Variability in HIV-1 Integrase Gene and 3′-Polypurine Tract Sequences in Cameroon Clinical Isolates, and Implications for Integrase Inhibitors Efficacy
Abstract
:1. Introduction
2. Results
2.1. Patient Demographics
2.2. HIV-1 Subtyping
2.3. INSTI Resistance Mutations in Cohort Samples
2.4. INSTI Resistance Mutations in Database Samples
2.5. INSTIs Resistance-Associated Mutations in Subjects Infected with HIV-1 CRF02_AG and Non-AG Viruses
2.6. Integrase Natural Polymorphisms in Subjects Infected with HIV-1 CRF02_AG and Non-AG Viruses
2.7. Effects of ART and Immune Function on Integrase RAMs and Natural Polymorphisms
2.8. Analysis of 3′-PPT and 5′ Terminal Nucleotides of 3′ Long Terminal Repeat
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Specimen Collection, HIV Serology, CD4 Cell Counts, and Viral Loads
4.3. RNA Extraction and PCR Amplification
4.4. Gene Sequencing
4.5. Sequence Analysis of the Study Cohort Samples
4.6. Sequence Analysis of the Database Samples
4.7. Statistical Analyses
4.8. Data Availability
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
HIV | Human immunodeficiency virus |
INSTIs | Integrase strand-transfer inhibitors |
ART | Antiretroviral therapy |
3′-PPT | 3′polypurine tract |
RAMs | Resistance-associated mutations |
CRF | Circulating recombinant form |
T | Threonine |
A | Alanine |
N | Asparagine |
K | Lysine |
E | Glutamic Acid |
Q | Glutamine |
M | Methionine |
I | Isoleucine |
S | Serine |
R | Arginine |
L | Leucine |
D | Aspartic Acid |
V | Valine |
G | Glycine |
C | Cysteine |
DNA | Deoxyribonucleic acid |
kDa | Kilodalton |
Aa | Amino acids |
AIDS | Acquired immunodeficiency syndrome |
FDA | United States Food and Drug Administration |
RAL | Raltegravir |
EVG | Elvitegravir |
DTG | Dolutegravir |
WHO | World Health Organization |
SSA | Sub-Saharan Africa |
PLWH | People living with HIV |
DRMs | Drug-resistance mutations |
URF | Unique recombinant form |
LTR | Long terminal repeats |
UNMC | University of Nebraska Medical Center |
ELISA | Enzyme-linked immunosorbent assay |
CD4 | cluster of differentiation 4 |
FACS | Fluorescence-Activated Cell Sorting |
RT-PCR | Reverse transcription polymerase chain reaction |
RNA | Ribonucleic acid |
cDNA | complementary DNA |
PCR | Polymerase chain reaction |
NCBI | National Center for Biotechnology Information |
FDR | False discovery rate |
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Characteristics | Male | Female | p Value |
---|---|---|---|
N (%) | 39 (30%) | 91 (70%) | |
Age (years; mean ± SD) | 37.28 ± 9.94 | 35.43 ± 8.75 | 0.29 |
Age range (years) | 18–58 | 20–56 | |
Education (years; mean ± SD) | 10.50 ± 3.47 | 9.09 ± 4.03 | 0.062 |
CD4 (mean ± SD) (cells /µL) | 360.3 ± 320.4 | 311.2 ± 184.5 | 0.285 |
CD4 range (cells /µL) | 4–1657 | 5–894 | |
Viral load (mean ± SD) (log copies/mL) | 4.64 ± 1.54 | 4.24 ± 1.5 | 0.174 |
Viral load range (log copies/mL) | 1.60–7.56 | 1.60–7 | |
ART-Naïve (N) | 33 | 66 | |
ART-Experienced (N) | 5 | 25 |
Mutations | HIV-1 Subtype/CRFs | |||||||
---|---|---|---|---|---|---|---|---|
A1 | CRF01_AE | CRF02_AG | CRF09_cpx | CRF11_cpx | CRF18_cpx | CRF36_cpx | Total (%) | |
INSTIs Accessory Resistance Mutations: | ||||||||
T97A | 1 (1.00) | 1 (1.00) | 2 (2.00) | |||||
E157Q | 6 (6.00) | 6 (6.00) | ||||||
Other Mutations: | ||||||||
M50I | 2 (2.00) | 1 (1.00) | 2 (2.00) | 1 (1.00) | 6 (6.00) | |||
S119R | 4 (4.00) | 4 (4.00) | ||||||
L74M | 8 (8.00) | 1 (1.00) | 9 (9.00) | |||||
L74I | 1 (1.00) | 22 (22.00) | 1 (1.00) | 1 (1.00) | 25 (25.00) |
Mutation | HIV-1 Subtypes/CRFs | ||||||||||
A1 | A2 | D | F2 | G | K | CRF01_AE | F2/CRF01_AE | CRF02_AG | CRF11_cpx | CRF13_cpx | |
INSTIs Major Resistance Mutations | |||||||||||
T66A | |||||||||||
N155K | |||||||||||
INSTIs Accessory Resistance Mutations | |||||||||||
T97A | 1 (0.47) | 1 (0.47) | 1 (0.47) | 1 (0.47) | 5 (2.32) | ||||||
A128T | |||||||||||
E157Q | 1 (0.47) | 1 (0.47) | |||||||||
Other Mutations | |||||||||||
M50I | 9 (4.18) | 1 (0.47) | 1 (0.47) | 5 (2.32) | 4 (1.86) | 1 (0.47) | 1 (0.47) | 1 (0.47) | 5 (2.32) | 6 (2.79) | 2 (0.93) |
L74M | 1 (0.47) | 6 (2.79) | 1 (0.47) | 1 (0.47) | |||||||
L74I | 1 (0.47) | 1 (0.47) | 17 (7.91) | 1 (0.47) | |||||||
S119R | 1 (0.47) | ||||||||||
S230N | 1 (0.47) | ||||||||||
E138D | 1 (0.47) | ||||||||||
Mutation | HIV-1 Subtypes/CRFs | ||||||||||
CRF18_cpx | CRF22_01A1 | CRF36_cpx | CRF45_cpx | URF | M/O recombinant | CRF01/A/D/F2 | N | O | P | Total | |
INSTIs Major Resistance Mutations | |||||||||||
T66A | 1 (0.47) | 1 (0.47) | |||||||||
N155K | 1 (0.47) | 1 (0.47) | |||||||||
INSTIs Accessory Resistance Mutations | |||||||||||
T97A | 1 (0.47) | 1 (0.47) | 4 (1.86) | 1 (0.47) | 16 (7.44) | ||||||
A128T | 1 (0.47) | 1 (0.47) | |||||||||
E157Q | 1 (0.47) | 3 (1.39) | |||||||||
Other Mutations | |||||||||||
M50I | 1 (0.47) | 13 (6.05) | 2 (0.93) | 2 (0.93) | 1 (0.47) | 2 (0.93) | 10 (4.65) | 67 (31.16) | |||
L74M | 9 (4.18) | ||||||||||
L74I | 3 (1.39) | 1 (0.47) | 1 (0.47) | 24 (11.16) | 49 (22.79) | ||||||
S119R | 1 (0.47) | ||||||||||
S230N | 1 (0.47) | 2 (0.93) | |||||||||
E138D | 1 (0.47) |
Cohort Samples | ||||
Gene M | AG (n = 75) (% with M) | Non-AG (n = 25) (% with M) | p Value Fisher | p Value FDR |
T97A | 1.33 | 4.00 | 0.43939 | 0.53221 |
E157Q | 8.00 | 0.00 | 0.33214 | 0.53221 |
M50I | 2.67 | 16.00 | 0.03294 | 0.19764 |
L74M | 10.67 | 4.00 | 0.44351 | 0.53221 |
L74I | 29.33 | 12.00 | 0.11097 | 0.33291 |
S119R | 5.33 | 0.00 | 0.56949 | 0.56949 |
Database Samples | ||||
Gene M | AG (n = 61) (% with M) | Non-AG (n =1 19) (% with M) | p Value Fisher | p Value FDR |
T66A | 0.00 | 1.40 | 0.54941 | 0.73722 |
N155K | 0.00 | 1.40 | 0.54941 | 0.73722 |
T97A | 17.62 | 15.36 | 0.67020 | 0.73722 |
A128T | 0.00 | 1.40 | 0.54941 | 0.73722 |
E157Q | 3.52 | 2.79 | 1.00000 | 1.00000 |
M50I | 17.62 | 86.56 | 0.00000 | 0.00000 |
L74M | 21.15 | 4.19 | 0.00091 | 0.00501 |
L74I | 59.92 | 44.67 | 0.05837 | 0.21402 |
S119R | 0.00 | 1.40 | 0.54941 | 0.73722 |
E138D | 0.00 | 1.40 | 0.54941 | 0.73722 |
S230N | 3.52 | 1.40 | 0.60510 | 0.73722 |
Cohort Samples | ||||
Gene Polymorphism | AG (n = 75) (% with P) | Non-AG (n = 25) (% with P) | p Value Fisher | p Value FDR |
E11D | 29.33 | 40.00 | 0.33308 | 0.40878 |
K14R | 77.33 | 56.00 | 0.06957 | 0.11049 |
R20K | 9.33 | 12.00 | 0.70761 | 0.73483 |
A21T | 5.33 | 24.00 | 0.01449 | 0.03913 |
S24N | 13.33 | 24.00 | 0.21976 | 0.28255 |
V31I | 68.00 | 60.00 | 0.47471 | 0.53405 |
L63I | 12.00 | 4.00 | 0.44389 | 0.52109 |
I72V | 16.00 | 44.00 | 0.00638 | 0.02462 |
L101I | 89.33 | 72.00 | 0.05136 | 0.08666 |
T112I | 12.00 | 32.00 | 0.03120 | 0.06479 |
T112V | 84.00 | 64.00 | 0.04702 | 0.08666 |
T124A | 90.67 | 68.00 | 0.01034 | 0.03103 |
T125A | 90.67 | 80.00 | 0.16850 | 0.22748 |
G134D | 10.67 | 28.00 | 0.05136 | 0.08666 |
G134N | 90.67 | 28.00 | 0.00000 | 0.00000 |
I135V | 81.33 | 28.00 | 0.00000 | 0.00001 |
K136Q | 0.00 | 44.00 | 0.00000 | 0.00000 |
K136T | 92.00 | 36.00 | 0.00000 | 0.00000 |
D167E | 8.00 | 28.00 | 0.01662 | 0.04079 |
V201I | 96.00 | 100.00 | 0.57096 | 0.61664 |
T206S | 97.33 | 52.00 | 0.00000 | 0.00000 |
I208L | 8.00 | 20.00 | 0.13639 | 0.19381 |
T218I | 28.00 | 32.00 | 0.80002 | 0.80002 |
L234I | 97.33 | 88.00 | 0.09808 | 0.14712 |
D256E | 20.00 | 48.00 | 0.00946 | 0.03103 |
R269K | 30.67 | 0.00 | 0.00068 | 0.00305 |
S283G | 64.00 | 88.00 | 0.02489 | 0.05601 |
Database Samples | ||||
Gene polymorphism | AG (n = 61) (% with P) | Non-AG (n = 119) (% with P) | p Value Fisher | p Value FDR |
E11D | 26.23 | 46.10 | 0.01023 | 0.01726 |
K14R | 75.41 | 57.79 | 0.02262 | 0.03393 |
R20K | 1.64 | 5.84 | 0.26882 | 0.31558 |
A21T | 8.20 | 25.97 | 0.00530 | 0.01022 |
S24N | 9.84 | 5.19 | 0.22585 | 0.27718 |
V31I | 80.33 | 50.65 | 0.00010 | 0.00024 |
L63I | 9.84 | 4.55 | 0.18737 | 0.24090 |
I72V | 31.15 | 39.61 | 0.32761 | 0.36856 |
L101I | 91.80 | 55.84 | 0.00000 | 0.00000 |
T112I | 9.84 | 15.58 | 0.36293 | 0.39196 |
T112V | 90.16 | 76.62 | 0.02780 | 0.03950 |
T124A | 95.08 | 78.57 | 0.00262 | 0.00545 |
T125A | 95.08 | 66.23 | 0.00001 | 0.00002 |
G134D | 11.48 | 29.22 | 0.00863 | 0.01554 |
G134N | 80.33 | 39.61 | 0.00000 | 0.00000 |
I135V | 75.41 | 9.09 | 0.00000 | 0.00000 |
K136Q | 9.84 | 51.30 | 0.00000 | 0.00000 |
K136T | 85.25 | 18.83 | 0.00000 | 0.00000 |
D167E | 14.75 | 42.21 | 0.00020 | 0.00045 |
V201I | 98.36 | 89.61 | 0.06340 | 0.08559 |
T206S | 91.80 | 46.10 | 0.00000 | 0.00000 |
I208L | 3.28 | 14.29 | 0.02228 | 0.03393 |
T218I | 44.26 | 12.34 | 0.00000 | 0.00002 |
L234I | 98.36 | 96.10 | 0.66536 | 0.69095 |
D256E | 3.28 | 29.22 | 0.00001 | 0.00003 |
R269K | 50.82 | 5.84 | 0.00000 | 0.00000 |
S283G | 70.49 | 68.83 | 0.86579 | 0.86579 |
Primer # | Position ^ | Sequence (5′ to 3′) |
---|---|---|
Integrase gene 1st PCR | ||
IN12 | 4007 → 4026 | GCAGGATTCGGGATTAGAAG |
IN13 | 5270 → 5251 | CTTTCTCCTGTATGCAGACC |
Integrase gene 2nd PCR | ||
IN1 | 4137 → 4157 | AAGGTCTATCTGGCATGGGTA |
BH4 | 5222 → 5200 | TCCCCTAGTGGGATGTGTACTTC |
Nef gene 1st PCR | ||
Nef5-1e F1 | 8513 → 8533 | GTGCCTCTTCAGCTACCACCG |
Nef3-3e R1 | 9488 → 9508 | AGCATCTGAGGGTTAGCCACT |
Nef gene 2nd PCR | ||
Nef5-1e F2 | 8698 → 8717 | TGGACAGAYAGGGTTATAGAA |
Nef3-7e R2 | 9448 → 9468 | CACCTCCCCTGGAAAGTCCCC |
Integrase gene sequencing primers | ||
IN1 | 4137 → 4157 | AAGGTCTATCTGGCATGGGTA |
BH4 | 5222 → 5200 | TCCCCTAGTGGGATGTGTACTTC |
IN4764AS | 4764 → 4743 | CCATTTGTACTGCTGTCTTAA |
IN4542S | 4542 → 4558 | GCAGGAAGATGGCCAGT |
Nef gene sequencing primers | ||
Nef5-1e F2 | 8698 → 8717 | TGGACAGAYAGGGTTATAGAA |
Nef3-7e R2 | 9448 → 9468 | CACCTCCCCTGGAAAGTCCCC |
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Acharya, A.; Tagny, C.T.; Mbanya, D.; Fonsah, J.Y.; Nchindap, E.; Kenmogne, L.; Jihyun, M.; Njamnshi, A.K.; Kanmogne, G.D. Variability in HIV-1 Integrase Gene and 3′-Polypurine Tract Sequences in Cameroon Clinical Isolates, and Implications for Integrase Inhibitors Efficacy. Int. J. Mol. Sci. 2020, 21, 1553. https://doi.org/10.3390/ijms21051553
Acharya A, Tagny CT, Mbanya D, Fonsah JY, Nchindap E, Kenmogne L, Jihyun M, Njamnshi AK, Kanmogne GD. Variability in HIV-1 Integrase Gene and 3′-Polypurine Tract Sequences in Cameroon Clinical Isolates, and Implications for Integrase Inhibitors Efficacy. International Journal of Molecular Sciences. 2020; 21(5):1553. https://doi.org/10.3390/ijms21051553
Chicago/Turabian StyleAcharya, Arpan, Claude T. Tagny, Dora Mbanya, Julius Y. Fonsah, Emilienne Nchindap, Léopoldine Kenmogne, Ma Jihyun, Alfred K. Njamnshi, and Georgette D. Kanmogne. 2020. "Variability in HIV-1 Integrase Gene and 3′-Polypurine Tract Sequences in Cameroon Clinical Isolates, and Implications for Integrase Inhibitors Efficacy" International Journal of Molecular Sciences 21, no. 5: 1553. https://doi.org/10.3390/ijms21051553