Analysis of Human Immunodeficiency Virus Type 1 Vif Gene Sequences Among Men Who Have Sex with Men in Heilongjiang Province of China

T he prevalence of AIDS has been continually increasing in Heilongjiang province since the first case was detected in 1993. According to a cross-sectional study conducted in four cities of Heilongjiang province, the prevalence of HIV infection among men who have sex with men (MSM) was 2.3% in 2008. ¹ Another five consecutive cross-sectional surveys conducted in Harbin (capital of Heilongjiang province) have demonstrated that the prevalence of HIV infection has increased from 1.0% in 2006 to 7.5% in 2010. ²

As is well known, human immunodeficiency virus, the agent responsible for AIDS disease, belongs to the retroviridae family, which produces structural proteins such as Gag, Pol, and Env; regulatory proteins such as Tat, Rev, and Nef; and auxiliary proteins such as Vif (Virion infectivity factor), Vpr, and Vpu. HIV-1 Vif is a 23-kDa protein and is localized in the cytoplasm of host cells. It is expressed late in the viral cycle and has essential functions for replication and infectivity. It is known that Vif protein neutralizes the antiviral activity of the cytidine deaminase family of agents, such as APOBEC3F and APOBEC3G, by recruiting the Cullin5–ElonginB–ElonginC E3 ubiquitin ligases. ³ Vif-deleted (ΔVif) mutants are 100–1,000 times less infective than the wild-type viruses in vitro. ⁴ HIV has a well-documented characteristic of sequence variability. Different areas and populations have a diversity of epidemic HIV subtypes. However, the detailed subtype characterization and genetic variation of HIV circulating among MSM in Heilongjiang remain unknown. Therefore, we analyzed the characteristics of epidemic subtypes of HIV-1 and their variation in Heilongjiang province. This study also reveals the general biochemical properties of Vif protein and its interactions with host cellular and viral partners from a structural point of view.

In the present study, we isolated and sequenced the Vif gene of HIV-1 strains from drug-naive seropositive MSM residing in Heilongjiang province, China, in 2011. Viral DNA was extracted from the anticoagulant peripheral blood samples collected from 17 MSM newly diagnosed with HIV-1 infection, whose age ranged from 22 to 55 years. At the same time, the CD4⁺ T cell counts were determined when the patients were diagnosed for the first time, which ranged from 49 to 454 cells/μl, and 58.82% of them had >200 cells/μl.

Full-length Vif sequences were amplified by two rounds of nested primer polymerase chain reaction (PCR) to obtain sufficient amounts of DNA for sequencing. Two sets of primers were designed: the outer primers were 5'-CGGGTTTATTACAGGGAC-3', 5'-GCTATGTCGACACCCAATTCTG-3', 5'-CAAAATTTTCGGGTTTATTACAGGGAC-3', and 5'-CTGCTATGTTGACACCCAATTCTG-3', and the inner primers were 5'-GGAAAGGTGAAGGGGCAGTA-3', 5'-CCCAAGTATCCCCATAAGTTTC-3', 5'-GGAAAGGTGAAGGGGCAGTAG-3', and 5'-TCTTAAGCTCCTCTAAAAGCTCTA GTG-3'. The first amplification round was performed at 94°C for 5 min with initial denaturation, followed by 25 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, and a final extension of 10 min at 72°C. The PCR product from the first amplification round was diluted 2.5-fold and subjected to the second round of PCR amplification with inner primers using the same conditions as in the first PCR.

Nucleotide sequences were aligned by using CLUSTAL W (included in Mega 5.05 software), and the alignments were edited manually and amino acid sequences were deduced by BioEdit (version 7.00). Genetic distances were computed by the Kimura two-parameter method including both transitions and transversions, and phylogenetic analyses were performed with the neighbor-joining method as implemented by Mega 5.05 software with reference sequences obtained from the Los Alamos HIV sequence database. The reliability of the topologies was estimated by performing bootstrap analysis with 1,000 replicates.

The sequences analyzed encompassed the entire coding region of the Vif gene. Phylogenetic analysis demonstrated that HIV-1 subtypes were as follows: CRF01_AE (23.53%), CRF07_BC (47.06%), subtype B (17.65%), and subtype B′ (Thailand variant of subtype B) (11.76%). Subtypes of HIV-1 demonstrated diversity among MSM in Heilongjiang province, China (Fig. 1). More strikingly, CRF07_BC had become dominant among MSM in this locality, with distinctive characteristics from subtypes in MSM from other areas in China, ^{5 –7} and from local major epidemic strains (subtype B′) in the general population. ⁸ A larger mean genetic distance was found among subtype B (10.39±1.12%); a similar mean genetic distance between CRF01_AE (5.58±0.74%) and subtype B′ (5.57±0.99%); and a smaller mean genetic distance in CRF07_BC (3.06±0.45%).

OPEN IN VIEWER

FIG. 1.

Phylogenetic analysis of full-length Vif sequences in HIV-1 strains from men who have with men (MSM) in Heilongjiang province. The bootstrap probability (>70%, 1,000 replicates) is indicated at the corresponding nodes of the tree. The scale bar indicates the evolutionary distance of 1% (0.01 substitutions per site). The reference sequences of subtypes A–D, F–H, J, K, CRF01_AE, CRF07_BC, and CRF08_BC were obtained from the HIV database (www.hiv.lanl.gov/). The black solid circles represent the Vif sequences analyzed in this study. The subtypes of the Vif genes are indicated on the right.

According to the mutation rate of 0.5%–1% for HIV-1 each year, we estimated that subtype B may have been prevalent among MSM in Heilongjiang province for 18–23 years, CRF07_ BC for 5–8 years, and subtypes B′ and CRF01_AE for 9–14 years. Thus, subtype B has had the longest epidemic time of all discovered subtypes of HIV-1 among MSM in Heilongjiang province. To explore further the relationship between our strains and those from other high-risk populations in different areas of China, we constructed another dendrogram with our sequences and reference sequences from MSM (Hebei province), and other populations such as intravenous drug users (IDUs), heterosexual people, commercial blood donors, and those with mother-to-fetus infection from different areas (including Henan, Xinjiang, Guangxi, Fujian, Sichuan, and Hubei), available at the Los Alamos HIV sequence database (Fig. 2).

OPEN IN VIEWER

FIG. 2.

Evolutionary relationship among HIV-1 sequences from MSM in Heilongjiang province (●) and other areas in China. Phylogenetic trees were constructed with full-length Vif genes. The reference sequences were downloaded from the Los Alamos database (www.hiv.lanl.gov/). Each reference sequence is labeled with the HIV-1 subtype, followed by country of isolation, isolation time, and sequence name. The bootstrap probability (>70%, 1,000 replicates) was also indicated at the corresponding nodes of the tree. Reference strains from different areas of China, including Hubei (▽), Hebei (■), Henan (△), Guangxi (▼), Sichuan (◯), Fujian (◊), and Xinjiang (□), are labeled with different symbols.

As indicated in the phylogenetic tree, most of our strains (10/17; black dots) clustered together with those from Hebei province (black squares), which is geographically close to Heilongjiang. However, a few strains clustered with the strains from different populations in geographically distant areas (i.e., Henan, Xinjiang, Fujian, Guangxi, Sichuan, and Hubei). This suggests that MSM in Heilongjiang province had multiple introductions of HIV and there were frequent sexual communications among MSM populations within the geographically close regions by certain HIV-carrying migrant MSM.

The nucleotide sequences of Vif described in the present study were translated and the corresponding putative products were aligned and compared with a reference sequence HXB2 available at the Los Alamos HIV sequence database, to establish whether there were some differences from our sequences. As shown in Fig. 3, the N-terminal tryptophan-rich stretch (residues 1–21, including W5, 11, and 21) of Vif in our sequences was well conserved. Different highly conserved tryptophan residues are required for efficient recognition and suppression of both APOBEC3G and APOBEC3F. Among the conserved tryptophan residues located at the N-terminus of Vif: Trp 11 and 79 were critical for interaction with APOBEC3F, while other residues including Trp 5, 21, 38, and 89 were important for Vif activity against APOBEC3G. The Trp 5, 11, 21, 38, 79, and 89 were all highly conserved in our sequences. The D¹⁴RMR¹⁷ and Y⁴⁰RHHY⁴⁴ motifs of Vif are essential for binding to APOBEC3F and APOBEC3G, respectively, and inducing their proteasomal degradation. ³ They were well conserved in our sequences, except that R17 was substituted by K in some sequences. Y40 and R41 were substituted by X and K, respectively, in HLJHM03 and HLJHM17. The first 64 residues in the N-terminal region were shown to have the strongest affinity with RNA binding and formed a 40S messenger ribonucleoprotein complex in the virus-producing cells.

OPEN IN VIEWER

FIG. 3.

Deduced amino acid residues of Vif sequences derived from HIV-1 strains from MSM in Heilongjiang province versus the reference sequences (HXB2). The deduced amino acids are shown by the single-letter code. Defined functional domains and regions are indicated on the consensus sequence and analyzed references by open boxes. The dots indicate amino acid identity and wavy lines correspond to gaps introduced to maintain the correct alignment. Scale plates at the top of figures indicate the position of each amino acid residue in the sequences. The asterisk indicates the stop codon in the Vif coding sequence.

The RNA-binding domain plays an important role in Vif function in the viral life cycle. In vitro binding assays have demonstrated that mutations of Vif at W11, Y30, and Y40 significantly decrease RNA-binding activity. ^3,9 Amino acids 63–70 and 86–89 have been predicted to be important for the formation of β-strand structures that are critical for maintaining a normal expression level of Vif and for viral infectivity. ³ The highly conserved glutamic acid at position 88 and the tryptophan at position 89 are located within the charged central hydrophilic region E⁸⁸ WRKKR⁹³ that is thought to enhance steady-state expression of Vif in host cells. ³ The H-x5-C-x17–18-C-x3–5-H motif (residues 108–139) consists of two conserved His/Cys pairs and is predicted to form a helix that contains a cluster of hydrophobic conserved residues. This HCCH motif forms a structural domain that has the potential to bind the Zn²⁺ ion through the universally conserved residues H108, C114, C133, and H139, and to bind Cullin5 selectively, which is distinct from other recognized classes of zinc-binding motif and is required for assembly of the Vif–Cul5–E3 ubiquitin ligase to degrade APOBEC3G proteins. A highly conserved stretch of hydrophobic residues F115-X4-I/V120-X2-A123-I/L124 within the Vif zinc-binding domain is required for the interaction with Cullin5.

Mutations of conserved hydrophobic residues (Ile-120, Ala-123, and Leu-124) located between the two Cys residues in the HCCH motif disrupt binding of the zinc-coordinating region to Cullin5 and inhibit APOBEC3G degradation. ^10,11 In addition, there is an asparagine insertion between residues 140 and 141 among HLJHM3–5, 7, 9, 11, 13, and 16 (not shown in Fig. 3), which is also found in some Chinese reference sequences available at the Los Alamos HIV sequence database. Whether the asparagine insertion only occurred in Chinese strains and how it may affect the properties of the Vif protein need further research. The highly conserved ¹⁴⁴SLQYLA¹⁴⁹ motif in our sequences defines the so-called BC-box motif and is crucial for inactivation of APOBEC3 proteins. This BC-box motif is responsible for the binding to ElonginC, which further targets APOBEC3 antiviral factors to the proteasome.

The amino acids 145–155 are predicted to form an α-helix, which is predicted to be fitted into the hydrophobic pocket of ElonginC. ^3,12 Vif neutralizes the antiviral activity of APOBEC3G and APOBEC3F predominantly by forming an E3 ubiquitin ligase with Cullin5, ElonginB, and ElonginC, and targets these proteins for degradation by the ubiquitin–proteasome pathway. Vif can bind directly to ElonginC via its BC-box motif and to Cullin5 via hydrophobic residues within a zinc-binding region formed by a conserved HCCH motif. ³ The conserved ¹⁶¹PPLP¹⁶⁴ domain of the Vif protein plays a key role in multimerization (Vif–Vif interaction). The deletion of these proline-enriched peptides of the Vif protein significantly impairs the ability of Vif proteins to interact with each other.

In HIV-1, Vif multimerization is crucial for viral infectivity and for preventing APOBEC3G incorporation into virions. ^3,13 Amino acids 85–99 and 169–192 have also been shown to interact strongly to mediate Vif–APOBEC3G binding. ³ In addition, some phosphorylation sites were identified: T96, S144, T155, and T188. T96 and S144 were highly conserved in our sequences. Mutations of T96 and/or S144 resulted in significant loss of Vif activity and inhibition of HIV-1 replication, suggesting that phosphorylation at this site plays an important role in regulating HIV-1 replication and infectivity. ³

MSM are vulnerable to HIV infection because of their unprotected anal intercourse and multiple sexual partners. They play a bridging role in the spread of HIV from high-risk groups to the general population. Genetic analysis based on HIV DNA sequences is an effective way to explore the relationship between HIV strains from different areas or populations. Our study shows a close relationship between our HIV strains and those from the MSM population in Hebei, which is geographically close to Heilongjiang province, instead of different populations from other geographically distant areas. Therefore, we hypothesized that the intrapopulation transmission of HIV among MSM was more frequent in a confined region. However, because of the lack of Vif sequences from other local high-risk populations in Heibei and from MSM in distant regions for comparison, we do not know whether there was also frequent sexual communication between MSM from the different regions. In this regard, Li et al. ⁶ have also found that HIV strains can be transmitted among the same MSM population from adjacent regions instead of among different populations in the locality, which confirms our hypothesis.

CRF07_BC has been reported to be distributed widely among IDUs, ¹⁴ while CRF01_AE is prevalent among heterosexual people in China. ¹⁵ High subtype diversity demonstrates that HIV in the MSM population in Heilongjiang province may have resulted from multiple introductions. MSM in Heilongjiang province may have various risk behaviors such as taking intravenous drugs or heterosexual activity. However, we cannot rule out the possibility of HIV-1 transmission from IDUs to MSM via heterosexual transmission (from female IDUs to MSM) or homosexual intercourse (from male IDUs to MSM); to determine this requires further epidemiological studies. Generally, most functional domains of the Vif protein were well conserved in our sequences.

Elucidating the distribution of the Vif gene subtype and the corresponding structure of the Vif protein will help us better understand the characteristics of the HIV epidemic and its interaction with host cellular partners, and allow us to develop an effective preventive and therapeutic strategy against HIV-1 infection by blocking the epidemiological or biological pathway of HIV.

Nucleotide Sequence Accession Numbers

The nucleotide sequences that we obtained for the Vif genes from each patient have been submitted to GenBank with accession numbers JX999596–JX999612.