Nef HIV-1 (Retroviridae: Orthoretrovirinae: Lentivirus: Human immunodeficiency virus-1), multifunctional protein: features of genetic virus variants circulating in Russia

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Introduction

The Nef protein of human immunodeficiency virus type 1 (HIV-1; Lentivirus, Orthoretrovirinae, Retroviridae) is an important factor in the progression of HIV infection: starting to be expressed in the earliest stages of infection, it creates optimal conditions for viral replication both inside the infected cell and outside of it, circulating in the blood plasma of HIV-infected patients [1, 2].

Nef is a 27–35 kDa protein consisting of approximately 206 amino acid residues (a.a.), N-terminally myristoylated, which allows it to bind to the cytosolic surface of cell membranes and perform most intracellular functions [1, 3, 4]. It has been shown that the three-dimensional structure of Nef, determined about 30 years ago based on the analysis of the NL4-3 variant of HIV-1 Nef using X-ray crystallography and nuclear magnetic resonance methods, contains: an N-terminal, a central, and a C-terminal region [1, 5–8]. The difficulty in studying the functionally significant domains of the Nef protein lies in the overlap of regions that interact with several cellular proteins; some of the most well-known motifs of the Nef protein are indicated in Fig. 1.

 

Fig. 1. Functional motifs of the Nef protein.

¹MGXXXS⁶ – myristoylated motif at the N-terminus, interaction with cell membranes [9]; length variable region – the region containing different amino acid insertions [10]; ⁵⁵CAWLEAQ⁶¹ – downregulation of CD4 [11] and a proteolytic cleavage site [12]; ⁶²EEEE⁶⁵ – interaction with AP-1, PACS-1/2, downregulation of MHC I, CCR5 and CXCR4 [1, 13]; ⁷²PxxPxR⁷⁷ – activation of Scr kinases (increased HIV production) [14] and downregulation of CCR5 and CXCR4 [13]; ⁹²KEKGG⁹⁶ – PPT LTR tract, where mutations of drug resistance to dolutegravir could be located [10, 15]; ¹²³D – is necessary for dimerization [16, 17]; ¹⁶⁰ExxxLL¹⁶⁵ – interaction with AP1 and AP2 (downregulation of MHC-I and CD4) [1, 13]; ¹⁷⁴DD¹⁷⁵ – downregulation of CD4 [13], ¹⁹¹F – interaction with PAK2, modification of cytoskeletal dynamics [18, 19].

Рис. 1. Функциональные мотивы белка Nef.

¹MGXXXS⁶ – миристоилированный мотив на N-конце, взаимодействие с клеточными мембранами [9]; length variable region – область белка с разными вариантами вставок аминокислот [10]; ⁵⁵CAWLEAQ⁶¹ – подавление экспрессии CD4 [11] и сайт протеолитического расщепления [12]; ⁶²EEEE⁶⁵ – взаимодействие с AP-1, PACS-1/2, снижение уровня экспрессии молекул МНС-Ι, CCR5 и CXCR4 [1, 13]; ⁷²PxxPxR⁷⁷ – активация Scr-киназ (усиление продукции ВИЧ) [14] и снижение экспрессии CCR5 и CXCR4 [13]; ⁹²KEKGG⁹⁶ – PPT LTR тракт, где могут находиться мутации лекарственной устойчивости к долутегравиру [10, 15]; ¹²³D – необходим для димеризации [16, 17]; ¹⁶⁰ExxxLL¹⁶⁵ – взаимодействие с AP1 и AP2 (снижение уровня экспрессии MHC-I и CD4) [1, 13]; ¹⁷⁴DD¹⁷⁵ – подавление экспрессии CD4 [13]; ¹⁹¹F – взаимодействие с PAK2, модификация динамики цитоскелета [18, 19].

 

The dimerization process is necessary for the proper functioning of the Nef protein. The studies conducted showed that viruses expressing Nef variants defective in their ability to dimerize were similar to viruses unable to express Nef [16].

Nef alters the composition of cell membrane proteins. HIV-infected cells through the modulation of the expression levels of these proteins. Thus, Nef enhances the surface expression of certain cytokines, such as TNF (tumor necrosis factor), which, on the one hand, can make cells more susceptible to apoptosis induction, and on the other hand, promotes the removal of TNF from cells, which, as mentioned earlier, mediates infection depletion [1, 20]. Simultaneously, Nef suppresses the surface expression of the following and many other molecules:

• CD4 receptors: inhibiting the expression of CD4 molecules prevents superinfection, i.e., the cell being infected by another strain of the virus, and also hinders the CD4-Env interaction within the producing cell, facilitating the release of virions from the surface of the cell membrane [1, 4, 13, 21];
• CCR5 and CXCR4 – HIV-1 co-receptors which also prevents superinfection [1, 13, 22];
• MHC-I (major histocompatibility complex class I molecules) и MHC-II (major histocompatibility complex class II molecules), preventing antigen presentation on the cell surface and the development of immune response mechanisms [4, 23, 24];
• CD8, which act as co-receptors during T-cell antigen recognition and can increase antigen recognition by almost 1 million times [25, 26];
• CD28, which initiate a costimulatory signal during the development of antigen-specific T-cell responses [27];
• SERINC 3/5, transmembrane cellular proteins that are incorporated into virions and, acting as cellular inhibitors of HIV-1 infectivity, limit viral replication [28, 29].

The mechanisms of suppression of CD4 and MHC-I molecule expression are the best studied. CD4 and MHC-I molecules are synthesized in the endoplasmic reticulum, transported to the Golgi apparatus, then reach the trans-Golgi network, from where they are transported to the plasma membrane. Nef activates clathrin-dependent endocytosis, which delivers CD4 and MHC-I molecules to endosomes and lysosomes for subsequent disposal. Clathrin-dependent endocytosis is mediated by adapter protein complexes, AP1 and AP2 [1, 30]. Nef binds to the cytoplasmic tail of the CD4 molecule located on the plasma membrane and attaches it to the AP2 complex. This results in the formation of a Nef:AP-2:CD4 triple complex, which activates clathrin-dependent endocytosis. Similarly, Nef induces the formation of a three-component Nef:MHC-I:AP-1 complex on the membrane of the trans-Golgi network [1]. Nef can also induce endocytosis of MHC-I molecules via an alternative pathway: the PACS-2 protein, which supports the functioning of the endoplasmic reticulum, directs Nef to the trans-Golgi network, where Nef interacts with a Src family kinase (SFK), after which a cascade of reactions is triggered, leading to Arf6-dependent endocytosis of MHC-I molecules [1, 31–33]. In early endosomes, Nef activates the AP1 complex through interaction with PACS-1, a sorting cell protein, and directs MHC-I molecules to the trans-Golgi network via the retrograde pathway (Fig. 2) [1, 34].

 

Fig. 2. Nef suppresses the expression of CD4 and MHC 1 molecules.

ER – endoplasmic reticulum; TGN – Trans-Golgi apparatus; PACS-2 – protein supporting endoplasmic reticulum function; SFK – Src-family kinase; PACS-1 – the sorting protein; AP1 and AP2 – adapter complexes mediating clathrin-dependent endocytosis; ARF6 –the factor mediating alternative pathway for endocytosis; clathrin – clathrin-dependent endocytosis.

Рис. 2. Nef подавляет экспрессию молекул CD4 и MHC1.

ER – эндоплазматический ретикулум; Golgi Apparatus – аппарат Гольджи; TGN – транс-отдел аппарата Гольджи; PACS-2 – клеточный белок, поддерживающий функционирование эндоплазматического ретикулума; SFK – киназа Src-семейства (Src- family kinase); PACS-1 – сортировочный клеточный белок; AP1 и AP2 – адаптерные комплексы, опосредующие клатрин-зависимый эндоцитоз; early endosome – ранние эндосоммы; lysosome – лизосома; ARF6 – фактор, опосредующий альтернативный путь эндоцитоза; clathrin – клатрин-зависимый эндоцитоз.

 

Fig. 3. Nef alters the dynamics of the cytoskeleton in the HIV-infected cells.

Membrane – cell membrane; Nef – Nef protein; PAK2 – cellular kinase.

Рис. 3. Nef изменяет динамику цитоскелета ВИЧ-инфицированной клетки.

Membrane – клеточная мембрана; Nef – белок Nef; PAK2 – клеточная киназа; actin – актин; cofilin – кофилин.

 

Nef alters cytoskeletal dynamics in infected cells: it causes disruption of cell polarity stabilization and reduced mobility of infected T cells in lymphoid tissue [35]. One end of Nef attaches to the lipid membrane and simultaneously forms a complex that includes several cellular proteins, including PAK2, a cellular kinase. Nef directs PAK2 to phosphorylate cofilin (a protein that controls the dynamics of actin filaments), which leads to the blocking of actin dynamics [18, 36].

Nef suppresses RNA interference – a protective mechanism of the cell that limits virus replication by suppressing gene expression using microRNA. Nef stimulates the proliferation of multivesicular bodies, which are the site of virus assembly in macrophages. It is believed that multivesicular bodies are also the site of assembly of the complex necessary for RNA interference. Thus, the multivesicular bodies contain Argonaut-2 and GW182 proteins, which are critical components of this complex. Nef binds to Argonaut-2 and disrupts the distribution of GW182 in exosomes, thereby suppressing RNA interference [37].

Nef inhibits immunoglobulin class switching. Switching immunoglobulin classes is necessary for the formation of immunity against viruses. In this process, stimulation of B cells by CD4⁺ cells plays a central role: the CD154 molecule located on the surface of CD4⁺ cells interacts with the CD40 receptor located on the surface of B cells, resulting in the production of pro-inflammatory cytokines. Nef induces the expression of CD154 inhibitors and signaling cytokines, preventing immunoglobulin class switching [38].

Nef plays an important role in the pathogenesis of HIV infection. Even with successful antiretroviral therapy (ART), Nef continues to be expressed and circulate in the blood plasma of HIV-infected patients. The exact mechanism by which Nef is released from HIV-infected cells into the extracellular space is still not fully understood. However, it has been shown that Nef is released as part of microvesicles, which are found in the plasma of HIV-infected patients in fairly high concentrations [2]. Internalization of exogenous Nef leads to the release of pro-inflammatory cytokines, the so-called “bystander effect.” Contact between immature dendritic cells and exogenous Nef causes the proliferation of neighboring CD4⁺ T lymphocytes, an increase in the formation of immunological synapses between immature dendritic cells and CD4⁺ T lymphocytes, and a disruption in the functions of CD8⁺ T lymphocytes [39]. Particular attention is paid to the neurotoxicity of the Nef protein and the role of Nef in the development of HIV-associated neurodegenerative diseases [40, 41].

Studies of the genetic variability of the Nef protein have revealed peculiarities in the structure of the Nef protein in patients with long-term non-progression of the disease. It has been noted that variations in protein sequences may be associated with disease stages, and variations in sequences associated with the development of HIV-associated neurodegenerative disorders and pulmonary hypertension have been identified [42–45]. In addition, mutations have been identified in the Nef protein that confer drug resistance of HIV-1 to antiretroviral drugs of the integrase inhibitor class, including the currently widely used drug dolutegravir (DTG) [15].

Nef and new approaches to HIV therapy. Given the special role of the Nef protein in enhancing HIV-1 replication and evading the host immune system, the Nef protein is being considered as a target for new approaches to HIV therapy: Nef protein antagonist molecules are being developed [46, 47], Nef is being studied as a candidate vaccine antigen [48], and designs for therapeutic vaccines are being tested based on it [49].

Since the first case of HIV infection was registered in Russia, and for many years thereafter, HIV-1 sub-subtype A6 has dominated in our country, but its prevalence has gradually declined from 94.9% in 2001–2002 to 61.4% in 2021–2022. HIV-1 subtype B ranked second in prevalence in our country: in 2002, it accounted for 2.3% of the total number of infections and gradually increased to 7.6% (in 2020); in 2023, the share of subtype B was 4.4%. At the same time, an increase in the prevalence of recombinant forms of HIV-1 was noted: CRF63_02A6 – from 0.4 to 23.9%, CRF02_AG – from 0.2 to 3.1%, while the circulation of the recombinant form CRF03_A6B remains stable [50]. The formation of new recombinant forms of HIV-1, CRF133_A6B and CRF157_A6C, and an increase in the proportion of circulating unique recombinants have also been recorded in Russia [51–53].

Previous studies analyzing the characteristics of the Nef protein in HIV-1 genetic variants circulating in Russia have identified characteristic substitutions for sub-subtype A6 [54]. A reference sequence of the Nef protein for HIV-1 variants of sub-subtype A6 was obtained, and the presence of Nef protein characteristics in variants of the virus sub-subtype A6 circulating in different regions of Russia was demonstrated [55]. It was also shown that nef variability depends on the genetic variant of HIV-1 and that in patients with more advanced stages of HIV infection, RNA splicing encoding nef is observed in cell nuclei, which is absent in the early stages of infection [56].

The aim of this study is to investigate the characteristics of the Nef protein of non-A6 genetic variants of HIV-1 characteristic of Russia, as well as to study the features of the Nef protein in HIV-1 variants of sub-subtype A6 circulating in the Moscow region, including a comparative analysis of genetic diversity in patients with different stages of the disease and an analysis of the presence of drug resistance mutations to DTG in the nef gene.

Materials and methods

Samples of peripheral blood mononuclear cells (n = 216) from individuals living with HIV (PLHIV) who had not previously received treatment were collected with the informed consent of the patients and analyzed. All patients were monitored at the Moscow Region State Healthcare Institution “Center for AIDS Prevention and Control” (hereinafter referred to as the AIDS Center). Blood samples were collected once between August 2019 and July 2020 as part of the CARE project (https://www.careresearch.eu/, accessed June 29, 2024). Bioethical approval for the study was obtained from the Biomedical Ethics Committee of the N.F. Gamaleya National Research Center for Epidemiology and Microbiology of the Ministry of Health of the Russian Federation (protocol No. 16 of February 8, 2019). Demographic, epidemiological, and clinical laboratory data were collected from patients. Patients were grouped according to the stage of HIV infection (Table 1) [57].

 

Table 1. Main characteristics of the studied cohort of HIV-infected patients

Таблица 1. Основные характеристики исследуемой когорты ВИЧ-инфицированных пациентов

Characteristics Характеристики	Stage II, stage of initial manifestations, n II стадия/стадия начальных проявлений, n	Stage III/Subclinical stage, n III стадия/субклиническая стадия, n	Stage IV/secondary manifestation stage, n IV стадия/стадия вторичных проявлений, n
Total patients Всего пациентов	32	85	99
Gender / Пол
М / M	21	43	70
F / Ж	11	42	29
Median age, years (range) Возраст (медиана лет, диапазон)	40 [19–58]	41 [18–70]	41 [24–64]
Infection route / Путь инфицирования
Hetero Гетеро	20	57	66
IDU ПИН	3	19	31
MSM МСМ	9	7	2
Nosocomial Нозокомиальный	0	0	0
Unknown Неизвестно	0	2	0
CD4, colonies/ml (range) CD4, кл/мкл	615.13 (108–1062)	427.44 (65–1658)	244.79 (8–1062)
Viral load lg RNA, copies/ml (range) Вирусная нагрузка lg РНК, копий/мл	5.1 (3.4–7.0)	4.7 (3.3–6.2)	5.1 (3.1–6.4)

Note. IDU – injecting drug users; MSM – men having sex with other men.

Примечание. ПИН – потребители инъекционных наркотиков; МСМ – мужчины, имеющие секс с мужчинами.

 

Proviral DNA was isolated from whole blood samples using a commercial HiPure Blood DNA Mini Kit (manufacturer: Magen Biotechnology, China) according to the manufacturer’s instructions. The resulting proviral DNA was then amplified by a two-round polymerase chain reaction (PCR). In the first round of PCR, the following primers were used: 5’-GTAGCTGGGTGGACAGATAGGGTTAT-3’ and 5’-GCACTCAAGGCAAGCTTTATTGAGGC-3’, and in the second round of PCR: 5’-ACATACCTAGGAGAATCAGACAGGGC-3’ and 5’-GCAGCATCTGAGGGTTAGC-3’. A PCR product of ~761 bp was obtained, with fragment coordinates in the HIV-1 genome 8749-9510 relative to the reference strain HXB2 (K03455). Sequencing of amplified DNA fragments was performed with second-round PCR primers, followed by detection on an ABI Prism 3130 automated genetic analyzer (Applied Biosystems, USA).

Preliminary determination of HIV-1 genetic variants was performed using specialized programs: COMET HIV-1, REGA HIV-1 Subtyping Tool (Version 3.46) and jpHMM, confirmation of genetic variants – with subsequent phylogenetic analysis using the maximum likelihood method according to the algorithm described earlier [58].

In the next stage of the study, consensus sequences of the Nef protein of HIV-1 sub-subtype A6 variants prevalent in the Moscow region and non-A6 genetic variants of HIV-1 characteristic of Russia were generated and analyzed: B, CRF02_AG, CRF63_02A6, as well as the new circulating recombinant form CRF133_A6B. The Nef-A6 consensus sequence was formed on the basis of the sequences obtained in the study, and for the other genetic variants from the international database of the Los Alamos Laboratory (Los Alamos database), USA (https://www.hiv.lanl, accessed on February 14, 2025).

The number of CRF03_A6B and CRF157_A6C variant sequences in the Los Alamos database did not allow for the reliable formation of consensus sequences; therefore, the analysis of Nef characteristics in these variants was not performed. The nucleotide sequences of the nef gene were divided into groups according to their genetic variant, then multiple alignment was performed within the groups and translated into amino acid sequences using the AlieView program [59]. If problematic regions were found in the sequences, additional alignment and editing were performed manually. For each group (i.e., each HIV-1 genetic variant), consensus sequences were generated based on the obtained amino acid sequences using the Simple Consensus Maker tool available at https://www.hiv.lanl.gov/content/sequence/CONSENSUS/SimpCon.html, accessed on 21.02.2025). When forming consensus sequences, deletions and insertions were taken into account only if they occurred in more than 50% of cases.

The consensus sequences obtained were aligned relative to the reference sequence NL4-3_Nef (HIV-1 subtype B, GenBank accession number AF324493.2) and compared with each other and with the reference sequence: amino acid sequences – in MEGA v. 10.2.2. (https://www.megasoftware.net/, accessed on 21.02.2025), predicted locations of unstructured regions – in the IsUnstruct program [60], predicted spatial structure using the AlphaFold 3 program (AlphaFold Protein Structure Database, https://alphafold.ebi.ac.uk/, accessed on 21.02.2025), predicted dimeric structures were superimposed on each other in Chimera (https://www.rbvi.ucsf.edu/chimera/).

When studying the genetic variability of Nef-A6 in patients with different stages of the disease, the conservatism and frequency of amino acid substitutions were compared. The conservatism analysis was performed similarly to the previously described algorithm [58]. Conservativeness was compared relative to the Nef-A6 consensus sequence. When comparing the frequency of amino acid substitutions, the Nef NL4-3 sequence was used as a reference, and the analysis was also performed according to the previously described algorithm [58]. Sites with statistically significant differences in frequency in patients with different stages of the disease were identified using the χ² criterion (p < 0.05) with subsequent additional correction – the introduction of the Bonferroni correction for multiplicity.

At the end of the study, the nucleotide sequences of HIV-1 variants circulating in Russia were analyzed: subtype A6, subtype B, CRF02_AG, CRF63_02A6, and CRF133_A6B, for the presence of drug resistance mutations to integrase inhibitors, including DTG: GCAGT sequences, positions 9068–9072 relative to strain NL4-3 (AF324493.2), in the G-track, at the 3’ end of the Nef PPT region [15].

Results

All HIV-1 nef gene nucleotide sequences (216) obtained in this study were deposited in the GenBank international genotype database under the following numbers: PV207057–PV207272. In this study, the numbers of clinical samples were indicated by a ten-digit number when presenting the results (e.g., 1311000491).

Genotyping of the obtained nucleotide sequences. Based on the results of the initial analysis of nucleotide sequences, it was determined that three samples (1.39%, 3/216) belonged to HIV-1 subtype B. Four samples (1.85%, 4/216) were identified as HIV-1 recombinant form CRF63_02A6. Eight samples (3.7%, 8/216) were identified as CRF02_AG. HIV-1 subtype A1 (1.39%, 3/216) and an undefined genetic variant (0.46%, 1/216) were also noted. The remaining 197 nucleotide sequences (91.2%) belonged to HIV-1 sub-subtype A6.

Overall, the results of the phylogenetic analysis confirmed the results of the preliminary subtyping. However, the sample previously identified as “A1 (check for 02_AG)” entered a reliable cluster formed by HIV-1 nucleotide sequences of the CRF02_AG recombinant form. Conversely, one sample identified as “CRF02_AG” entered the HIV-1 subtype A6 cluster (SH-aLRT = 0.97, Shimoda-Hasegawa approximate likelihood ratio criterion). The sample with an undefined genetic variant also belonged to the HIV-1 sub-subtype A6 cluster. One sequence, previously labeled as “A6” based on phylogenetic analysis, was identified as a potential unique recombinant form formed by fragments of viruses of genetic variants A6 and CRF63_02A6. Another sequence was identified as a unique URF A6/B recombinant based on preliminary genotyping and phylogenetic analysis (Fig. 4).

 

Fig. 4. Phylogenetic analysis of nucleotide sequences of the HIV-1 nef gene (n = 248, nucleotide substitution model – GTR + I + G4).

Reference sequences are highlighted in red, and sequences under study are highlighted in black. The asterisk indicates the URF sequences.

Рис. 4. Филогенетический анализ нуклеотидных последовательностей гена nef ВИЧ-1 (n = 248, модель замещения нуклеотидов – GTR + I + G4).

Референсные последовательности выделены красным цветом, исследуемые – черным. Звездочкой отмечена последовательность URF.

 

Thus, the final ratio (based on the results of preliminary subtyping and phylogenetic analysis) of HIV-1 Nef genetic variants was as follows: A6 – 90.74% (196/216), CRF02_AG – 4.17% (9/216), CRF63_02A6 – 1.85% (4/216), B – 1.39% (3/216), A1 – 0.93% (2/216), URF_A6/CRF63 – 0.46% (1/216), URF_A6/B – 0.46% (1/216).

Alignment of the obtained amino acid sequences relative to the reference sequence. When aligned to the NL4-3_Nef reference sequence, the following amino acid insertions were detected: 1311001075 – 33GVGAA34 (stage II, CRF02_AG), 1311000757 – 21RAPAPGAPAPAA22 (stage III, sub-subtype A6), 1311000491 – 7R8 (stage 2, subtype B), 1311000640 – 49AAAA50 (stage IV, sub-subtype A6), 1311000675 – 11GW12 (stage IV, sub-subtype A6), 1311000852 – 21RS22 (stage IV, sub-subtype A6), in 28 sequences – insertions of varying lengths (1–7 bp) between positions 28 and 29 bp (all stages of the disease, subtype A1, subtype A6, CRF02_AG), in 19 sequences – insertions (E or EE) at the position between the 66th and 67th bp (all stages of the disease, sub-subtype A6, CRF02_AG). Deletions were detected in 43 sequences (19.9%) obtained from patients with different stages of the disease and genetic variants of HIV-1, with no statistically significant differences between stages – the percentage of sequences containing deletions was approximately 20%. Most deletions were observed in positions up to 30 bp.

Formation of consensus sequences. To form consensus sequences from the Los Alamos database, the following were additionally downloaded: 46 sequences of subtype B, 2 of CRF02_AG, 30 of CRF63_02A6, and 8 of CRF133. At the same time, 9 sequences of subtype B downloaded from the Los Alamos database were excluded from further analysis due to the presence of long sections with deletions and premature stop codons. As a result, 77 non-A6 sequences from the Los Alamos database were included in the study, and consensus sequences were formed: for subtype A6 – based on 196 sequences obtained, for subtype B – 3 obtained and 37 downloaded from the Los Alamos database, for CRF02_AG – 9 obtained and 2 downloaded from the Los Alamos database; for CRF63_02A6 – 4 obtained and 30 downloaded from the Los Alamos database, for CRF133 – 8 downloaded from the Los Alamos database. The length of the consensus sequences of Nef-A6 and the recombinant form CRF133_A6B was 207 bp (due to the insertion of 24A25), Nef-B, Nef-CRF02_AG, and Nef-CRF63 – 206 bp. The formed consensus sequences are shown in Fig. 5, 24-25insA in the consensus sequence of A6 and CRF133_A6B is not shown in the figure.

 

Fig. 5. Consensus sequences of Nef HIV-1 sub-subtype A6, B, CRF63_02A6, CRF02_AG and CRF133_ A6B genetic variants aligned with the NL4-3_Nef (sequence of the Nef protein analyzed in determining the spatial structure [3–6]).

The dots indicate amino acid residues (a.a.r.) positions in which the a.a.r. in the consensus corresponded to the reference. Non-polar amino acids: G (glycine), A (alanine), V (valine), L (leucine), I (isoleucine), P (proline) – are marked in blue; Polar uncharged amino acids: S (serine), T (threonine), C (cysteine), M (methionine), N (asparagine), Q (glutamine) – green; aromatic amino acids: F (phenylalanine), Y (tyrosine), W (tryptophan), H (histidine) – yellow; Polar acidic, negatively charged, amino acids: D (aspartic acid) and E (glutamic acid) – orange; Polar basic, positively charged amino acids: K (lysine), R (arginine) – in red [61, 62].

Рис. 5. Консенсусные последовательности Nef ВИЧ-1 суб-субтипа А6, субтипа В и рекомбинантных форм CRF63_02A6, CRF02_AG и CRF133_A6B, выравненные относительно NL4-3_Nef (последовательность белка Nef анализируемого при определении пространственной структуры [3–6]).

Точками обозначены позиции а.о., в которых а.о. в консенсусах соответствовали референсу. Аминокислоты классифицированы на основе полярности радикалов. Неполярные аминокислоты: G (глицин), A (аланин), V (валин), L (лейцин), I (изолейцин), P (пролин) отмечены синим цветом; полярные незаряженные аминокислоты: S (серин), T (треонин), C (цистеин), M (метионин), N (аспарагин), Q (глутамин) – зеленым; ароматические аминокислоты: F (фенилаланин), Y (тирозин), W (триптофан), H (гистидин) – желтым; отрицательно заряженные аминокислоты: D (аспарагиновая кислота) и E (глутаминовая кислота) – оранжевым; положительно заряженные аминокислоты: K (лизин), R (аргинин) – красным [61, 62].

 

Comparative analysis of spatial structures of consensus sequences. Subsequently, to study spatial structures when analyzing the consensus sequence of subtype B, we analyzed the sequence variant containing D in position 54 and E in position 15; CRF02_AG – the sequence variant containing Q in position 15 and T in position 133; CRF63_02A6 – a sequence variant containing I at position 153; CRF133_A6B – a sequence variant containing I at position 173.

Fig. 6 shows the predicted profiles for unstructured unfolded regions without a clearly defined secondary structure for NL4-3, consensus sequences of sub-subtype A6, subtype B, CRF63_02A6, CRF02_AG, and CRF133_A6B.

 

Fig. 6. The comparison of profiles of unstructured areas in Nef consensus sequences of sub-subtype A6, subtype B, CRF63_02A6, CRF02_AG, CRF133_ A6B и NL4-3_ Nef, predicted by the IsUnstruct program.

A – NL4-3_Nef: unfolded regions from 1–75, 150–178, 200–206 a.a.r.; B – sub-subtype A6 consensus: unfolded regions from 1–76 and 150–164 и 174–176 и 193–207 a.a.r.; C – subtype B consensus: unfolded regions from 1–75, 147–176, 199–204 a.a.r.; D – CRF63_02A6 consensus: unfolded regions from 1–77, 197–206 a.a.r.; E – CRF02_AG consensus: unfolded regions from 1–76, 149–161, 172–175, 193–205 a.a.r.; F – CRF133_A6B consensus: unfolded regions from 1–77, 154–163, 196–207 a.a.r.

Рис. 6. Сравнение профилей неструктурированных участков Nef для консенсусных последовательностей суб-субтипа А6, субтипа В, CRF63_02A6, CRF02_AG, CRF133_A6B и NL4-3_Nef, предсказанные программой IsUnstruct.

A – NL4-3: развернутые участки c 1–75, 150–178, 200–206 а.о.; B – консенсус суб-субтипа А6: развернутые участки c 1–76 и 150–164 и 174–176 и 193–207; C – консенсус субтипа В: развернутые участки с 1–75, 147–176, 199–204 а.о.; D – консенсус CRF63_02A6: развернутые участки с 1–77, 197–206 а.о.; E – консенсус CRF02_AG: развернутые участки c 1–76, 149–161, 172–175, 193–205 а.о.; F – консенсус CRF133_A6B: развернутые участки c 1–77, 154–163, 196–207 а.о.

 

Figures 7 and 8 show the results of predicting the monomeric and dimeric spatial structures of the analyzed Nef protein sequences.

 

Fig. 7. Monomeric structures of Nef protein in NL4-3_Nef, sub-subtype A6, subtype B and CRF63_02A6, CRF02_AG, CRF133_A6B variants predicted by the AlphaFold 3 program.

A – monomeric structures of Nef protein, colored from N (blue) to C (red) end of the protein; B – monomeric structures of Nef protein predicted by AlphaFold 3 and colored according to IsUnstruct predictions for unstructured regions (see Fig. 6).

Рис. 7. Мономерные структуры белка Nef вариантов NL4-3_Nef, суб-субтипа А6, субтипа В, рекомбинантных форм CRF63_02A6, CRF02_AG, CRF133_A6B, предсказанные программой AlphaFold 3.

A – мономерные структуры белка Nef, покрашены от N (синий цвет) к C (красный цвет) концу белка; B – мономерные структуры белка Nef, предсказанные программой AlphaFold 3 и покрашенные согласно предсказаниям программы IsUnstruct для неструктурированных участков (см. рис. 6).

 

Fig. 8. Dimeric structures of Nef protein in NL4-3_Nef, sub-subtype A6, subtype B and CRF63_02A6, CRF02_AG, CRF133_A6B variants predicted by the AlphaFold 3 program.

A – structures are colored from N (blue) to C (red) end of Nef protein; B – dimeric structures of Nef protein, where one chain is colored green and the other is colored pink.

Рис. 8. Димерные структуры белка Nef вариантов NL4-3_Nef, суб-субтипа А6, субтипа В, рекомбинантных форм CRF63_02A6, CRF02_AG, CRF133 A6B, предсказанные программой AlphaFold 3.

A – структуры покрашены от N (синий цвет) к C (красный цвет) концу белка Nef; B – димерные структуры белка Nef, где одна цепь покрашена зеленым, а вторая – малиновым цветом.

 

It turned out that all predicted dimer structures are different for the Nef protein sequences under consideration. In subtype B, the dimer core is formed by 3 and 5 helices from two chains, resulting in a tetramer, a structure consisting of 4 helices, in the center. The N-terminal helix interacts with the globular part of the structure. In sub-subtype A6, dimers are formed by the interaction of β-regions, resulting in a more elongated structure where the first two helices interact with the second chain (swapped structures). In CRF02_AG, as in subtype B, the dimer is formed by the interaction of 3 and 5 helices. In CRF63_02A6, the dimer is formed by the interaction of the C-terminal parts and the 4th helix, while the N-terminal helix interacts with the second chain, resulting in another exchange of structural segments. For CRF133, the dimer is formed by the interaction of the C-terminal parts and 5 helices, with the N-terminal helix interacting with the second chain, again resulting in an exchange of structural segments. The dimer structure for NL4-3 is formed by the interaction of the C-terminal helices. Therefore, when superimposing dimer structures, we obtain a significant root mean square deviation for Cα atoms. The most similar (with the smallest root mean square deviation for Cα atoms, 21.5 Å) were the dimer structures for CRF02_AG and subtype B (Fig. 9), in which the dimer core is formed by the interaction of the 3rd and 5th helices from two chains.

 

Fig. 9. Pairwise superposition of all dimeric Nef structures studied in this study.

The standard deviation between Cα atoms for different dimer pairs is shown in the figure, which ranges from 21.5 Å to 42.6 Å.

Рис. 9. Попарное совмещение всех изучаемых димерных структур Nef.

Среднеквадратичное отклонение между Cα-атомами для разных пар димеров изменяется от 21.5 Å до 42.6 Å.

 

The results of pairwise superposition of predicted dimer structures are shown in Fig. 9 (“Å” is the unit of measurement for distances, angstrom; 1 Å = 0.1 nm).

Comparison of Nef-A6 conservatism in groups of patients with different stages of the disease. Consensus sequences of Nef-A6 in groups of patients with different stages (II – 29 samples, III – 78 samples, and IV – 89 samples) of the disease mainly contained amino acid residues similar to those in the general consensus sequence, but with different frequencies of detection (Fig. 10, Table 2).

 

Fig. 10. Conservatism of amino acid sequences in Nef-A6 in groups of patients with different stages of the disease.

Amino acids are designated by a general consensus with a one-letter code: A, alanine; C, cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine.

Рис. 10. Консервативность аминокислотных последовательностей белка Nef-A6 в группах пациентов с разными стадиями заболевания.

Аминокислоты в общем консенсусе обозначены однобуквенным кодом: А – аланин; С – цистеин; D – аспарагиновая кислота; Е – глутаминовая кислота; F – фенилаланин; G – глицин; H – гистидин; I – изолейцин; К – лизин; L – лейцин; М – метионин; N – аспарагин; P – пролин; Q – глутамин; R – аргинин; S – серин; Т – треонин; V – валин; W – триптофан; Y – тирозин.

 

Table 2. Distribution of conserved positions in the Nef-A6 protein in patients with different stages of the disease

Таблица 2. Распределение консервативности позиций в белке Nef-A6 у пациентов с разными стадиями заболевания

Conservatism (%) Консервативность (%)	Number of sites (n) Число сайтов (n)			p*
	Stage IV IV стадия	Stage III III стадия	Stage II II стадия	IV compared to III IV против III	III compared to II III против II	II compared to IV II против IV
100	115	79	35	0.0005**	< 0.0001**	< 0.0001**
90–99	35	77	114	< 0.0001**	0.0004**	< 0.0001**
76–89	32	27	29	0.5470	0.8858	0.7816
51–75	19	17	22	0.8618	0.5013	0.7424
≤ 50	5	6	6	1.0000	1.0000	1.0000

Note. * – indicates p-value for difference between groups is indicated (Fisher’s exact two-tailed test with Bonferroni multiple test correction, p = 0.0033); ** – p < 0.0033.

Примечание. * – указано значение p для разницы в показателе между группами (точный двусторонний тест Фишера с коррекцией множественного теста 

 

An assessment of the distribution of positions with varying degrees of conservatism in the Nef-A6 protein in patients with different stages of the disease showed that the number of sites with 100% conservatism increased significantly between patient groups: from stage II to stage III and from stage III to stage IV (Table 2).

Comparison of Nef-A6 genetic diversity in patients with different stages of the disease. When comparing the frequency of amino acid substitutions in Nef-A6 in patients with different stages of HIV infection, 21 amino acid substitutions were identified that had statistically significant differences (p < 0.05) in their frequency of occurrence (Table 3).

 

Table 3. Nef-A6 amino acid substitutions with statistically significant differences in frequency of occurrence in groups of HIV-infected patients with different stages of the disease

Таблица 3. Аминокислотные замены Nef-A6 со статистически значимыми различиями по частоте встречаемости в группах ВИЧ-инфицированных пациентов с разными стадиями заболевания

Position Позиция	Mutation Мутация	Stage II Стадия II	Stage III Стадия III	Stage I Стадия IV	pII–III	pII–IV	pIII–IV
9	S9K	3	1	1	0.028	0.017	–
11	I11–	2	0	4	0.019	–	–
14	P14T	0	0	8	–	–	0.007
38	E38D	29	75	77	–	0.037	0.03
39	K39X	0	0	5	–	–	0.034
50	A50D	3	0	1	0.004	0.017	–
81	Y81F	13	28	22	–	0.04	–
125	Q125H	2	3	0	–	0.012	–
133	V133I	14	29	48	–	–	0.03
146	V146E	1	5	0	–	–	0.015
148	V148I	2	0	2	0.019	–	–
151	D151S	3	0	7	0.004	–	0.011
155	E155A	2	9	2	–	–	0.016
155	E155K	6	5	9	0.031	–	–
158	K158G	2	0	2	0.019	–	–
169	S169C	29	77	81	–	–	0.028
176	P176E	23	73	72	0.031	–	0.016
176	P176D	5	2	12	0.006	–	0.011
178	R178K	7	25	41	–	0.037	–
182	E182R	7	20	7	–	0.019	0.002
197	E197D	2	0	1	0.019	–	–

Note. The p-values are presented for items with p < 0.05; items with p ≥ 0.05 are marked with ‘–’. Differences with p-value with Bonferroni correction (p < 0.0008) were considered to be significantly significant.

Примечание. Значения p представлены для позиций с p < 0,05; позиции с p ≥ 0,05 отмечены знаком «–». Достоверно значимыми считали различия с p с поправкой Бонферрони (p < 0,0008).

 

Taking into account the Bonferroni correction (p < 0.0008), no sites with statistically significant differences in frequency of occurrence in patients with different stages of the disease were identified.

Identification of drug resistance mutations to integrase inhibitors (DTG) in the nef gene. To analyze the presence of DTG drug resistance mutations in HIV-1 genetic variants circulating in Russia, we analyzed the same nucleotide sequences of the HIV-1 nef gene that were used to construct consensus amino acid sequences (see “Results,” formation of consensus sequences). A total of 289 nef gene sequences obtained from patients in Russia were analyzed: 196 for sub-subtype A6, 40 for subtype B, CRF02_AG – 11, CRF63_02A6 – 34, and CRF133_A6B – 8. No drug resistance mutations to DTG were found.

Discussion

The composition of HIV-1 genetic variants circulating in Russia is unique. It is known that its formation was influenced by the history of the virus’ entry and subsequent large-scale spread throughout our country. For decades, sub-subtype A6 has remained the dominant variant, with subtype B still being the second most common. However, recent studies indicate an increase in the genetic diversity of HIV-1 in our country: the emergence and spread of recombinant forms – CRF03_A6B, CRF02_AG, CRF133_A6B, and CRF157_A6C [51–53]. Previous studies have identified Nef protein features in HIV-1 sub-subtype A6 variants that may influence the replicative properties of the virus [54]. Foreign studies have found associations between amino acid substitutions in the Nef protein and stages of HIV infection in patients, and have also shown that the Nef protein may contain mutations conferring drug resistance to dolutegravir, an integrase inhibitor that is currently part of the first-line therapy for HIV infection in Russia [15, 43, 57]. This study focuses on investigating the possible influence of the characteristics of various genetic variants of HIV-1 circulating in Russia on the functional properties of the Nef protein, determining the presence/absence of drug resistance mutations to dolutegravir in virus variants circulating in Russia, and analyzing the genetic diversity of the Nef protein of the most widespread subtype A6 in Russia in patients with various stages of HIV infection.

With regard to NL4-3, the consensus sequence of subtype B contained the smallest number of substitutions—28 positions, including two deletions—which can be explained by the fact that the NL4-3 strain also belongs to subtype B. The consensus sequence of the A6 sub-subtype of HIV-1 variants circulating in the Moscow region contained 43 substitutions, including one insertion. The consensus sequences CRF63_02A6 and CRF133_A6B also contained a comparable number of substitutions relative to the reference sequence – 44 and 44 positions, including one insertion, respectively. This similarity can be explained by the fact that these variants are recombinant forms formed in Russia based on sub-subtype A6 (https://www.hiv.lanl.gov/components/sequence/HIV/crfdb/crfs.comp, accessed on 22 August 2025). The largest number of substitutions was found in the consensus sequence of the recombinant form CRF02_AG – in 52 positions, including 1 deletion. At the same time, the recombination point of subtypes A and G in the recombinant form CRF02_AG is located in the nef gene region (https://www.hiv.lanl.gov/ components/sequence/HIV/crfdb/crfs.comp, accessed on 22.08.2025), and therefore the presence of this type of difference is probably associated with the characteristics of subtype G. In a functionally significant position, sub-subtype A6 and CRF133_A6B contained the F191L substitution, which led to a change in the chemical properties of amino acids at a given position, which may affect the interaction of the Nef protein with the cellular protein p21-activated kinase 2, and, as a result, on the function of modulating the dynamics of the infected cell’s cytoskeleton [18, 19]. The insertion detected in sub-subtype A6 and CRF133_A6B, 24-25insA, as well as the deletion CRF02_AG in position 24 are located near a region of high variability in the Nef protein and, therefore, are unlikely to affect the functional properties of the protein [10]. The possible functional significance of deletions in subtype B at positions 10 and 11, according to the available literature, is not yet clear.

When comparing the monomeric and dimeric structures of the sequences studied, a difference in mutual orientation can be noted. Thus, dimers A6, B, CRF63_02A6, and CRF133_A6B have a more closed shape, while dimers NL4-3 and CRF02_AG have an open shape.

By comparing the dimer forms, it was determined that the largest root mean square deviation between Cα atoms was found for the consensus sequences CRF02_AG and CRF133_A6B (42.6 Å), and the smallest for the consensus sequences B and CRF02_AG (21.5 Å). When comparing the dimer forms of the consensus sequences and the reference sequence NL4-3, the smallest root mean square deviation was detected for CRF02_AG (25.7 Å), and the largest for CRF63_02A6 (40.8 Å). Thus, the existing features of the amino acid sequences of the Nef protein of different HIV-1 variants may influence the formation of dimeric forms and, given the importance of the Nef protein dimerization process, potentially influence the functional properties.

A comparison of the genetic diversity of the Nef-A6 protein in patients with different stages of HIV infection did not reveal any amino acid substitutions with a statistically significant difference, which is inconsistent with data previously obtained for subtype B [43], but identified a statistically significant sequential increase in the conservatism of the Nef protein between groups of patients with stages II, III, and IV of the disease. Taking into account our results and the results of previous studies, including those that showed that in more advanced stages of the disease, there are 120 fewer fragments of the nef gene in the nuclear mRNA fraction than in earlier stages of the disease [56], it can be assumed that for further study of Nef protein variability in patients with different stages of the disease, it would be advisable to compare the products of nef gene expression.

In HIV-1 variants circulating in Russia, no drug resistance mutations to integrase inhibitors were found at the 3’ end of the PPT region in the Nef protein, which indicates that there is no risk in using drugs of this class in first-line therapy.

A limitation of this study is the absence of HIV-1 CRF03_A6B and CRF157_A6C variants in the analysis, as well as the small number of CRF02_AG and CRF133_A6B sequences.

Conclusion

For the first time, a comparison of the genetic diversity of the Nef protein variants of HIV-1 circulating in Russia was conducted. The results revealed the characteristics of the Nef protein in circulating virus variants and identified differences that could potentially affect the functional properties of the protein and could be taken into account in the future when developing Nef protein-based therapies. The conservatism of Nef-A6 was significantly higher in groups of patients with later stages of the disease. To compare Nef variability in patients with different stages of the disease in the future, it would probably be advisable to study the products of nef expression. No mutations conferring drug resistance to the first-line ART drug in Russia, dolutegravir, were detected in nef in HIV-1 of different genetic variants circulating in Russia, which indicates that there are no risks associated with the use of modern treatment protocols.

About the authors

Anna I. Kuznetsova

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: a-myznikova@list.ru
ORCID iD: 0000-0001-5299-3081

head of laboratory of T-lymphotropic viruses, PhD, leading researcher

Russian Federation, 123098, Moscow

Anastasiia A. Antonova

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: anastaseika95@mail.ru
ORCID iD: 0000-0002-9180-9846

PhD, Researcher, Laboratory of T-lymphotropic viruses

Russian Federation, 123098, Moscow

Larisa A. Protasova

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: larisa.protasova.03@mail.ru
ORCID iD: 0009-0001-0430-1578

research assistant, Laboratory of T-lymphotropic viruses

Russian Federation, 123098, Moscow

Anna V. Glyakina

Institute of Mathematical Problems of Biology RAS – the Branch of Keldysh Institute of Applied Mathematics of Russian Academy of Sciences

Email: quark777a@gmail.com
ORCID iD: 0000-0002-6352-2880

PhD, Researcher

Russian Federation, 142290, Pushchino

Kristina V. Kim

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: kimsya99@gmail.com
ORCID iD: 0000-0002-4150-2280

junior researcher, Laboratory of T-lymphotropic viruses

Russian Federation, 123098, Moscow

Iana M. Munchak

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: yana_munchak@mail.ru
ORCID iD: 0000-0002-4792-8928

junior researcher, Laboratory of T-lymphotropic viruses

Russian Federation, 123098, Moscow

Ekaterina N. Mezhenskaya

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: belokopytova.01@mail.ru
ORCID iD: 0000-0002-3110-0843

PhD, Researcher, Laboratory of T-lymphotropic viruses

Russian Federation, 123098, Moscow

Elena A. Orlova-Morozova

Center for the Prevention and Control of AIDS and Infectious Diseases

Email: orlovamorozova@gmail.com
ORCID iD: 0000-0003-2495-6501

PhD, Head of outpatient department

Russian Federation, 140053, Kotelniki

Alexander Yu. Pronin

Center for the Prevention and Control of AIDS and Infectious Diseases

Email: alexanderp909@gmail.com
ORCID iD: 0000-0001-9268-4929

PhD, Chief Physician

Russian Federation, 140053, Kotelniki

Alexey G. Prilipov

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya

Email: a_prilipov@mail.ru
ORCID iD: 0000-0001-8755-1419

Doctor of Biological Sciences, leading researcher, head of the laboratory of molecular genetics

Russian Federation, 123098, Moscow

Oxana V. Galzitskaya

D.I. Ivanovsky Institute of Virology of National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya; Institute of Theoretical and Experimental Biophysics RAS

Author for correspondence.
Email: ogalzit@vega.protres.ru
ORCID iD: 0000-0002-3962-1520

Doctor of Physical and Mathematical Sciences, Head of the Bioinformatics Laboratory, Chief Researcher

Russian Federation, 123098, Moscow; 142290, Pushchino

References

Supplementary files