amino Acid Length Polymorphisms among Infected Family Members
to this hypothesis, shorter variant with fewer glycans are
expected during earlier time points of infection whilst
longer V1 - V5 forms with more glycans are expected to
evolve at a later stage in response to prolonged immune
pressures [14,15], making it possible to assign the direc-
tionality of transmission.
Regardless of the HIV-1 subtype, the number of poten-
tial N-glycosylation site(s) (PNGs) within the gp120 env
gene (env) is conserved at about 25 [10,16]. However,
there is a tendency for heterosexually and perinatally
transmitted viruses to have shorter env variable loops and
fewer PNGs [14,17-21]. Changes in the number of PNGs
and env gp120 V1-V5 amino acid lengths have been as-
sociated with striking a balance between transmission
competence and resistance to immune challenges [14,21-
23]. Whilst glycosylation within the HIV-1 env V3 loop
may change the viral co-receptor usage [24], loss of
PNGs within the V4 region has been correlated with de-
mentia [25]. Intra-host HIV-1 PNGs diversity varies, with
some PNGs being evolutionarily conserved while others
may be present in some hosts but absent in others and
some may even appear or disappear during the course of
infection [3,10]. HIV-1 subtype B env gp120 V4 loop has
been shown to appear as swarms of heterogeneous
N-linked glycosylation variants due to mutations, inser-
tions and deletions (indels) which consequently affect
amino acid sequence lengths and PNGs distribution pat-
terns [26,27].
Although glycosylation of HIV-1 remains the main
obstacle to viral control and eradication, it is possible to
exploit the protective glycosylation profiles of gp120
against the virus for the development of an env based
vaccine candidate [26]. For this reason, understanding
glycan shield glycosylation patterns is critical. There is a
paucity of data in Zimbabwe regarding the number and
distribution patterns of HIV-1 subtype C gp120 env
PNGs and sequence length polymorphisms in adults fol-
lowing heterosexual transmission, let alone in vertically
infected children. More so, gp120 env characteristics of
the unusually prolonged survival of HIV-1 infected but
drug naïve pediatric patients are not well documented.
Our study population of HIV-1 infected family members
provided a unique opportunity to investigate gp120 env
PNGs patterns and amino acid sequence lengths poly-
morphisms following heterosexual and subsequent parent
to child transmission (PTCT) events, a unique scenario
that has not been reported elsewhere.
2. M at erials and Method
Study Population and Procedures
Described are HIV-1 transmission events of close con-
tacts of whom the time and directionality of vertical
transmission was known but unknown for heterosexual
transmission. The unit of analysis was a family. Four
families were willing to participate in our study. HIV-1
infected families labeled 205, 366, 375 and 567 consisted
of biological parent(s) and children, index child
(older/first sibling) and index child’s sibling
(younger/second sibling), constituted the study popula-
tion. The index child was defined as the first child to be
recruited into our study. Two families comprised each of
both parents and a respective biological index child. The
other two families were composed of parent(s) and two
subsequent biological children, the first and second sib-
lings. For Family 567 the father figure was missing as he
was working in another regional country. Each family
member was HIV-1 infected and none had received anti-
retroviral therapy at the time of sample collection.
Consent was obtained from the respective pregnant
mother of each family participating in the national
PMTCT programme in Harare peri-urban mother and
child clinics that were known to be HIV-1 positive at 36
weeks gestation. Spouses also consented to participate in
the family HIV genetic study. Similar recruitment and
procedures were followed as previously described [27].
All the infants were breast fed for at least nine months.
First siblings’ blood samples were collected at 60 ± 10
months of age as there were insufficient sample volumes
from their respective first available HIV-1 positive sam-
ples. In cases of second siblings the first available HIV-1
positive samples were genotyped and sampling time was
at about 15 ± 3 months of age. Nucleic acid extraction,
PCR amplification, cloning and DNA sequencing meth-
ods for the HIV-1 env gp120 C2V5 region were done as
previously described and so was HIV-1 subtype determi-
nation[28].
3. Data Analysis
Data were entered and analysed using Stata version 10.
The number and distribution of PNGS including C2V5
sequence length polymorphisms were determined for
both the parent(s) and children. To compare the se-
quences’ lengths and number of PNGs between family
members, a non parametric Wilcoxon rank-sum (Mann-
Whitney) test was used. Tests of statistical significance
included the 95% confidence interval of relative risks and
two sided p-values. Similar analysis was done after
stratifying the study population by age and gender for
both heterosexual and vertical transmission events. Indels
assessment along the sequences was done manually. The
520 base pair nucleotide sequences were translated to
amino acid sequence using the Gene Doc program. The
amino acid sequences in their fasta formats were entered
into a glycosylation an alysis site:

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and
Amino Acid Length Polymorphisms among Infected Family Members
3
http://www.hiv.lanl.gov /content/hiv-db/GLYCOSITE/gly
cosite where PNGs were marked and counted. Variations
in the number and location of PNGs within the entire
C2V5 region an d sub-regions w ere analysed. Median and
range values of sequence lengths and PNGs were calcu-
lated for each family member as previously described by
others [14].
4. Ethical Consideration
The study was approved by the Medical Research Coun-
cil of Zimbabwe (MRCZ) and the Ethical Review Com-
mittee in Norway. Written consent to participate in the
HIV-1 genetic research study was obtained from the par-
ent(s) who also consented on behalf of their minors.
Study participants were free to discontinue at any time
without any prejudice. Parents also consented to have
their blood samples and those of their children to be used
for other future HIV related research studies.
5. Results
5.1. Characteristics of the Four Families
Three of the four couples were in a monogamous mar-
riage except for Mother 375 who was in a polygamous
relationship. All parents had at least seven years in school
and were of low economic status. Mean age of the moth-
ers and their spouses were 34.8 years ± 3.2 years and 38
years ± 2 years, respectively.
5.2. HIV Infections
Parents of the four families were HIV-1 positive at en-
rolment but did not know when and how they got in-
fected. Mode of HIV-1 acquisition was most likely het-
erosexually as none mentioned any history of blood
transfusion, drug abuse or homosexuality except for
mother 366 who had a history of blood transfusion hence
it could not be substantiated whether she got infected
heterosexually or through blood transfusion. Index chil-
dren of families 366, 375 and 567 and second siblings of
families 205 and 567 were HIV-1 DNA PCR negative at
delivery and six weeks postpartum but later got infected
through breastfeeding. Index child 205 was HIV-1 DNA
PCR negative at delivery. However, he did not turn up
for his 6 week postpartum visit hence his time of infec-
tion was not definite.
5.3. Global Analysis of Families’ C2V5 Sub-Re-
gions Nucleotide Sequences
Consistent feature of families sequence analysis showed
the highest variation within the env gp120 V4 and V5 and
interestingly also within the C3 sub-regions. Indels char-
acterized the genetic diversity of these three sub-regions.
In contrast amino acid substitutions constituted the main
cause of variability within the constant domains C2, C4
and to a lesser extent V3 region, see Figure 1. Of note
was a 12 base pair nucleotide in sertion observed in Fam-
ily 366 Mother-Infant pair viral sequences that was ab-
sent in the purported father`s HIV sequence.
5.4. Glycosylation Patterns and Amino Acid
Lengths of the Variable Loops, V3-V5
Of the variable regions, V3 was relatively conserved re-
gardless of family member ag e or sex with no insertions,
deletions or shifts in PNGs that ch aracterized V4 and V5
domains. Families 205, 366 and 375 V3 amino acid
lengths were all 35, see Tables 1-3. Contrary, most se-
quences of 567 family members were of 33 amino acids
in length; consequently they had the least number of
PNGs. Despite having the optimal amino acids length
within the V3 region, Family 375 had a just single PNGs,
see Tables 1-6. Remarkably was the presence of the
conserved GPGQ motif within the V3 crown of all family
members’ viral sequences that was preserved following
both heterosexual and vertical transmission events. On
the contrary, 2/5 of Mother 366 HIV-1 clones had GPGR
motif instead.
V4 region showed extensive amino acid length polymor-
phism and PNGs variation ranging from median (range)
28(24 - 35) and 3(2 - 6) respectively, see Tables 1 and 6.
There were numerous deletions in this reg ion resulting in
variants with different V4 region amino acid length poly-
morphisms as shown by sequence lengths of fathers 205
and 375 including Mother 567 and to some extent Mother
366. The first two PNGs had more fixed locations in the
distribution whilst the other two were randomly distrib-
uted. Interestingly, Father 205 lost a PNGs resulting in
his major HIV-1 variants having just 3 PNGs. Insertions
within Mother 366 HIV-1 sequences resulted in her
gaining one more PNGs. Of note was the loss of the first
PNGs within the V4 region observed in 3/4 of the index
children, see Figure 1. V5 region PNGs varied from me-
dian (range) 2 (1 - 3) between families, with Family 205
HIV-1 variants having an average of 3 per family mem-
ber whilst just one PNGs was a common observation with
Family 567.
5.5. Glycosylation Patterns and Amino Acid
Lengths of the Constant Regions
5.5.1. Heterosexual Transmission
There were no significant gender differences with respect
to amino acid lengths between fathers and mothers for the
entire C2V5 length, 207 (199 - 215); 208 (198 - 216),
p = 0.734. However, after stratifying by sub-regions,
there was a general tendency for mothers of families 366
and 375 to have longer C3 region amino acid lengths

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and 4 Amino Acid Length Polymorphisms among Infected Family Members

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and 5
Amino Acid Length Polymorphisms among Infected Family Members

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and
6 Amino Acid Length Polymorphisms among Infected Family Members
Figure 1. HIV subtype C Env C2-V5 Regions.

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and
Amino Acid Length Polymorphisms among Infected Family Members
7
Table 1. C2V5 and subregions amino acid (AA) lengths following Heterosexual transmission.
Pairs C2V5
Median (Range)
C2
Median
(Range)
V3
Median
(Range)
C3
Median
(Range)
V4
Median
(Range)
C4
Median
(Range)
V5
Median
(Range)
205
Father
Mother
P-value
200 (199 - 202)
202 (200 - 203)
0.156
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
48 (47 - 49)
47 (47 - 47)
0.028
26 (26 - 26)
28 (26 - 29)
0.079
36 (36 - 36)
36 (36 - 36)
-
13 (13 - 14)
14 (14 - 14)
0.025
366
Father
Mother
P-value
211 (210 - 214)
214 (213 - 216)
0.045
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
49 (49 - 51)
54 (54 - 54)
0.006
34 (33 - 35)
32 (30 - 33)
0.017
36 (36 - 36)
36 (36 - 36)
-
15 (15 - 15)
16 (15 - 16)
0.074
375
Father
Mother
P-value
211 (203 - 215)
212 (212 - 213)
1.000
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
49 (49 - 51)
51 (51 - 51)
0.025
33 (25 - 35)
28 (28 - 28)
0.178
36 (36 - 36)
36 (36 - 36)
-
16 (15 - 17)
20 (20 - 21)
0.012
567
Father
Mother
P-value
-
198 (198 - 200)
-
-
42 (42 - 42)
-
-
34 (33 - 35)
-
-
48 (47 - 48)
-
-
25 (24 - 25)
-
-
36 (36 - 36)
-
-
14 (14 - 15)
-
. Table 2. C2V5 and sub-regions amino acid (AA) lengths following vertical transmission.
Pairs C2V5
Median (Range)
C2
Median
(Range)
V3
Median
(Range)
C3
Median
(Range)
V4
Median
(Range)
C4
Median
(Range)
V5
Median
(Range)
205
Mother
1st sibling
p-value
2nd Sibling
p-value
202 (200 - 203)
207 (206 - 207)
0.013
200 (199-202)
0.058
42 (42 - 42)
42 (42 - 42)
-
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
35 (35 - 35)
-
47 (47 - 47)
52 (52 - 52)
0.005
48 (47 - 52)
0.264
28 (26 - 29)
26 (26 - 26)
0.079
26 (25 - 26)
0.039
36 (36 - 36)
36 (36 - 36)
-
36 (36 - 36)
-
14 (14 - 14)
16 (15 - 16)
0.007
13 (12 - 13)
0.028
366
Mother
1st sibling
p-value
214 (213 - 216)
216 (216 - 216)
0.105
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
54 (54 - 54)
54 (54 - 54)
-
32 (30 - 33)
32 (32 - 32)
1.000
36 (36 - 36)
36 (36 - 36)
-
16 (15 - 16)
17 (17 - 17)
0.040
375
Mother
1st sibling
p-value
212 (212 - 213)
209 (209 - 209)
0.040
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
51 (51 - 51)
49 (49 - 49)
0.014
28 (28 - 28)
28 (28 - 28)
-
36 (36 - 36)
36 (36 - 36)
-
20 (20 - 21)
19 (19 - 19)
0.040
567
Mother
1st sibling
p-value
2nd Sibling
p-value
198 (198 - 200)
201 (201 - 202)
0.043
201 (198 - 202)
0.239
42 (42 - 42)
42 (42 - 42)
-
42 (42 - 42)
-
34 (33 - 35)
33 (33 - 33)
0.317
33 (33 - 33)
0.317
48 (47 - 48)
49 (49 - 49)
0.034
48 (48 - 49)
0.197
25 (24 - 25)
27 (27 - 27)
0.034
30 (25 - 30)
0.099
36 (36 - 36)
36 (36 - 36)
-
36 (36 - 36)
-
15 (14 - 15)
14 (14 - 15)
0.456
13 (13 - 13)
0.034
relative to their respective spou ses; 49(49-51); 54(54-54),
and 49(49 - 51); 51(51 - 51), p=0.006 and 0.025 respec-
tively whilst the opposite was true for Mother 205, see
Table 1. Mother 375 consistently had lower numbers of
PNGs compared to the spouse, yet the opposite was true
regarding amino acid lengths. See Table 2. No clear
trend was observed regarding the number of PNGs
following the 3 heterosexual transmission events. There
were also no significant gender differences with respect
to PNGs between mothers and fathers 14(9 - 16); 15(12 -
19) p = 0.156. Howeve r, for sub-regionV4 fathers tend ed
to have higher PNGs, being significant for heterosexual
partners of family 375.5(4 - 6), 3(3 - 3) p = 0.007, see
Table 2.

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and 8 Amino Acid Length Polymorphisms among Infected Family Members
Table 3. C2V5 and sub-regions amino acid (AA) lengths variations following PTC transmission.
Pairs C2V5
Median (Range)
C2
Median
(Range)
V3
Median
(Range)
C3
Median
(Range)
V4
Median
(Range)
C4
Median
(Range)
V5
Median
(Range)
205
Father
1st sibling
p-value
2nd Sibling
p-value
200 (199 - 202)
207 (206 - 207)
0.019
200 (199 - 202)
0.350
42 (42 - 42)
42 (42 - 42)
-
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
35 (35 - 35)
-
48 (47 - 49)
52 (52 - 52)
0.013
48 (47 - 52)
0.439
26 (26 - 26)
26 (26 - 26)
-
26 (25 - 26)
0.127
36 (36 - 36)
36 (36 - 36)
-
36 (36 - 36)
-
13 (13 - 14)
16 (15 - 16)
0.017
13 (12 - 13)
0.617
366
Father
1st sibling
p-value
211 (210 - 214)
216 (216 - 216)
0.057
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
49 (49 - 51)
54 (54 - 54)
0.046
34 (33 - 35)
32 (32 - 32)
0.057
36 (36 - 36)
36 (36 - 36)
-
15 (15 - 15)
17 (17 - 17)
0.025
375
Father
1st sibling
p-value
211 (203 - 215)
209 (209 - 209)
1.000
42 (42 - 42)
42 (42 - 42)
-
35 (35 - 35)
35 (35 - 35)
-
49 (49 - 51)
49 (49 - 49)
0.480
33 (25 - 35)
28 (28 - 28)
0.340
36 (36 - 36)
36 (36 - 36)
-
16 (15 - 17)
19 (19 - 19)
0.057
567
Father
1st sibling
p-value
2nd Sibling
p-value
-
201 (201 - 202)
0.043
201 (198 - 202)
0.239
-
42 (42 - 42)
-
42 (42 - 42)
-
-
33 (33 - 33)
0.317
33 (33 - 33)
0.317
-
49 (49 - 49)
0.034
48 (48 - 49)
0.197
-
27 (27 - 27)
0.034
30 (25 - 30)
0.099
-
36 (36 - 36)
-
36 (36 - 36)
-
-
14 (14 - 15)
0.456
13 (13 - 13)
0.034
Table 4. Heterosexual patterns following transmission (PNGS).
Pairs C2V5 Median
(Range)
C2
Median
(Range)
V3
Median
(Range)
C3
Median
(Range)
V4
Median
(Range)
C4
Median
(Range)
V5
Median
(Range)
205
Father
Mother
p-value
13 (12 - 15)
16 (15 - 16)
0.014
3 (3 - 3)
3 (3 - 3)
-
2 (1 - 2)
2 (1 - 2)
0.371
3 (2 - 3)
3 (3 - 3)
0.091
3 (3 - 3)
3 (3 - 3)
-
2 (2 - 2)
2 (2 - 2)
-
1 (1 - 2)
3 (3 - 3)
0.006
366
Father
Mother
p-value
16 (14 - 19)
15 (14 - 16)
0.379
3 (3 - 3)
2 (2 - 2)
0.005
1 (0 - 1)
2 (2 - 2)
0.006
3 (3 - 4)
3 (3 - 4)
0.866
5 (4 - 6)
4 (3 - 5)
0.302
2 (1 - 2)
2 (2 - 2)
0.264
3 (2 - 3)
2 (1 - 2)
0.058
375
Father
Mother
p-value
16 (15 - 18)
13 (13 - 13)
0.007
3 (3 - 3)
3 (3 - 3)
-
1 (1 - 1)
1 (1 - 1)
-
3 (3 - 4)
2 (2 - 2)
0.006
5 (4 - 6)
3 (3 - 3)
0.007
2 (2 - 2)
2 (2 - 2)
-
2 (2 - 2)
2 (2 - 2)
-
567
Father
Mother
p-value
-
10 (9 - 11)
-
-
3 (3 - 3)
-
-
1 (1 - 2)
-
-
1 (1 - 3)
-
-
3 (3 - 3)
-
-
0 (0 - 0)
-
-
1 (1 - 1)
-
C2 and C4 regions sequence lengths were exactly the
same for all families‟ heterosexual partners but differed
with respect to C3, V4 and V5 sub-regions. Applying the
postulated hypothesis to C3 and to a lesser extent V5
sub-region sequence lengths [14], it is tantamount to
tempting to propose the direction of heterosexual trans-
mission, a very sensitive issue and should be handled
carefully. A male to female (MTF) heterosexual trans-
mission was suspected for family 205 with the father
probably infecting his wife whilst female to male (FTM)
transmission events were possible for families 366 and
375. Interestingly, though not statistically significant, an
exact opposite trend was observed for the V4 variable
region, see Table 1.
5.5.2. PTCT and Sequence Lengths and PNGs
There were no significant V4 region amino acid lengths
differences with respect to the C2, V3 and to some extent
C4 sub-regions between mothers and first siblings.
However, for the entire C2V5 region there was a tendency

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and 9
Amino Acid Length Polymorphisms among Infected Family Members
Table 5. PNG patterns following transmission.
Pairs C2V5 Median
(Range)
C2
Median
(Range)
V3
Median
(Range)
C3
Median
(Range)
V4
Median
(Range)
C4
Median
(Range)
V5
Median
(Range)
205
Mother
1st sibling
p-value
2nd Sibling
p-value
16 (15 - 16)
11 (10 - 13)
0.010
14 (12 - 16)
0.080
3 (3 - 3)
2 (2 - 2)
0.005
3 (3 - 3)
-
2 (1 - 2)
1 (1 - 2)
0.120
2 (1 - 2)
0.866
3 (3 - 3)
2 (1 - 2)
0.007
3 (1 - 3)
0.264
3 (3 - 3)
3 (3 - 3)
-
3 (2 - 3)
0.264
2 (2 - 2)
2 (2 - 2)
-
2 (1 - 2)
0.264
3 (3 - 3)
2 (1 - 2)
0.007
2 (2 - 3)
0.025
366
Mother
1st sibling
p-value
15 (14 - 16)
14 (13 - 15)
0.320
2 (2 - 2)
3 (2 - 3)
0.114
2 (2 - 2)
2 (2 - 2)
-
3 (3 - 4)
3 (3 - 3)
0.527
4 (3 - 5)
4 (3 - 4)
0.306
2 (2 - 2)
2 (2 - 2)
-
2 (1 - 2)
1 (1 - 1)
0.180
375
Mother
1st sibling
p-value
13 (13 - 13)
12 (11 - 13)
0.114
3 (3 - 3)
3 (3 - 3)
-
1 (1 - 1)
1 (1 - 1)
-
2 (2 - 2)
2 (2 - 2)
-
3 (3 - 3)
3 (3 - 3)
-
2 (2 - 2)
1 (0 - 2)
0.114
2 (2 - 2)
2 (2 - 2)
-
567
Mother
1st sibling
p-value
2nd Sibling
p-value
10 (9 - 11)
12 (12 - 12)
0.037
13 (12 - 13)
0.046
3 (3 - 3)
3 (3 - 3)
-
3 (3 - 3)
-
1 (1 - 2)
1 (1 - 1)
0.317
1 (1 - 1)
0.317
1 (1 - 3)
2 (2 - 2)
0.480
2 (2 - 2)
0.480
3 (3 - 3)
2 (2 - 2)
0.025
4 (3 - 4)
0.114
0 (0 - 0)
2 (2 - 2)
0.025
2 (2 - 2)
0.025
1 (1 - 1)
2 (2 - 2)
0.025
1 (1 - 1)
-
Table 6. PNG patterns following PTC transmission.
Pairs C2V5 Median
(Range)
C2
Median
(Range)
V3
Median
(Range)
C3
Median
(Range)
V4
Median
(Range)
C4
Median
(Range)
V5
Median
(Range)
205
Father
1st sibling
p-value
2nd Sibling
p-value
13 (12 - 15)
11 (10 - 13)
0.080
14 (12 - 16)
0.457
3 (3 - 3)
2 (2 - 2)
0.008
3 (3 - 3)
-
2 (1 - 2)
1 (1 - 2)
0.495
2 (1 - 2)
0.495
3 (2 - 3)
2 (1 - 2)
0.061
3 (1 - 3)
0.739
3 (3 - 3)
3 (3 - 3)
-
3 (2 - 3)
0.317
2 (2 - 2)
2 (2 - 2)
-
2 (1 - 2)
0.317
1 (1 - 2)
2 (1 - 2)
0.495
2 (2 - 3)
0.040
366
Father
1st sibling
p-value
16 (14 - 19)
14 (13 - 15)
0.240
3 (3 - 3)
3 (2 - 3)
0.157
1 (0 - 1)
2 (2 - 2)
0.046
3 (3 - 4)
3 (3 - 3)
0.480
5 (4 - 6)
4 (3 - 4)
0.134
2 (1 - 2)
2 (2 - 2)
0.480
3 (2 - 3)
1 (1 - 1)
0.053
375
Father
1st sibling
p-value
16 (15 - 18)
12 (11 - 13)
0.060
3 (3 - 3)
3 (3 - 3)
-
1 (1 - 1)
1 (1 - 1)
-
3 (3 - 4)
2 (2 - 2)
0.046
5 (4 - 6)
3 (3 - 3)
0.057
2 (2 - 2)
1 (0 - 2)
0.157
2 (2 - 2)
2 (2 - 2)
-
567
Father
1st sibling
p-value
2nd Sibling
p-value
-
12 (12 - 12)
0.037
13 (12 - 13)
0.046
-
3 (3 - 3)
-
3 (3 - 3)
-
-
1 (1 - 1)
0.317
1 (1 - 1)
0.317
-
2 (2 - 2)
0.480
2 (2 - 2)
0.480
-
2 (2 - 2)
0.025
4 (3 - 4)
0.114
-
2 (2 - 2)
0.025
2 (2 - 2)
0.025
-
2 (2 - 2)
0.025
1 (1 - 1)
-

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and 10 Amino Acid Length Polymorphisms among Infected Family Members
for the index children to have longer amino acid lengths
compared to the mothers p = 0.013, 0.040 and 0.043 for
families 205, 375 an d 567 respectively, see Table 2. Dif-
ferences were also observed within the V5 sub-region
with p values =0.007, 0.040, 0.040 and 0.456 for Fami-
lies 205, 366, 375 and 567 respectively, but without any
clear trend. Family 205 second siblings’ V4 and V5
sub-regions amino acid lengths were both significantly
shorter relative to the maternal ones; p=0.039 and 0.028
respectively. Similar observations were also noted for
567 second sibling sequences which were significantly
shorter relative to maternal ones p=0.034. Significant
differences were also observed in the C3 region but with
no clear trends. See Table 2. Interestingly a similar trend
like the one observed in the mothers with first siblings
was also observed with paternal sequences with respect
to the C2V5, C3, and V5 regions. However, there were
no differences in sequence lengths between paternal ones
and those from second sibling, see Table 3. There was a
tendency for parents to have longer amino acid lengths
for the regions C2V5 and V3 being; 208(198 - 216); 205
(198 - 216): p = 0.138 and 35(33 - 35); 34(33 - 35): p =
0.018. Following stratification by age, there was also a
tendency for parents to have denser glycans within the
C2V5, C3 and V4 reg ions which were 14(9 - 19); 13 (10
- 16), 3(1 - 4); 2(1 - 3) and 4(3 - 6); 3(2 - 4) p= 0.0025,
0.005 and 0.008 respectively.
For both the entir e C2V5 region and sub-region C3 the
first siblings had significantly longer amino acid lengths
relative to the second sib lings; 207(201 - 216), 200(198 -
202) and 51(49 - 54), 48(47 - 52); p = 0.005 and 0.007
respectively. However, the opposite was true for the V5
region where the second siblings had much longer amino
acid lengths 13(12 - 14), 11(14 - 19); p= 0.0006. After
gender stratification no differences were observed re-
garding amino acid lengths whilst female siblings tended
to have a denser glycan shield relative to their male
counterparts, 14(11 - 16); 12 (10 - 13); p = 0.039. Gly-
cosylation patterns found in siblings had similar patterns
to those found in the parents, see Table 6.
6. Discussion
N-glycosylation is an essential co-translational modifica-
tion process where a sugar, glycan, is covalently attached
to the amide group of asparagine (N) residues [29].The
N-linked glycosylation of the HIV-1 env gp120 is one of
the most important viral protective mechanisms to over-
come in order to develop an effective neutralizing anti-
body-inducing vaccine. Since several PNGs are relatively
constant across HIV-1 subtypes, there is a great deal of
interest in developing a carbohydrate based antigen de-
signed to elicit a humoral immune response. Studies have
shown a subtype env sequence length dependency fol-
lowing transmission with subtypes A and C significantly
shifted towards shor ter lengths though untrue for subtype
B [14, 20, 30]. Our results are similar to previous subtype
A and D heterosexual transmission where trends were
observed for HIV-1 env lengths but no significant differ-
ences were observed with PNGs [19].
For PTCT, our study observed children being infected
with shorter sequences demonstrated by significantly
shorter sequences in the second sibling relative to the
maternal or paternal sequences which tended to increase
with age or disease progression, probably due to immune
selection as shown in the first siblings who had signifi-
cantly longer sequ ences and PNGs relative to the pa rents.
Our results are suggestive of the fact that a child acquires
more or less the same number of PNGs like the mother
and these tend to decrease with time or disease progres-
sion as evidenced by the presence of the first V4 region
PNGs observed amongst the second siblings who were
about 14 months old but was absent or lost in 3 out of 4
of the first siblings who were about five years old. This
observation may have something to do with functional
elements of V4 region which exist solely to facilitate
viral escape during the evolving glycan shield [41]. PNGs
number differences were also observed in chronic HIV-1
infection [31-33]. Similar to our findings some pediatric
HIV-1 env subtype C studies have also demonstrated no
clear pattern of change in the number PNGs [34,35].
Mother 567 with the least number of PNGs has since died
during beginning of this year suggestive of the fact that
loss of PNGs may be associated with advanced disease
and probably a risk factor of dying. It would be interest-
ing to correlate PNGs with markers of disease progres-
sion.
A consistent feature of families’ sequence analysis
showed the highest levels of variation within the env
gp120, V4 and V5 variable including the C3 regions
challenging whether the so-called “constant region” with
respect to subtype B is really also a constant one when it
comes to subtype C. More so, atypically, our families’
HIV-1 subtype C V3 region was relatively constant,
again querying the currently common practice of ex-
trapolating subtype B findings to non-subtype B ones.
Traditionally, V3 region has been considered a variable
domain based on analysis of subtype B sequences. How-
ever, the small numbers of clones sequenced may not
have been sufficient for statistically sound conclusions. A
larger sample size is recommended to substantiate these
findings. Interestingly, similar results of a relatively con-
stant subtype C V3 region have been obtained elsewhere
[36-39]. Conserved PNGs within the V3 region amongst
all family members regardless of age or sex points to

HIV-1 Env gp120 C2V5 Potential N-Linked Glycosylation Site(s) (PNGs) Variations and 11
Amino Acid Length Polymorphisms among Infected Family Members
their potentially important functional elements which
may function primarily to mask the large number of neu-
tralizing antibody ep itopes defined in this region. As pre-
viously reported by others, emergence of deletions within
the V4 region was also observed among all family mem-
bers both adults and children [40]. These coupled with
amino acid substitutions affected the number and distri-
bution of PNGs resulting in the coexistence within some
family members V4 variants each characterized by dif-
ferent amino acid sequences and PNGs patterns. Previous
subtype C studies have showed extensive V4 length
polymorphism where shorter lengths have been shown to
enhance infectivity at the cost of exposing neutralizing
epitopes [41].
Some countries including Zimbabwe now seek to in-
stitute legislation to hold persons criminally responsible
for willfully infecting others with HIV. This law has been
unsuccessfully applied as it is very difficult to prove
criminal intent on the part of the alleged transmitter and
currently there is no supporting forensic evidence to
prove directionality of infection. For successful prosecu-
tion of willful transmission, ascertaining the association
of number of PNGs or the HIV-1 env domain length
polymorphism and age of infection may be the first step
to provide a line of eviden ce to that effect, hence the need
for bigger studies.
7. Conclusions
Highest env gp120 region variation characterised by in-
dels were observed within theC3, V4 and V5 sub-regions.
Indels character ized s. Our study su ppor ts th e ob serv ation
that HIV-1 env C2V5 sequences and PNGs tend to in-
crease with age and disease progression in children.
Though sensitive and should be handled carefully, with
respect to C3 and V5 sequence lengths it is tempting to
propose a possible male to female and female to male
heterosexual transmission events for families 205 and
366 including 375 respectively. Ascertaining the associa-
tion of PNGs or env sequence length polymorphism and
period of infection may be the first step towards a possi-
ble line of forensic evidence. Hence bigger studies are
warranted to substantiate the authenticity of this poten-
tially useful application.
8. Acknowledgements
Acknowledgements are due to Drs. Kurewa NE and
Munjoma MW including all the support staff for facili-
tating sample collection. Many thanks are due to the
families who participated in this study. Special mention
goes to the Letten Foundation and Professor Letten F
Saugstad herself for funding the study.
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