Short Communication: An Insertion of Seven Amino Acids in the Envelope Cytoplasmic Tail of HIV-2 Selected During Disease Progression Enhances Viral Replication

The envelope (Env) glycoproteins of primate lentiviruses play several key roles in the viral replication cycle. The Env proteins recognize and bind the cellular receptors and allow the fusion and virus entry processes in the early steps of infection. In the late stages of infection, Env glycoproteins interact with cellular and viral proteins to stabilize the matrix formation, and these proteins are incorporated into the envelope of budding virions. ¹ In human immunodeficiency viruses type 1 and 2 (HIV-1 and -2), Env glycoproteins are expressed from the env gene and consist of two subunits, the extracellular glycoprotein (gp120 or gp105) and the transmembrane glycoprotein (gp41 or gp36), noncovalently linked to constitute heterodimers. In turn, heterodimers form trimeric Env glycoprotein spikes as the functional and mature Env glycoprotein on the surface of virions and infected cells. ^1,2 The transmembrane glycoprotein is divided into three domains: an extracellular, a transmembrane, and an intracellular domain. The latter is also called the cytoplasmic tail (CT). Retroviral CTs diverge in size from 20 to 200 amino acids, and the CTs of HIV-1 and HIV-2 display approximatively a length variability of 160 amino acids. Immediately downstream the transmembrane domain, CT is structurally composed of an endocytic motif (YxxΦ), the Kennedy epitope, and three lentiviral lytic peptides (LLP-2, LLP-3, and LLP-1) involved in the induction of apoptosis in infected cells. ^{1 –3}

The CTs of HIVs are dispensable for binding and fusion activities but these domains are essential for antero- and retrograde trafficking, viral assembly, incorporation in newly synthesized virions, and conformational structure of Env glycoprotein. Interestingly, Env glycoprotein has also been reported to impact viral replication and release via, for instance, activation of NF-κB pathway and antagonism of BST-2/Tetherin in the case of HIV-2. ^4,5

HIV-2 infection is characterized by lower transmission rates, lower plasma viral loads, and better immune control compared with HIV-1 disease. In contrast to HIV-1 patients, the majority of HIV-2 patients do not progress to AIDS and 70%–80% are called long-term nonprogressors. ^6,7 In a previous study, our research group observed insertions of 3 or 7 amino acids (aa) in env sequences deduced from plasma viral RNA of several HIV-2-infected individuals. A cohort of 72 HIV-2 patients from Belgium and Luxembourg was established and among these, 23 patients were selected because of their detectable viral load (>100 copies/mL). Among these 23 progressors, the insertions of 7 (QRALTAI) or 3 (TAI) amino acids were observed in the Env CT from four different patients (17%). Noticeably, the patients infected with virus harboring the 7 aa insertion in the Env CT displayed the highest viral load of this cohort (>35,000 copies/mL). ⁸ The only exception was a patient infected with a virus harboring an insertion of 7 aa LRALTAT in Env CT and showing a low viral load (405 copies/mL). Patient characteristics and available clinical information are summarized in Table 1.

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Table 1.

Patient Characteristics and Clinical Information for the Four HIV-2 Patients Infected with Viruses Harboring an Insertion in the Env Cytoplasmic Tail

Patient	Insertion	Viral load (copies/mL)	CD4 count (cell/mL)	ART	Gender	Age
1	QRALTAI	515,000	100	Yes	M	33
2	QRALTAI	47,435	N.A.	No	F	40
3	QRALTAI	35,750	266	Yes	M	46
4	TAI	14,800	N.A.	Yes	M	10

ART, antiretroviral therapy; N.A., not available.

To confirm the impact of these insertions on viral replication in vitro, we reproduced the insertions by site-directed mutagenesis using a molecular clone of HIV-2_ROD encoding a complete envelope cytoplasmic domain (pKP59-HIV-2_ROD, ⁹ GenBank: M15390.1). We generated two different mutants of HIV-2_ROD: a clone containing the insertion L₇₉₁ TAI and another clone containing the insertion L₇₉₁ QRALTAI in the envelope CT (Fig. 1A). The sequences of primers used in inverse PCR were as follows: F Env 7aa 5′-cag aga gca cta aca gca atc aga gac tgg ctg aga ctt aga aca gcc ttc ttg-3′ or F Env 3aa 5′-aca gca atc aga gac tgg ctg aga ctt aga aca gcc ttc ttg caa tat ggg tgc-3′ (inserted nucleotides are in italic) and R Env: 5′-gag att ctg gta gat gag ttg gag ggt cag gaa gct cct gga tag-3′. Molecular clones were transfected in HEK293T cells, and the produced viral particles were purified by using a sucrose cushion 3 days post-transfection. We used our quantitative reverse transcription PCR (RT-qPCR) assay to quantify the number of viral RNA copies from a fraction of viral stocks following a protocol previously described. ⁹ H9 lymphocyte T cells were infected with wild-type HIV-2_ROD (WT), HIV-2_ROD Env L₇₉₁ TAI, or HIV-2_ROD Env L₇₉₁ QRALTAI viruses at a multiplicity of infection of three (MOI = 3, based on the number of viruses calculated by the RT-qPCR assay). After 3 h of infection, inocula were removed; infected cells were carefully washed three times with PBS and then incubated in complete RPMI culture medium. At 3 and 6 days postinfection, virus-containing supernatants were harvested and the number of viral copies released from infected cells was determined by RT-qPCR assay. We observed that viral replication and release ability of the HIV-2_ROD Env L₇₉₁ QRALTAI virus were enhanced by 8- to 11-fold compared with that of wild-type HIV-2_ROD virus (Fig. 1B). We also investigated the expression of viral proteins by western blot analysis to check the enhanced viral production in H9 cells. To this end, H9 cells were infected with the different viruses at a multiplicity of infection of three (MOI = 3) and Maraviroc and bicyclam AMD-3100 (a CCR5 and a CXCR4 competitive antagonist, respectively) were added the next day. Two days postinfection, cells were harvested and lysed. The cell lysates were clarified by centrifugation at 20,000 × g for 15 min at 4°C, and protein sample loading buffer was added. Samples were boiled, resolved by electrophoresis on 12% polyacrylamide gels, and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA in TBS supplemented with 0.1% of Tween-20 for 1 h and probed with a rabbit polyclonal anti-HIV-2 gp120 antibody (AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH), a mouse monoclonal anti-HIV-2 gag p26 antibody (USBiological), or a rabbit monoclonal GAPDH antibody to detect endogenous proteins (Cell Signaling Technology). As shown in Figure 1C, HIV-2 Env and p26 expression levels were increased in H9 cells infected with HIV-2_ROD Env L₇₉₁ QRALTAI viruses compared with HIV-2_ROD WT.

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FIG. 1.

Impact of 3 and 7 amino acid insertions in the HIV-2 envelope cytoplasmic tail on viral replication and release. (A) Schematic representation of the insertions (3 or 7 amino acids) in the HIV-2 envelope cytoplasmic tail. Site-directed mutagenesis using the HIV-2_ROD molecular clone (pKP59-HIV-2_ROD) was performed to introduce insertions of TAI or QRALTAI sequences in position 791 in the Env protein (MSD: membrane-spanning domain). (B) Absolute quantification of the number of viral particles released at 3 and 6 days postinfection. Amounts of wild-type HIV 2_ROD (WT), HIV-2_ROD Env L₇₉₁ TAI, or HIV-2_ROD Env L₇₉₁ QRALTAI viruses released from H9 cells were evaluated by using an RT-qPCR protocol. (C) Expression levels of viral proteins were investigated by western blot analysis. H9 cells were mock-infected or infected with HIV-2_ROD Env (WT), Env L₇₉₁ TAI, or Env L₇₉₁ QRALTAI viruses and were lysed 2 days postinfection. Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies to HIV-2 Env (gp105/gp140) or HIV-2 p26. Endogenous GAPDH proteins were detected as loading control. (D) Amino acid sequences of wild-type Rev and Rev with 7 aa insertion. This insertion (PESTNSN) is shown in italic. (E) Impact of the 7 aa insertion in HIV-2 Rev proteins on viral replication and release. HEK293T cells were transfected with full-length infectious molecular clones for HIV-2_ROD Δrev or HIV-2_ROD Δrev Env L₇₉₁ QRALTAI (500 ng) alone, or they were transcomplemented with plasmids encoding wild-type Rev or Rev with 7 aa insertion (100 ng). Differences in the amount of plasmid DNA in each transfection were offset by the addition of empty pcDNA3.1 vector. Virus yield determined by RT-qPCR assay 48 h post-transfection. Graph presents the number of viral copies on a logarithmic scale. Error bars indicate mean ± SD derived from four independent experiments for all data presented in this figure [n = 4; *p < .05, **p < .01, ns = nonsignificant; Kruskal–Wallis test for (B, E)]. RT-qPCR, quantitative reverse transcription PCR.

It is important to note that these insertions also modify the second exon of the HIV-2 rev gene. Since the Rev protein is implicated in viral RNAs exportation from the nucleus to the cytoplasm, the insertion of 7 aa (showing the most important effect on the HIV-2 replication) may conceivably enhance viral RNAs transport, thereby improving virus production. To test this hypothesis, we constructed molecular clones for HIV-2_ROD Δrev and HIV-2_ROD Δrev Env L₇₉₁ QRALTAI, and we obtained vectors encoding the synthetic gene of wild-type rev or rev with the corresponding 7 aa insertion (Fig. 1D; Eurofins Genomics, Germany). The different molecular clones for HIV-2_ROD Δrev were transfected alone in HEK293T cells or transcomplemented with plasmids encoding wild-type Rev or Rev with 7 aa insertion. Forty-eight hours later, accumulated viral particles in culture supernatants were quantified by RT-qPCR assay. Of note, HEK293T cells transfected with the molecular clone for HIV-2_ROD Δrev generated nearly no viral particles, given the absence of Rev proteins. Interestingly, the production of HIV-2_ROD Δrev (encoding wild-type Env protein) transcomplemented with Rev with the 7 aa insertion did not enhance viral production and release, whereas the production of HIV-2_ROD Δrev Env L₇₉₁ QRALTAI viruses along with the expression in trans of wild-type Rev boosted viral production and release (Fig. 1E). These results demonstrate that the enhanced viral production is only due to the modification in the HIV-2 Env sequence, and not in the Rev sequence. Further, these results corroborate previous observations that all the HIV-2 patients infected with HIV-2 virus harboring this 7 aa insertion QRALTAI in the Env CT exhibit a high viral load and disease progression. ⁸

Given that insertions are located in the putative Env LLP-2 region of HIV-2, we next sought to determine whether cell viability could be impacted after expression of the Env CT mutants. Indeed, it has been reported in HIV-1 that the LLPs, three conserved amphipathic α-helical segments in the Env CTs of HIVs, induce apoptosis or cytolysis of infected cells by disturbing cell membrane integrity. ^3,10 Therefore, at day 4 postinfection, we evaluated cell viability of the infected H9 T cells by using a CASY cell counter that was able to discriminate living cells from dead ones. Whereas 77% of cells infected with HIV-2_ROD WT virus were still living, only 55% of cells infected with HIV-2_ROD Env L₇₉₁ QRALTAI virus were living cells. In the case of HIV-2_ROD Env L₇₉₁ TAI, there were no significant differences regarding cell viability compared with the infection with wild-type HIV-2_ROD virus (Fig. 2A). Thus, the 7 aa insertion in the HIV-2 Env CT causes a higher mortality, possibly due to the presence of additional charged residues in the putative LLP-2 region or may be due to enhanced viral transmission followed by cell death.

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FIG. 2.

Impact of 3 and 7 amino acid insertions in the HIV-2 envelope cytoplasmic tail on cell viability, viral infectivity, Env incorporation, and NF-κB activation. (A) Cell viability of H9 cells infected with wild-type HIV-2_ROD (WT), HIV-2_ROD Env L₇₉₁ TAI, or HIV-2_ROD Env L₇₉₁ QRALTAI viruses at 4 days postinfection was monitored by using a CASY cell counter. (B) Virus infectivity capacities for the three different viruses were assessed by infecting TZM-bl reporter cells at a multiplicity of infection of three (MOI = 3), and luciferase assays were performed 28 h later. (C) Envelope incorporation per virion of the wild-type HIV-2_ROD Env (WT), Env L₇₉₁ TAI, or Env L₇₉₁ QRALTAI viruses was evaluated and compared by investigating the gp105/p26 ratios. (D) NF-κB activation by the HIV-2_ROD Env (WT), Env L₇₉₁ TAI, or Env L₇₉₁ QRALTAI proteins. Overall, 2.5 × 10⁵ HEK293T-NF-κB reporter cells were transfected with 100 ng of pcDNA3.1 (empty vector, used as a negative control), pcDNA3.1-HIV-2_ROD Env, pcDNA3.1-HIV-2_ROD Env L₇₉₁ TAI, or pcDNA3.1-HIV-2_ROD Env L₇₉₁ QRALTAI. Luciferase tests were performed 24 h post-transfection. As a positive control, we added TNFα (10 ng/mL) in the culture medium to validate this assay. Error bars indicate mean ± SD derived from four independent experiments for (A) and from three independent experiments for (B, D) [*p < .05, ANOVA test for (A, B, D)].

To better define mechanisms involved in the enhanced viral replication and release observed with the HIV-2_ROD Env L₇₉₁ QRALTAI virus, we hypothesized that the 7 aa insertion can promote fusogenicity and quantity of Env glycoproteins at the virion surface and, consequently, may favor binding and entry processes during early steps of infection. Therefore, we tested viral infectivity of HIV-2_ROD Env L₇₉₁ TAI and HIV-2_ROD Env L₇₉₁ QRALTAI viruses and we compared this ability with that of wild-type HIV-2_ROD virus. We used TZM-bl reporter cells to test viral infectivity of the three different viruses. These cells contain a luciferase reporter gene under the control of an HIV long terminal repeat (LTR) promoter and can be used to assess viral infectivity. TZM-bl cells were seeded in a 24-well plate and infected with wild-type HIV-2_ROD, HIV-2_ROD Env L₇₉₁ TAI, or HIV-2_ROD Env L₇₉₁ QRALTAI viruses at a multiplicity of infection of three (MOI = 3) during 3 h. After this step of infection, inocula were removed, adherent TZM-bl cells were gently washed three times with PBS, and fresh culture medium was added to the wells. To avoid multiple cycles of infection and test only viral infectivity capacity, we added Maraviroc and bicyclam AMD-3100 in the culture medium, and we performed luciferase reporter assays 28 h later. As presented in Figure 2B, viral infectivity of the three viruses remained equal, suggesting that the insertions did not promote infectivity. However, insertions in the Env CT may impede or promote the Env incorporation per virion, thereby explaining the improved viral fitness of the HIV-2_ROD Env L₇₉₁ QRALTAI viruses. To observe and compare the Env incorporation ratio of these different viruses, we performed a western blot of the infection-derived viral fraction. Of note, we used equivalent amounts of virus to complete the western blot analysis. No real differences were observed in Env incorporation ratios, although a slight decrease was seen with HIV-2_ROD Env L₇₉₁ QRALTAI viruses (Fig. 2C). As fusogenicity and Env incorporation may compensate each other, it could still be that a lower Env incorporation may balance a higher fusogenicity to maintain a similar viral infectivity.

Our results suggest that the improvement in viral replication induced by the insertion of 7 aa in Env CT is due to a postintegration mechanism. Indeed, Env glycoproteins may impact and promote viral replication. In respect to this, we recently reported an important role of HIV-2 Env in activation of the NF-κB signaling pathway and this effect can induce LTR-driven proviral genes transcription. ⁵ Therefore, it is tempting to speculate that insertions in Env CT could lead to a higher activation of NF-κB, resulting in a higher viral production in infected cells. We used our HEK293T reporter cell line stably expressing the luciferase gene under the control of NF-κB binding sequences to assess the NF-κB activation ability of the different Env glycoproteins. ⁵ We cloned wild-type HIV-2_ROD Env, Env L₇₉₁ TAI, or Env L₇₉₁ QRALTAI coding sequences into pcDNA3.1 expression vector and we transfected these plasmids in HEK293T-NF-κB cells. Twenty-four hours later, we performed a luciferase reporter assay, allowing us to evaluate the effect of these insertions on NF-κB activity. We observed no increased capability of the mutated Env glycoproteins in activation of NF-κB (Fig. 2D).

Overall, our in vitro results validate in vivo observations that the 7 aa insertion in HIV-2 Env CT induces enhanced viral replication and cell mortality but the mechanisms underpinning this viral fitness and phenotype remain to be defined. We believe that this study provides new insights into the role of the Env CT in the HIV-2 replication and pathogenesis.