Co-evolution of retroviruses and their vertebrate hosts have occurred for tens of millions of years.1 During the course of evolution, vertebrate hosts developed varying approaches to suppress retroviruses replication, such as innate immunity, adapted immunity, and restriction factors. In 2004, Stremlau et al2 identified TRIM5α as a restriction factor in rhesus monkey cells that blocked HIV-1 replication at a postentry, preintegration stage of the HIV-1 life cycle. TRIM5α proteins from other primates also have restriction activity for different retroviruses,3-6 and the human TRIM5α also restricts HIV-1 though to a lesser extent when compared with that found in rhesus monkey.3-5
Although the initial report indicated that TRIM5α might be a trimer, subsequent studies now claim that it is a dimmer.7 TRIM5α contains RING domain, B-box 2 domain, coiled-coil domain, and B30.2/SPRY domain.8 The RING domain has E3 ubiquitin ligase activity, but it is not absolutely required for retrovirus restriction; whereas B-box 2 domain, whose function was unknown, seems to be essential for efficient retrovirus restriction.9,10 The coiled-coil domain is indispensable for TRIM5α multimerization.11 Both the coiled-coil and the B30.2/SPRY domains are required for retroviruses restriction, such that a single amino acid substitution (R332P) in the human TRIM5α B30.2/SPRY domain was shown to be essential for the ability to restrict HIV-1.11-13 Our and others' studies revealed that B30.2/SPRY domain experienced a positive selection during primate evolution history.14-16
The potential role of human TRIM5α nucleotide variants on HIV-1 susceptibility and AIDS progression has been studied in white, African, and Asian population, but the results were not consistent.17-23 Previous studies indicate that H43Y, located in the RING domain of TRIM5α, was not associated with HIV-1 susceptibility in white,19,20,23 although was more frequent in HIV-1-resistant subjects in Asian and African-American.20,22
We hypothesized that some TRIM5α variants may be positively selected by HIV-1 so that they enriched among HIV-1-seronegative (resistant) subjects from the population having high-risk exposures to HIV, such as intravenous drug users (IDUs). Meanwhile, interspecies differences in exon8 show that it is an essential domain determining antiretroviruses activities although intraspecies variants in human mainly locate in exon2. Therefore, in this study, we recruited 1294 IDUs to investigate the relationship between the polymorphism in TRIM5α exon2 and exon8 and HIV-1 susceptibility. We found 43Y homozygote in exon2 is likely to account for HIV-1 resistance due to TRIM5α in Chinese IDUs.
MATERIALS AND METHODS
We recruited 1294 IDUs from Yunnan Province, China. All participants answered a standard questionnaire about history of intravenous drug usage and other risk factors. Blood was collected using vacutainer tubes. The plasma and DNA were then prepared and stored in a −70°C freezer until use according to standard procedures. HIV-1 status testing was carried out using enzyme-linked immunosorbent assay (Beijing Wantai Biological Pharmacy Enterprise Co, Ltd, Beijing, China), and positive results were confirmed by another enzyme-linked immunosorbent assay kit (Shanghai Kehua Bio-engineering Co, Ltd, Shanghai, China). The protocol of the study was approved by the Ethics Committee of Kunming Institute of Zoology, Chinese Academy of Sciences.
Resequencing TRIM5 Exon2 and Exon8
Genomic DNA was isolated from blood for all participants using Gentra Puregene Blood Kit (QIAGEN, Inc, MD) after preparing plasma. For analysis of variations in the TRIM5 exon2 and exon8, DNA samples were amplified by polymerase chain reaction using ExTaq DNA polymerase (Takara, Inc, Dalian, China). Theses primer pairs were used: Exon2-F (5′-GCAGGGATCTGTGAACAAGAGG-3′) and Exon2-R (5′-CCCGGGTCTCAGGTCTATCATG-3′), Exon8-F (5′-ACAGTTGATGTGACAGTGGC-3′), and Exon8-R (5′-CTTGGTGAGCACAGAGTCAT-3′). Direct sequencing of polymerase chain reaction product was carried out by BigDye Terminator v3.1 Cycle Sequencing Kits and Applied Biosystems 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA).
Automated sequence analysis was performed in a 3730xl DNA analyzer (Applied Biosystems). Sequence data were aligned using CLUSTAL W.24
Demographic and genotype comparison of HIV-1 seronegatives (resistant) and infected IDUs was performed by using χ2 test, Fisher exact test, or t test, as appropriate. Genetic risks were calculated as odds ratios (OR) with 95% confidence intervals (CIs). The Mantel-Haenszel test was used for stratified analysis of Han and Dai ethnicity, and exact confidence limits were obtained by Epi Info Version 3.5.1 software package (CDC, Atlanta, GA; and World Health Organization, Geneva, Switzerland).25 The statistical significance was fixed at P = 0.05. Other statistical analysis was performed using the statistical software SPSS 16.0 for Windows (SPSS, Chicago, IL).
Basic Information of the Chinese IDUs
In total, there were 666 (51.5%) HIV-1 seronegatives and 628 (48.5%) HIV-1-infected individuals (Table 1). On average, HIV-1 seronegatives (median, 31.2 ± 8.3 years; range 19-56 years) was 1.1 years younger than those in the HIV-1-infected individuals (median 32.3 ± 6.4 years; range 14-68 years). Otherwise, there was no significant difference in age and gender between seronegatives and HIV-1-infected population.
Genetic Polymorphisms in TRIM5 by Resequencing 2 Exons
To identify potential TRIM5 variations in Chinese IDUs, we sequenced TRIM5 exon2 and exon8 coding region from genomic DNA of 1151 subjects (666 HIV-1 seronegatives and 485 HIV-1 infected). Because all variants with high frequencies were located in exon2, exon2 were resequenced in additional 143 HIV-1-infected IDUs. We identified a total of 14 different nucleotide variants, including nine were located in the exon2 and 5 in the exon8 (Table 2). However, most variants, except for 4 single-nucleotide polymorphisms (SNPs), were observed at low frequencies [minor allele frequency (MAF) < 0.05]. Four common SNPs were −2C/G (rs3824949, MAF = 0.432), H43Y (rs3740996, MAF = 0.179), V112F (rs11601507, MAF = 0.100), and R136Q (rs10838525, MAF = 0.070).
Association of Common SNPs and Susceptibility to HIV-1 Infection
Associations between common SNPs of TRIM5α and the susceptibility to HIV-1 infection were listed in Table 3. No significant differences in 4 common SNPs' genotype frequencies and allele frequencies were observed between HIV-1-infected IDUs and seronegative (resistant) IDUs, except H43Y (P = 0.006, χ2 analysis of genotype distribution, df = 2). For the H43Y, the frequency of homozygote in resistant seronegative IDUs was significantly higher than that in the HIV-1-infected IDUs [OR (95% CI) = 0.48 (0.23 to 0.97), P = 0.028, recessive model]. This result implies that this polymorphism might confer resistance to HIV-1 infection in IDUs.
Because all participants belong to either Han or Dai ethnicity, we perform a stratified analysis using Mantel-Haenszel test, which again confirmed the significant P value (0.033), Mantel-Haenszel weighted OR = 0.46 and exact confidence limits (95% CI = 0.22 to 0.94). It indicates that the association between homozgyote and the susceptibility to HIV-1 infection in Chinese IDUs remains strong after controlling for ethnicity (Table 4). We conclude that the 43rd amino acid change of TRIM5α (His→Tyr) may be a resistant variant to HIV-1 infection in Chinese IDUs.
A species-specific difference in infection potency of HIV is recognized, such that HIV-1 can infect human but not in most other nonhuman primates.26 But the mechanism was not understood until the TRIM5α was identified as an inhibiting factor restricting HIV-1 replication in rhesus monkey cells.2 Subsequently, many reports showed that TRIM5α proteins from different primate species could also restrict HIV-1 infection,3-6 whereas human TRIM5α only weakly restrict HIV-1.3-5 Our and other groups revealed that TRIM5α, especially for B30.2/SPRY domain, experienced a positive selection in primates evolution history which may also account for such a difference in HIV restriction property.14-16 Meanwhile, species-specific genetic polymorphisms in the human TRIM5α have also been shown to affect anti-HIV-1 activity. Therefore, we recruited 1294 IDUs from Yunnan Province, China and resequencing the TRIM5 putative functional domains of exon2 and exon8 to investigate the relationship between genetic polymorphisms and anti-HIV-1 activity. Though we found 14 different nucleotide variants, there were 10 rare nucleotide variants (MAF < 0.05) (Table 2). All rare variants in exon8 did not located in the positive selection clusters in the B30.2/SPRY domain and half of them were synonymous substitution, which indicated that human TRIM5α might have been selected by viruses unrelated to HIV-1.
Two of the 4 SNPs showed strong linkage disequilibrium (LD) with each other (rs11601507 and rs10838525, D' = 1). As we were focused on 1 single candidate gene and the presence of high LD in a pair of them, so we believe that multiple comparison is not a major issue here. We demonstrated the association between 43Y homozygote and reduced HIV-1 susceptibility in Chinese IDUs. Though there were controversies about the association H43Y with the HIV-1 infection in several studies (Table 5),17-23 our result provided additional data to support the presence of an association. Reason to explain for the different results may include the following situations. First, it is well known that HIV-1 infected different kinds of cells due to different transmission routes, and expression level of TRIM5α is not always same among different kinds of cells, therefore, TRIM5α may have different effect for HIV-1 infection due to different transmission mechanism.27 Sewram et al28 reported that high expression of human TRIM5α was associated with reduced susceptibility to HIV-1 infection. On the other hand, results from Richardson et al29 also confirmed that mode of HIV-1 transmission could affect the anti-HIV-1 activity of TRIM5α. Hence, we only recruited IDUs (1 single type of predisposition) to avoid sample heterogeneity in this study because HIV-1-infected IDUs was theoretically considered to be infected by the same mechanism. Meanwhile, China is a multiethnicity country, so we recruited individuals belonging to either Han or Dai ethnicity to see if there is a general cross-ethnic effect. We found that the 43Y/43Y genotype was less frequent among HIV-1-infected IDUs than in the seronegative (resistant) IDUs and obtained results which were robust to stratification analysis, therefore, the effect of HIV-1 resistant in 43Y homozygote is likely to be a general feature in Chinese IDUs. Compared with other 4 reports about the association between H43Y with HIV-1 infection or AIDS progression, only 2 studies enrolled participants infected by a single HIV-1 transmission method. For example, our results in this study was similar to Nakajima et al22 study in 94 HIV-1-infected Japanese hemophiliac patients; whereas van Mannen et al23 studied the effect of the TRIM5α H43Y and R136Q polymorphisms on the clinical of HIV-1 infection in participants of the Amsterdam Cohort studies, in which all participants are homosexual men, and observed an accelerated disease progression in the group of 43Y homozygote. These data are in keeping with an association between genetic polymorphisms in the human TRIM5α at codon 43 with HIV-1 infection or AIDS progression. However, we could not exclude the possibility that H43Y was not the primary causative SNP but was only associated with susceptibility due to LD with another genuine functional variant in other part of the gene, such as promoter or even in adjacent gene.
The two important functional domains of TRIM5α are B30.2/SPRY and RING. B30.2/SPRY domain of TRIM5α is essential for its anti-HIV-1 activity, Yap et al12 found a single amino acid substitution (R332P) in the human TRIM5α could confer the ability to restrict HIV-1.11-13 In our study, exon8 of TRIM5 were resequenced, and we observed few rare gene variations but frequencies of nonsynonymous substitutions were less than 0.01. Therefore, we could not determine the statistical significance of these variations in the B30.2/SPRY domain.
The RING domain, in which H43Y is located, has E3 ubiquitin ligase activity, but it is not absolutely required for retrovirus restriction.9,10 Disruption or deletion of the RING domain resulted in reduced activity against HIV-1.9 Anderson et al10 demonstrated that though blocking ubiquitination failed to prevent TRIM5α from restricting HIV-1 infection, proteasome inhibition rescued HIV-1 reverse transcriptase products from restriction, which implied TRIM5α might inhibit HIV-1 infection via a multistep mechanism. Because human TRIM5α is less potent at suppressing HIV-1 infection, the anti-HIV-1 activity of human TRIM5α will be strengthened through increasing E3 ubiquitin ligase activity. Residue 43 located in the “loop2” region of the RING domain, which may be involved in the interaction interface between E2 and E3 enzymes.17 Hence, altering positive charged residue (His) to uncharged residue (Tyr) is likely to enhance the interaction between E2 and E3 enzymes, which may enhance proteasomal degradation of HIV-1.
Previous in vitro transfection study showed that 43Y had a reduced viral restriction when 43Y was expressed in a stably transfected Crandel Feline Kidney (CRFK) cell as an exogenous expression plasmids. However, such effect was not found in endogenous expressed 43Y in human B cells.17 On the other hand, genetic association studies suggested a protective effect of 43Y.20,22 Such a paradoxical observation between transfection study and genetic association studies was also noted in a recent review.30 It is possible that in vitro expression study might not reveal the complete biological effect of TRIM5α molecule, and further studies are required to clarify this issue. However, TRIM5α need to form homo-oligomers to restrict HIV-1 infection, and Javanbakht et al9 showed that RING domain mutants (TRIM5α−HA C15A/C18A) exert minimal dominant-negative on anti-HIV-1 activity of wild-type TRIM5α, which indicates that mutants disrupting the RING domain might attenuate anti-HIV-1 activity of wild-type TRIM5α. TRIM5α is likely to form 43H/43Y heteroligomers in the IDUs with 43H/43Y heterzygosity, and this would lead to 43H, which might affect 43Y anti-HIV-1 ability, so that 43H/43Y heterzygosity has a weak or intermediate anti-HIV-1 ability compared with 43Y homozygosity, which may be the reason why 43Y is a protective allele in recessive genetic model.
In conclusion, our data confirm 43Y homozygosity reduce HIV-1 susceptibility in Chinese IDUs. In addition, we think the different route of HIV-1 transmission might influence the predisposition effect of host gene polymorphisms on the HIV-1 susceptibility. H43Y may alter the anti-HIV-1 activity of TRIM5α protein, as His of residue 43 is very conservative in primates.15,16,18 And it may also contribute to the species-specific HIV-1 restriction effect of TRIM5α protein.
The authors are grateful to the Yingjiang, Centers for Disease Control and Prevention; the Lincang, Centers for Disease Control and Prevention; the Zhaotong, Centers for Disease Control and Prevention; the Baoshan, Centers for Disease Control and Prevention; the Qujing, Centers for Disease Control and Prevention; and the Dehong, Centers for Disease Control and Prevention for providing the blood samples. We also thank the volunteers who participated in this research. We thank Ms Chen-Di Liao (Departments of Chemical Pathology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong) for helpful suggestion.
1. Johnson WE, Coffin JM. Constructing primate phylogenies from ancient retrovirus sequences. Proc Natl Acad Sci USA
2. Stremlau M, Owens CM, Perron MJ, et al. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature
3. Keckesova Z, Ylinen LM, Towers GJ. The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities. Proc Natl Acad Sci USA
4. Hatziioannou T, Perez-Caballero D, Yang A, et al. Retrovirus resistance factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proc Natl Acad Sci USA
5. Yap MW, Nisole S, Lynch C, et al. Trim5alpha protein restricts both HIV-1 and murine leukemia virus. Proc Natl Acad Sci USA
6. Nakayama EE, Miyoshi H, Nagai Y, et al. A specific region of 37 amino acid residues in the SPRY (B30.2) domain of African green monkey TRIM5alpha determines species-specific restriction of simian immunodeficiency virus SIVmac infection. J Virol
7. Langelier CR, Sandrin V, Eckert DM, et al. Biochemical characterization of a recombinant TRIM5alpha protein that restricts human immunodeficiency virus type 1 replication. J Virol
8. Takeuchi H, Matano T. Host factors involved in resistance to retroviral infection. Microbiol Immunol
9. Javanbakht H, Diaz-Griffero F, Stremlau M, et al. The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha. J Biol Chem
10. Anderson JL, Campbell EM, Wu X, et al. Proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse TRIM5 proteins. J Virol
11. Perez-Caballero D, Hatziioannou T, Yang A, et al. Human tripartite motif 5alpha domains responsible for retrovirus restriction activity and specificity. J Virol
12. Yap MW, Nisole S, Stoye JP. A single amino acid change in the SPRY domain of human Trim5alpha leads to HIV-1 restriction. Curr Biol
13. Stremlau M, Perron M, Lee M, et al. Specific recognition and accelerated uncoating of retroviral capsids by the TRIM5alpha restriction factor. Proc Natl Acad Sci USA
14. Sawyer SL, Wu LI, Emerman M, et al. Positive selection of primate TRIM5alpha identifies a critical species-specific retroviral restriction domain. Proc Natl Acad Sci USA
15. Song B, Gold B, O'Huigin C, et al. The B30.2(SPRY) domain of the retroviral restriction factor TRIM5alpha exhibits lineage-specific length and sequence variation in primates. J Virol
16. Liu HL, Wang YQ, Liao CH, et al. Adaptive evolution of primate TRIM5alpha, a gene restricting HIV-1 infection. Gene
17. Sawyer SL, Wu LI, Akey JM, et al. High-frequency persistence of an impaired allele of the retroviral defense gene TRIM5alpha in humans. Curr Biol
18. Goldschmidt V, Bleiber G, May M, et al. Role of common human TRIM5alpha variants in HIV-1 disease progression. Retrovirology
19. Speelmon EC, Livingston-Rosanoff D, Li SS, et al. Genetic association of the antiviral restriction factor TRIM5alpha with human immunodeficiency virus type 1 infection. J Virol
20. Javanbakht H, An P, Gold B, et al. Effects of human TRIM5alpha polymorphisms on antiretroviral function and susceptibility to human immunodeficiency virus infection. Virology
21. Nakayama EE, Carpentier W, Costagliola D, et al. Wild type and H43Y variant of human TRIM5alpha show similar anti-human immunodeficiency virus type 1 activity both in vivo and in vitro. Immunogenetics
22. Nakajima T, Nakayama EE, Kaur G, et al. Impact of novel TRIM5alpha variants, Gly110Arg and G176del, on the anti-HIV-1 activity and the susceptibility to HIV-1 infection. AIDS
23. van Manen D, Rits MA, Beugeling C, et al. The effect of Trim5 polymorphisms on the clinical course of HIV-1 infection. PLoS Pathog
24. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res
25. Mehta CR, Patel NR, Gray R. Computing an exact confidence-interval for the common odds ratio in several 2x2 contingency-tables. J Am Stat Assoc
26. Morrow WJ, Homsy J, Eichberg JW, et al. Long-term observation of baboons, rhesus monkeys, and chimpanzees inoculated with HIV and given periodic immunosuppressive treatment. AIDS Res Hum Retroviruses
27. Sawyer SL, Emerman M, Malik HS. Discordant evolution of the adjacent antiretroviral genes TRIM22 and TRIM5 in mammals. PloS Pathog
28. Sewram S, Singh R, Kormuth E, et al. Human TRIM5alpha expression levels and reduced susceptibility to HIV-1 infection. J Infect Dis
29. Richardson MW, Carroll RG, Stremlau M, et al. Mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus TRIM5alpha. J Virol
30. Nakayama EE, Shioda T. Anti-retroviral activity of TRIM5 alpha. Rev Med Virol