The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity.

Jawahar Raina
The V3 Loop of HIV-1 Env Determines Viral Susceptibility to IFITM3 Impairment of Viral Infectivity.
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Interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including HIV-1. IFITM proteins deter HIV-1 entry when expressed in target cells and also impair HIV-1 infectivity when expressed in virus producer cells. However, little is known about how viruses resist IFITM inhibition. In this study, we have investigated the susceptibilities of different primary isolates of HIV-1 to the inhibition of viral infectivity by IFITMs. Our results demonstrate that the infectivity of different HIV-1 primary isolates, including transmitted founder viruses, is diminished by IFITM3 to various levels, with strain AD8-1 exhibiting strong resistance. Further mutagenesis studies revealed that HIV-1 Env, and the V3 loop sequence in particular, determines the extent of inhibition of viral infectivity by IFITM3. IFITM3-sensitive Env proteins are also more susceptible to neutralization by soluble CD4 or the 17b antibody than are IFITM3-resistant Env proteins. Together, data from our study suggest that the propensity of HIV-1 Env to sample CD4-bound-like conformations modulates viral sensitivity to IFITM3 inhibition.

IMPORTANCE Results of our study have revealed the key features of the HIV-1 envelope protein that are associated with viral resistance to the IFITM3 protein. IFITM proteins are important effectors in interferon-mediated antiviral defense. A variety of viruses are inhibited by IFITMs at the virus entry step. Although it is known that envelope proteins of several different viruses resist IFITM inhibition, the detailed mechanisms are not fully understood. Taking advantage of the fact that envelope proteins of different HIV-1 strains exhibit different degrees of resistance to IFITM3 and that these HIV-1 envelope proteins share the same domain structure and similar sequences, we performed mutagenesis studies and determined the key role of the V3 loop in this viral resistance phenotype. We were also able to associate viral resistance to IFITM3 inhibition with the susceptibility of HIV-1 to inhibition by soluble CD4 and the 17b antibody that recognizes CD4-binding-induced epitopes.

KEYWORDS: IFITM, envelope protein, human immunodeficiency virus

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INTRODUCTION

The interferon-inducible transmembrane proteins (IFITMs) inhibit a broad spectrum of viruses, including influenza viruses, West Nile virus, dengue virus, yellow fever virus, Marburg virus, Ebola virus, severe acute respiratory syndrome (SARS) coronavirus, human immunodeficiency virus type 1 (HIV-1), and others (1,–5). The importance of IFITM proteins in antiviral defense is further demonstrated by the decreased survival of ifitm3 knockout mice upon infection by influenza A virus (6, 7). Furthermore, an ifitm3 single nucleotide polymorphism, rs12252-C, which leads to the expression of a truncated version of the IFITM3 protein with impaired antiviral activity, is associated with severe cases of influenza virus infection requiring hospitalization and rapid disease progression of HIV-1 patients (8, 9).

IFITM proteins exert their antiviral activity by impeding virus entry (10,–12). This mechanism of inhibition was first reported in studies showing that IFITM3, when expressed in target cells, hinders the fusion of virions with cellular membranes (3, 13). Subsequent experiments showed that IFITM3 prevents the hemifusion of the viral membrane and cellular membrane and/or obstructs the formation of the viral fusion pore (14, 15). Each of these two mechanisms of action may result from the ability of IFITM3 to increase the rigidity of cellular membranes (15). The latter activity of IFITM3 may be attributed to its unique intramembrane topology and oligomerization as well as its possible effect on cholesterol trafficking through an association with the vesicle-associated membrane protein-associated protein A (VAPA) protein (16,–22).

Apart from acting in target cells to block virus entry, IFITM proteins are also incorporated into HIV-1 particles and reduce viral infectivity (23,–25). Correlated with this impairment in viral infectivity is the impaired processing of HIV-1 Env into gp120 and gp41 by IFITM3 (25), which suggests that IFITM3 may undermine viral infectivity through impacting the viral Env protein. In addition to diminishing the infectivity of HIV-1, IFITM proteins also inhibit viruses that carry envelope proteins from Gibbon ape leukemia virus and feline leukemia virus RD114, although the mechanism of inhibition may differ from that for HIV-1 (24).

A number of different viral envelopes, including those of murine leukemia virus (MLV), Lassa virus, Machupo virus, and lymphocytic choriomeningitis virus, are relatively resistant to the inhibition of IFITM proteins when they are expressed in target cells (1). Different HIV and simian immunodeficiency virus (SIV) strains also show different degrees of susceptibility to IFITM inhibition in target cells (26, 27). However, it is not known whether any viral envelopes resist the inhibition of IFITM proteins that are incorporated into virus particles. The identification of this type of IFITM-resistant viral envelope is expected to help decipher how IFITM proteins impair viral infectivity. Here, we tested a number of HIV-1 primary isolates, including transmitted founder viruses, for their sensitivity to IFITM inhibition. By producing viral particles in HEK293T cells, we identified several HIV-1 strains that appear to be refractory to inhibition by IFITM proteins despite being incorporated into virus particles. Further experiments revealed that viral Env, and in particular the V3 loop, determines this resistance phenotype.

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RESULTS

 

HIV-1 strain AD8-1 is resistant to the inhibition of IFITM proteins that are incorporated into virus particles.

We first tested the sensitivity of different HIV-1 strains to inhibition by IFITM proteins when they are incorporated into virus particles. We started with laboratory-adapted strain NL4-3 and primary isolates 89.6, YU-2, and AD8-1 (28,–32). We transfected HEK293T cells with plasmid DNAs that express IFITM1, IFITM2, or IFITM3 together with different HIV-1 DNA clones. Different doses of IFITM plasmid DNAs were transfected to ensure that the levels of IFITM inhibitory activity fall in the linear range. The results of Western blotting showed that high doses of IFITM1 and -2, for example, 200 ng, diminished the expression of viral Gag/Pol and Env proteins for NL4-3, 89.6, and YU-2, whereas IFITM3 exerted much less inhibition (Fig. 1). Protein expression of the AD8-1 strain was not affected by IFITM proteins (Fig. 1). As a result, the level of production of NL4-3, 89.6, and YU-2, but not AD8-1, viruses was moderately reduced by IFITM1 and IFITM2 (Fig. 2A). We then measured the infectivity of virus particles with the same reverse transcriptase (RT) activity by infecting TZM-bl cells. The results showed that IFITM1 did not affect the infectivity of NL4-3, 89.6, and AD8-1 but enhanced the infectivity of YU-2 by 4-fold when 100 ng or 200 ng of IFITM1 plasmid DNA was transfected (Fig. 2A). In contrast, both IFITM2 and IFITM3 significantly diminished the infectivity of NL4-3 and 89.6 and moderately reduced the infectivity of YU-2 but did not affect the infectivity of AD8-1 (Fig. 2A and ​andB).B). We further measured the levels of IFITM3 in purified virus particles by Western blotting and observed significant levels of IFITM3 in all viruses that were examined, although IFITM3 levels vary among different viruses and do not directly correlate with the degree of inhibition of viral infectivity by IFITM3 (Fig. 2C).

 

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FIG 1

Effect of ectopic IFITM proteins on HIV-1 protein expression. HEK293T cells were cotransfected with different amounts of IFITM1, -2, or -3 DNA together with HIV-1 proviral DNA clones, including NL4-3, 89.6, YU-2, AD8-1, and NL(AD8). Cell lysates were harvested and examined by Western blotting with antibodies against HIV-1 gp120, viral p24, the FLAG tag, or tubulin.

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FIG 2

Effects of IFITM1, -2, or -3 on HIV-1 production and viral infectivity. (A) Viruses were produced by cotransfecting HEK293T cells with the viral DNA plasmids [NL4-3, 89.6, YU-2, AD8-1, and NL(AD8)] and different doses of IFITM1, -2, or -3 DNA (25, 50, 100, and 200 ng). Levels of produced viruses were determined by measuring viral RT activity. The infectivity of the viruses was assessed by infecting TZM-bl cells. The levels of virus RT activity or viral infectivity of each virus in the absence of IFITM proteins are arbitrarily set to a value of 1. (B) Relative infectivity of different HIV-1 strains that were produced in transfections with 100 ng IFITM DNA. The infectivity of each virus in the absence of IFITM proteins was arbitrarily set to a value of 1. Results shown are the averages of data from three independent experiments. (C) Incorporation of IFITM3 into particles of different HIV-1 strains. Samples from cells that were transfected with IFITM3 alone served as the control for IFITM3 proteins in the culture supernatants. Viruses were produced by transfecting HEK293T cells with proviral DNA and IFITM3 DNA and then harvested by ultracentrifugation through a 20% sucrose cushion. Levels of virion-associated IFITM3 were measured by Western blotting. (D and E) Effects of IFITM proteins on the infectivity of transmitted founder viruses, including CH040, CH058, CH077, CH106, REJO, RHPA, THRO, WITO, ZM246F_10, and ZM247Fv2. Viruses were produced by transfecting HEK293T cells with HIV-1 proviral DNA (500 ng) and IFITM DNA (100 ng). (D) Levels of virus particles were determined by measuring viral RT activity. (E) Viral infectivity was assessed by infecting TZM-bl cells. Levels of viruses from transfections with the empty vector pQCXIP are arbitrarily set to a value of 1. The data shown are the averages of results from three independent transfection experiments. The error bars represent the standard errors of the means. P values were calculated with an unpaired two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

We further tested 10 HIV-1 transmitted founder strains for their susceptibility to IFITM inhibition by performing cotransfection experiments. IFITM1 moderately reduced the production of some founder viruses (Fig. 2D). When viral infectivity was measured, IFITM1 increased the infectivity of 5 out of 10 founder viruses by 2- to 2.5-fold (Fig. 2E). IFITM2 diminished the infectivity of 5 out of 10 founder viruses by >2-fold, and 4 founder viruses were inhibited by IFITM3 by >2-fold (Fig. 2E). Two founder viruses, THRO and WITO, were resistant to inhibition by IFITM2 as well as by IFITM3. Together, these data suggest that different HIV-1 strains exhibit different levels of sensitivity to IFITM inhibition, with some strains showing strong resistance. This conclusion is further supported by the results of HIV-1 infection of MT4 cells that express ectopic IFITM3 (Fig. 3). In contrast to NL4-3, which was inhibited 7-fold by the ectopic expression of IFITM3 during infection of MT4 cells, AD8-1 was inhibited by 4-fold, and founder virus strains WITO, THRO, and ZM246F_10 showed almost complete resistance. This observation supports the recent finding of the resistance of transmitted founder HIV-1 strains to IFITM3 inhibition (33).

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FIG 3

Effect of IFITM3 in target cells on infection by different HIV-1 strains. Different HIV-1 strains were used to infect MT4/R5 cells that were transduced to express the ectopic IFITM3 protein. The number of infected cells was determined by scoring HIV-1 p24-positive cells using flow cytometry. (A) Representative results. (B) The inhibition of each HIV-1 strain by IFITM3 calculated from data from three independent experiments and summarized. MT4_pQCXIP indicates cells that were transduced with the empty retroviral vector. ***, P < 0.001.

 

AD8-1 Env confers resistance to IFITM3 inhibition.

 

We next asked which viral protein renders the AD8-1 virus resistant to IFITM-mediated inhibition. We started with an HIV-1 proviral DNA clone called NL(AD8), which has the Env sequence of AD8-1 substituted for that of the NL4-3 virus in the context of the NL4-3 proviral DNA (34). We performed cotransfection experiments in HEK293T cells with NL(AD8) DNA and IFITM1, IFITM2, or IFITM3 plasmid DNA. The results showed that the NL(AD8) virus was as resistant to IFITM inhibition of viral infectivity as the AD8-1 virus (Fig. 2A), suggesting that the AD8-1 Env protein is sufficient to confer resistance to IFITM inhibition.

In order to determine which domain of AD8-1 Env was responsible for this resistance phenotype, we changed each of the V1, V2, and V3 loop sequences of NL4-3 Env to those of AD8-1. The V1 and V2 substitutions generated noninfectious viral particles (data not shown). This incompatibility of different variable loops between different HIV-1 Env proteins has also been reported by others (35). In contrast, V3-substituted NL(AD8V3) produced viruses that were almost as infectious as wild-type NL4-3 and showed resistance to IFITM3 inhibition (Fig. 4A to ​toC).C). We further tested the V3 loops of HIV-1 strains YU-2, WITO, THRO, and RHPA by inserting these V3 sequences into NL4-3. These V3 loop sequences conferred different levels of resistance to IFITM3, with the WITO V3 loop generating as much resistance to IFITM3 as the AD8-1 V3 loop (Fig. 4A to ​toC).C). Since all of these V3 loop-substituted viruses use CCR5 as their coreceptor and have shown different levels of sensitivity to IFITM3 inhibition, coreceptor usage does not determine virus resistance to IFITM3.

 

FIG 4

The V3 loop sequences differentially modulate IFITM3 inhibition of HIV-1. (A to C) Viruses were produced by cotransfecting individual chimeric viral DNAs together with either the empty vector pQCXIP or IFITM3 DNA (100 ng) into HEK293T cells. The viruses were quantified by measuring their viral RT activity. Viral infectivity was then determined by infecting the TZM-bl cells using viruses with the same amounts of RT activity. (A) Results of one representative infection experiment. RLU, relative light units. Effects of IFITM3 on the infectivity of each virus were determined by calculating the ratio of luciferase activities of the IFITM3-free (pQCXIP) viruses to those of the IFITM3-bearing viruses (fold inhibition). (B) Averages of data from three independent transfection experiments. (C) Sequences of the V3 loop in Env from different HIV-1 strains. (D and E) Effects of IFITM3 on HIV-1 gp160 processing. Virus particles of different HIV-1 strains were produced by transfecting HEK293T cells in the absence or presence of IFITM3. Levels of virus particle-associated Gag, IFITM3-FLAG, and Env (gp160 and gp120) were assessed by Western blotting. The intensities of gp160 and gp120 signals for each virus were quantified by using the NIH ImageJ program. The ratios of gp120/(gp120 plus gp160) were calculated for each virus, and the results from four independent transfection experiments are summarized (E). (F) Correlation analysis of the effect of IFITM3 on gp160 processing and IFITM3 inhibition of HIV-1 infectivity (data from panel B). (G to I) Stability of Env proteins from different HIV-1 strains. HEK293K cells were transfected with proviral DNA of NL4-3, AD8-1, NL(AD8), or NL(AD8V3) and then labeled with [35S]methionine-cysteine. HIV-1 Env in the cell lysates as well as in the supernatants was immunoprecipitated and analyzed by SDS-PAGE followed by PhosphorImager analysis. (G) Results of one representative experiment from 3 independent experiments. (H and I) The processing index and the association index values of HIV-1 Env were calculated from three independent experiments, and the results are summarized. The values for NL4-3 were arbitrarily set to 1. ***, P < 0.001.

We previously observed that IFITM3 interferes with the processing of the viral Env precursor gp160 (25). Indeed, when Env proteins in virus particles that were produced from HEK293T cells were examined by Western blotting, the IFITM3-bearing NL4-3 virus contained less gp120 concurrently with the accumulated gp160 precursor than did the IFITM3-free NL4-3 virus (Fig. 4D and ​andE).E). In contrast, the IFITM3-resistant AD8-1 virus had similar levels of gp120 and gp160 regardless of virus incorporation of the IFITM3 protein. The NL(AD8), NL(AD8V3), and NL(WITOV3) viruses, which are also resistant to IFITM3, had nearly 50% of the Env protein processed into gp120 when IFITM3 was coexpressed. In contrast, upon virus incorporation of IFITM3, the NL(YU-2V3) and NL(THROV3) viruses had 14.9% and 23% of Env processed into gp120, respectively (Fig. 4D and ​andE).E). There is a significant correlation between IFITM3 inhibition of HIV-1 infectivity and gp160 processing (Fig. 4F). Further studies are warranted to investigate whether impaired gp160 processing directly diminishes HIV-1 infectivity. We also noted that there was no correlation between the inhibition of viral infectivity and the incorporation of IFITM3 into HIV-1 virions, similar to data in a previous report (25).

The results of Western blotting of virus samples also revealed lower levels of total Env proteins for NL4-3 and NL(YU-2V3) than for the rest of the viruses that are resistant to IFITM3. We therefore performed metabolic labeling of viral Env using [35S]methionine-cysteine and assessed the efficiency of Env gp160 processing and the stability of the gp120/gp41 complex by calculating the processing index (PI) and association index (AI) values. The results showed that all viruses tested had similar PI values but that the IFITM3-sensitive NL4-3 virus had a lower AI value than did the IFITM3-resistant viruses, including AD8-1, NL(AD8), and NL(AD8V3) (Fig. 4G to ​toI).I). These data suggest that the V3 loop sequences from AD8-1 and WITO confer resistance to IFITM3 by maintaining the stability of the trimeric Env complex.

IFITM3-bearing HIV-1 has been shown to be defective in entry (23, 25). Indeed, the results of a beta lactamase (BlaM)-Vpr virion fusion assay showed that the entry of IFITM3-bearing NL4-3 was reduced by 5-fold (Fig. 5). In contrast, the entry of IFITM3-resistant strains AD8-1, NL(AD8V3), and NL(WITOV3) was slightly affected by IFITM3 (Fig. 5). The entry of the NL(RHPAV3), NL(THROV3), and NL(YU-2V3) viruses was diminished by 2- to 4-fold, which correlates with their levels of resistance to IFITM3. Together, these data demonstrate that the V3 loop sequences are able to overcome IFITM3 inhibition of viral infectivity by stabilizing Env and therefore rescuing virus entry.

 

FIG 5

Effect of IFITM3 on the entry of V3 loop chimeric viruses. (A) The BlaM-Vpr-containing viruses were produced by cotransfecting HEK293T cells with BlaM-Vpr DNA, each HIV-1 DNA, and either the empty vector or IFITM3 DNA. Viruses with the same viral RT activity were used to infect C8166-R5 cells. Viral entry was determined by measuring the cleavage of CCF2 by BlaM. The results of one representative entry assay are shown. (B) The effects of IFITM3 on virus entry were determined by calculating the ratios of the entry efficiency of each virus in the absence of IFITM3 to that of the same virus in the presence of IFITM3. The results are presented as fold inhibition values and are the averages of data from four independent assays. *, P < 0.05; ***, P < 0.001.

 

IFITM3-resistant Env proteins are resistant to inhibition by soluble CD4 and the 17b antibody.

We further investigated how the substituted V3 loop causes resistance to IFITM3. Since the V3 loop determines coreceptor usage (36, 37), changing the V3 sequence may affect the ability of Env to use either CXCR4 or CCR5. A high affinity for either CXCR4 or CCR5 may compensate for impaired viral entry by IFITM3. We therefore measured the sensitivity of the NL4-3, NL(AD8), NL(AD8V3), NL(YU-2V3), NL(WITOV3), NL(THROV3), and NL(RHPAV3) viruses to inhibition by either the CXCR4 inhibitor AMD3100 or the CCR5 inhibitor maraviroc (38, 39). As expected, NL4-3 was inhibited by AMD3100 but not by maraviroc, whereas the other 6 viruses were resistant to AMD3100 and were inhibited by maraviroc to different levels, with NL(AD8), NL(YU-2V3), and NL(THROV3) exhibiting lower sensitivity to maraviroc inhibition and NL(RHPAV3) being the most inhibited (Fig. 6A). The data furthermore showed that the sensitivity of these viruses to maraviroc inhibition did not correlate with their susceptibility to IFITM3 inhibition (Fig. 6B), suggesting that the ability of Env to use the CCR5 coreceptor for entry does not determine virus susceptibility to IFITM3 that is incorporated into virus particles.

 

FIG 6

Effects of V3 loop substitution on viral responses to the coreceptor inhibitors AMD3100 and maraviroc. (A) TZM-bl cells were preincubated with different doses of the CXCR4 inhibitor AMD3100 or the CCR5 inhibitor maraviroc for 1 h at 37°C. NL4-3 and other V3 loop chimeric viruses with the same amount of RT activity were then added to infect TZM-bl cells. At 40 h postinfection, the cells were harvested for luciferase activity measurements. Levels of luciferase activity of each virus without any drug treatment are arbitrarily set to a value of 1. (B) Correlation analysis of sensitivities of HIV-1 strains to maraviroc inhibition and to IFITM3 inhibition (data from Fig. 4B). The inhibition data for maraviroc at a concentration of 0.00026 μg/ml were used for the analysis.

Next, we examined the possible conformational changes of HIV-1 Env as a result of replacing the V3 loop. The V3 loop chimeric viruses were tested for neutralization by a panel of antibodies (Abs) that recognize different epitopes in Env. We expected to observe a correlation between the sensitivity of HIV-1 Env to neutralization by certain antibodies and the sensitivity of HIV-1 Env to inhibition by IFITM3. Among the neutralizing antibodies that we tested, two antibodies, VRC03 and 17b, that recognize the CD4-binding site and CD4-induced (CD4i) epitopes (40,–42), respectively, strongly inhibited the NL4-3 virus (Fig. 7A). When the V3 loop of NL4-3 was changed to that of AD8-1, YU-2, WITO, THRO, or RHPA, the chimeric viruses showed different levels of resistance to these two antibodies, indicating that the V3 loop substitution affects the epitopes that are recognized by either VRC03 or 17b. The degrees of inhibition by 17b correlate well with the sensitivity of the tested viruses to IFITM3 inhibition (Fig. 7B).

 

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IFITM3-resistant HIV-1 is refractory to inhibition by the 17b antibody. (A) Viruses with the same RT activity were incubated with different doses of antibodies VRC03 (0.125, 0.25, 0.5, and 1.0 μg/ml), 17b, CH01 (0.25, 0.5, 1.0, and 2.0 μg/ml), PG16 (0, 0.02, 0.05, 0.1, and 0.2 μg/ml), 447-52D (0, 0.1, 0.2, 0.5, and 2.0 μg/ml), 7H6, 10E8, and 10-1074 (0, 0.1, 0.2, 0.5, and 1.0 μg/ml) for 1 h in a cell culture incubator at 37°C. The viruses were then used to infect TZM-bl cells. At 40 h postinfection, the cells were harvested, and luciferase activity was measured. Levels of luciferase activity for each virus without treatment are arbitrarily set to a value of 1. The results shown are the averages of data from two independent infections. (B) Correlation analysis of the inhibition of HIV-1 strains by the 17b antibody and by IFITM3 (data from Fig. 4B). Data for the inhibition of different HIV-1 strains by the 17b antibody at a concentration of 2 μg/ml were used for the analysis.

Since 17b recognizes a CD4i epitope that overlaps the coreceptor-binding site (40, 41), we therefore tested NL4-3 and its mutants for inhibition by soluble CD4 (sCD4). The results showed that NL4-3 was very sensitive to sCD4 inhibition, with a 50% inhibitory concentration (IC50) of 62 ng/ml (Fig. 8A). In contrast, the IFITM3-resistant virus strains AD8-1, NL(AD8V3), and NL(WITOV3) were refractory to sCD4 inhibition. The NL(YU-2V3), NL(THROV3), and NL(RHPAV3) viruses exhibited differential sensitivities to sCD4 neutralization, with IC50 values of 72.4 ng/ml, 226.5 ng/ml, and 387.5 ng/ml, respectively. The sensitivity of the tested HIV-1 strains to sCD4 neutralization correlated well with the inhibition of these viruses by IFITM3 (Fig 8B). We also observed that IFITM3-free and IFITM3-bearing viruses were equally sensitive to sCD4 neutralization (Fig.. 8A), suggesting that IFITM3 itself does not alter the binding of gp120 to sCD4. These results together suggest that the conformation of the CD4-binding site in HIV-1 Env is associated with viral susceptibility to IFITM3 inhibition.

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FIG 8

Inhibition of HIV-1 by soluble CD4. (A) The viruses used were produced by cotransfecting HEK293T cells with the virus DNA and the empty pQCXIP vector or IFITM3 DNA. Viruses with the same RT activity were incubated with different doses of sCD4 (0.025, 0.05, 0.1, 0.2, and 0.5 μg/ml) for 1 h at 37°C and were then used to infect TZM-bl cells. Levels of luciferase activity for each virus without sCD4 treatment are arbitrary set to a value of 1. (B) Correlation analysis of the sensitivity of HIV-1 strains to inhibition by sCD4 and to inhibition by IFITM3 (data from Fig.4B). Data for the inhibition of different HIV-1 strains by sCD4 at a concentration of 0.2 μg/ml were used for the analysis.

DISCUSSION

Viruses have evolved various mechanisms to evade host restrictions. One mechanism is exemplified by the HIV-1 Vif protein that antagonizes the restriction factor APOBEC3G by recruiting the E3 ubiquitin ligase complex that modifies APOBEC3G for proteasomal degradation (43, 44). Viruses can also change the sequence of viral proteins that are targeted by restriction factors and thereby escape from the host immune response. For example, HIV-1 has adapted its capsid protein to avoid recognition and inhibition by human TRIM5α but is restricted by Old World monkey TRIM5α (45). The results of our study suggest that resistance to IFITM3 inhibition is associated with the HIV-1 Env sequence, including the V3 loop, which agrees with data from a previous study by Foster et al. showing that changes in the Env sequence underlie the increased sensitivity of 6-month HIV-1 to IFITM3 inhibition compared to the transmitted founder virus (33).

Previous studies have shown that the envelope glycoproteins of some viruses, including murine leukemia virus (MLV), Lassa virus, Machupo virus, and lymphocytic choriomeningitis virus, are resistant to IFITM-mediated inhibition (1, 15, 46, 47). The involvement of viral envelope proteins in overcoming IFITM inhibition has also been supported by the results of our previous studies (25, 48, 49). The highly heterogeneous envelope protein sequences of different viral species make it difficult to identify which protein domain or sequence determines this IFITM resistance phenotype. This problem becomes approachable in light of our observation that different HIV-1 strains exhibit differential sensitivities to IFITM inhibition. The same domain structure and the relatively similar sequences of different HIV-1 Env proteins allow the conduction of mutagenesis studies to identify the Env sequence that modulates HIV-1 susceptibility to IFITM inhibition. We have now identified the V3 loop as one determinant in HIV-1 Env that regulates viral resistance to IFITM3. Amino acids outside the V3 loop may also contribute to viral resistance to IFITMs. For example, NL4-3 developed resistance to a truncated version of IFITM1, IFITM1Δ(117–125), through acquiring the Env mutation A539V that is located in gp41 (25). Similarly, HIV-1 strain BH10 was able to acquire the Env mutation E367E in the C3 region in order to escape from inhibition by IFITM1 (48).

The V3 loop determines the coreceptor usage of either CXCR4 or CCR5 by forming the coreceptor-binding pocket together with the bridging sheet (50,-52). In the unliganded state, the V3 loop resides within a cradle that is formed by the V1V2 domains from its own and adjacent protomers in the gp120 trimer and contributes to trimer stability (53). The V3 loop is also one of the frequent targets of host neutralizing antibodies (54,-56). To understand how the V3 loop modulates the sensitivity of HIV-1 to IFITM3 inhibition, we tested several possible mechanisms, including coreceptor usage as well as the response of HIV-1 Env to ligands and antibodies. We did not observe a strong correlation between IFITM3 inhibition of HIV-1 infectivity and HIV-1 coreceptor usage. Given the few CXCR4-tropic viruses that were tested in this study, a more systematic study of the CXCR4- and CCR5-tropic viruses is warranted to further investigate the possible correlation between HIV-1 coreceptor usage and virus susceptibility to IFITM3 restriction. A recent study reported a high sensitivity of CCR5-tropic HIV-1 strains, compared to CXCR4-tropic strains, to inhibition by IFITM3 that is expressed in target cells (33). It appears that coreceptor usage differentially affects HIV-1 sensitivity to inhibition by IFITM3 that is expressed in target cells or is incorporated into virus particles. Nonetheless, we observed that the IFITM3-sensitive virus NL4-3 is also much more sensitive to inhibition by the 17b antibody and soluble CD4 than is the IFITM3-resistant virus AD8-1 (Fig.7 and 8). By using the single-molecule fluorescence resonance energy transfer method, Munro and colleagues showed that HIV-1 Env proteins transit between dynamic conformations and that laboratory-adapted neutralization-sensitive HIV-1 Env tends to adopt the high-energy “open” state more frequently than does neutralization-resistant Env (57). Along with this finding, we suggest that IFITM3-resistant Env proteins adopt the low-energy “closed” ground state that is more resistant to inhibition by antibodies and other factors, including IFITM3. Since IFITM3 itself does not affect the inhibition of HIV-1 by soluble CD4, the role of the V3 loop in resisting IFITM3 may be indirect, including a possible modulation of the transition between the high- and low-energy states of Env and affecting the stability of Env, as shown by the data in Fig.4. It remains to be tested whether and how IFITM3, upon incorporation into virus particles, modulates HIV-1 sensitivity to inhibition by neutralizing antibodies and/or the efficiency of coreceptor usage.

Our data showed that HIV-1 strains that are resistant to virion-incorporated IFITM3, including the transmitted founder viruses WITO, THRO, and ZM246F_10, are also more resistant to IFITM3 that is expressed in target cells (Fig.3), which is in agreement with data from a study by Foster and colleagues reporting the resistance of transmitted founder HIV-1 strains to IFITM3 (33), suggesting a protective role of IFITM3 in HIV-1 transmission. In contrast to IFITM3, which impairs the infectivity of HIV-1, we observed that IFITM1 increases the infectivity of certain HIV-1 strains, including YU-2, CH058, CH106, RHPA, THRO, and WITO. This finding awaits further investigation under conditions of depleting endogenous IFITM1 in HIV-1 natural target cells, including primary CD4+ T cells, especially in light of the suppression of YU-2 viral infectivity by ectopic IFITM1 from Jurkat cells (25). All the same, it was previously reported that IFTM proteins promote, rather than inhibit, the infection of human coronavirus OC43 (58), which suggests that IFITM proteins may exert different, even opposing, effects on different viruses.

In summary, our data demonstrate the important role of the HIV-1 Env conformation in counteracting inhibition by IFITM3. Mutagenesis studies further illustrate the V3 loop as one determinant in viral Env that modulates virus sensitivity to IFITM3 inhibition. Given that HIV-1 Env is also targeted by other host antiviral factors such as guanylate-binding protein 5 (GBP5) and membrane-associated really interesting new gene C4HC3 8 (MARCH8) (59, 60) and that Env also determines HIV-1 susceptibility to host restriction factors, including serine incorporator 5 (SERINC5) (61, 62), our results further emphasize the important role of Env proteins in the “arms race” between host antiviral defense and viral antagonism.

MATERIALS AND METHODS

 

Cell culture.

HEK293T cells and TZM-bl cells were propagated in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin under 37°C with 5% CO2. C8166-R5 cells (63) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 μl/ml of puromycin. MT4/R5 cells (64) were cultured in RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 500 μg/ml G418. DMEM (catalog number 11965-092), RPMI 1640 (catalog number 11875-093), FBS (catalog number 11875-093), and penicillin-streptomycin (catalog number 15140-122) were purchased from Gibco by Life Technology. Puromycin (catalog number P8833) and G418 (catalog number A1720) were obtained from Sigma-Aldrich.

 

Plasmid DNA.

pNL4-3 (catalog number 114), p89.6 (catalog number 3552), pYU-2 (catalog number 1350), pWITO.c/2474 (catalog number 11739), pCH040.c/2625 (catalog number 11740), pCH058.c/2960 (catalog number 11856), pCH077.t/2627 (catalog number 11742), pCH106.c/2633 (catalog number 11743), pRHPA.c/2635 (catalog number11744), pTHRO.c/2626 (catalog number11745), pREJO.c/2864 (catalog number 11746), pZM246F_10 (catalog number 11828), and pZM247Fv2 (catalog number 11829) were obtained from the NIH AIDS Reagent Program. pAD8-1 and pNL(AD8) were kindly provided by Eric O. Freed (34). N-terminally FLAG-labeled IFITM1, -2, or -3 was cloned into pQCXIP as previously described (3). pQCXIP (catalog number 631516) was purchased from Clontech. NL4-3 env sequences that contain the V3 loop sequence of AD8-1, YU-2, WITO, THRO, or RHPA were synthesized by Invitrogen. The synthesized sequences were cloned into pNL4-3 via restriction enzyme digestion by SalI and NheI.

 

Virus production.

HEK293T cells were seeded into a 6-well plate at 0.6 × 106 cells/well 20 h prior to transfection. A total of 500 ng of a proviral DNA plasmid and 0, 25, 50, 100, and 200 ng of IFITM1, -2, or -3 plasmids were cotransfected into HEK293T cells by using polyethylenimine (PEI). The medium was changed at 6 h posttransfection. The supernatant and cells were harvested at 48 h posttransfection. The collected supernatant was centrifuged at 3,000 rpm (CS-6R; Beckman Coulter) for 20 min to remove the cell debris. The virus was then separated into 1-ml aliquots and stored in a −80°C freezer. The amount of virus was quantified by measuring viral RT activity.

 

Virus infectivity.

A total of 5.0 × 104 TZM-bl cells were seeded into each well of a 24-well plate 20 h prior to infection. Viruses with equal amounts of RT activity were used to infect TZM-bl cells. At 40 h postinfection, TZM-bl cells were washed once with cold Dulbecco's phosphate-buffered saline (PBS) (DPBS) (catalog number 14190-144; Gibco by Life Technology) and lysed in 100 μl of 1× passive lysis buffer (catalog number E1941; Promega). Ten microliters of the lysate from each sample was mixed with 40 μl of the luciferase substrate (catalog number E4530; Promega). Luciferase activity was measured by using a Glomax 20/20 luminometer.

 

Ultracentrifugation to harvest virus particles.

In order to produce enough HIV-1 particles for ultracentrifugation and Western blotting, transfection was performed with HEK293T cells that were seeded into a 10-cm cell culture plate at 4 × 106 cells/plate. Twenty hours after seeding, 5 μg of HIV-1 proviral DNA, 1 μg of IFITM3 DNA, or 1 μg of pQCXIP DNA was cotransfected into each dish by using PEI. The ratio of HIV-1 DNA to IFITM3 DNA (5 μg–1 μg) was kept the same as the one that was used in the transfection experiments to measure IFITM3 inhibition of HIV-1 (500 ng–100 ng). The medium was changed at 6 h posttransfection, and the viruses were harvested 48 h later. The harvested viruses were filtered through a 0.2-μm sterile polyethersulfone membrane syringe filter (catalog number 28145-501; VWR International) to remove the cell debris. Viruses were slowly transferred to 14- by 89-mm ultracentrifugation tubes (catalog number 344059; Beckman Coulter) that were preloaded with 2 ml of a 20% sucrose cushion. Virus particles were pelleted at 35,000 rpm for 1 h at 4°C by ultracentrifugation (Optima L-100XP ultracentrifuge; Beckman Coulter). The pelleted viruses were resuspended in 100 μl of DMEM. Five microliters of the pelleted viruses was used for measuring viral RT activity.

 

BlaM-Vpr viral entry assay.

A BlaM-Vpr assay was performed to measure HIV-1 entry (65, 66). The BlaM-Vpr-containing viruses were produced by the cotransfection of 5 μg of HIV-1 proviral DNA, 1 μg of pCMV-BlaM-Vpr DNA, and 1 μg of IFITM3 or the pQCXIP vector into HEK293T cells. The collected supernatant was filtered through a 0.2-μm filter and pelleted at 35,000 rpm for 1 h at 4°C. The viruses were resuspended in RPMI 1640 medium. Five microliters of each virus was used for RT activity quantification. The rest of the viruses were aliquoted and stored in a −80°C freezer. C8166-R5 cells were used for the viral entry assay. A total of 1 × 106 C8166-R5 cells were plated with 400 μl of 10% FBS–RPMI 1640 medium into each well of a 24-well plate. The volumes of the viruses were adjusted according to their RT measurements. Fresh medium was added to achieve a final volume of 100 μl for each virus. A total of 5 μg/ml of Polybrene (catalog number 107689; Sigma-Aldrich) was included in the medium, and a 45-min spinoculation at 1,800 rpm (CS-6R; Beckman Coulter) was carried out, followed by 2 h of incubation in a humidified environment at 37°C with 5% CO2. The cells were then transferred to Eppendorf tubes (EP tubes), and the viruses that did not succeed in entry were washed off by using CO2-independent medium (catalog number 18045-088; Gibco by Life Technology). The cells were loaded with CCF2-AM (catalog number K1032; Invitrogen) and incubated in the dark at room temperature for 1 h. The cells were washed with developing solution, transferred into a V-bottom 96-well cell culture plate (catalog number 651180; Greiner Bio-One International), and bathed in developing solution overnight (16 h) at room temperature in the dark. The next day, the cells were washed twice with cold DPBS containing 2% FBS, fixed with 2% paraformaldehyde (catalog number PAR070; BioShop) dissolved in 2% FBS–DPBS, and analyzed by flow cytometry (BD LSR Fortessa analyzer; BD Biosciences). The results obtained by flow cytometry were analyzed by using FlowJo software.

 

Neutralization assay.

Monoclonal neutralizing antibodies VRC03 (catalog number 12032), 17b (catalog number 4091), 7H6 (catalog number 12295), 10E8 (catalog number 1294), 447-52D (catalog number 4020), 10-1074 (catalog number 12477), and PG16 (catalog number 12150), together with sCD4 (catalog number 4615), were obtained from the NIH AIDS Reagent Program. The volumes of viruses were adjusted according to their RT activity, and 10% FBS–RPMI 1640 was used to adjust the volume to 100 μl. The viruses were then coincubated with each of the antibodies or sCD4 at various concentrations for 1 h in a cell culture incubator at 37°C with 5% CO2. After incubation, the viruses were used to infect TZM-bl cells that were preseeded in a 24-well plate 20 h prior to infection. Four hundred microliters of additional fresh medium was added to each well to achieve a final volume of 1 ml, and the cells were incubated in a cell culture incubator. The cells were harvested at 40 h postinfection, and the luciferase activity was measured.

 

Western blotting.

Harvested cells were lysed in Cytobuster protein extraction reagent (catalog number 71009; EMD Millipore Novagen) containing a protease inhibitor (catalog number 11836153001; Roche) on ice for 1 h. The cell debris were removed by centrifugation at 13,200 rpm (Microfuge 16; Beckman Coulter) for 20 min at 4°C. The lysates were denatured by the addition of 4× protein loading buffer followed by 5 min of boiling. The protein samples were loaded onto a 1% sodium dodecyl sulfate (SDS) (catalog number SDS001; BioShop)–12% polyacrylamide (catalog number ACR009; BioShop) gel for the detection of tubulin, Gag, and FLAG-IFITM3 and onto a 1% SDS–8% polyacrylamide gel for the detection of Env. The proteins were separated by electrophoresis at 100 V for ∼2 to 3 h and then transferred onto polyvinylidene difluoride (PVDF) membranes (catalog number 3010040; Roche). The membranes were blocked in 5% skim milk dissolved in 1× PBS with 0.1% Tween 20 (catalog number TWN510; BioShop) (PBST) for 1 h, followed by incubation with a 1:5,000 dilution of primary antibodies for 2 h at room temperature. After washing with PBST, the membranes were further incubated with a 1:10,000 dilution of secondary antibodies for 1 h at room temperature. The primary antibodies included monoclonal mouse antitubulin antibody (B-5-1-2) (catalog number sc-23948; Santa Cruz Biotechnology), monoclonal mouse anti-FLAG antibody (catalog number F1804-1MG; Sigma-Aldrich), polyclonal rabbit anti-p24 antibody (catalog number SAB3500946; Sigma-Aldrich), monoclonal anti-gp41 antibody (catalog number 526; NIH AIDS Reagent Program), and sheep anti-gp120 single-chain antibody (catalog number 11710; NIH AIDS Reagent Program). The secondary antibodies included enhanced chemiluminescence (ECL) rabbit IgG horseradish peroxidase (HRP)-linked whole antibody from donkey (catalog number NA934V; GE Health Care Life Science), Amersham ECL mouse HRP-linked IgG whole Ab from sheep (catalog number NA931; GE Health Care Life Science), and HRP-rabbit anti-sheep IgG (catalog number 618620; Invitrogen). The membranes were treated with the Western Lightening Plus-ECL substrate (catalog number NEL105001EA; PerkinElmer), and the chemiluminescent signals were detected by exposure to X-ray films (catalog number 6041768; Carestream).

 

Assay of HIV-1 inhibition by AMD3100 and maraviroc.

TZM-bl cells were incubated with 0, 5.14 × 10−5, 2.67 × 10−4, 1.28 × 10−3, 5.14 × 10−3, and 0.01 μg/ml of the CXCR4 inhibitor AMD3100 or the CCR5 inhibitor maraviroc for 1 h in a humidified incubator at 37°C with 5% CO2. The cells were then infected by the same amount of viruses. Infected cells were harvested at 40 h postinfection, and the luciferase activity was measured. Both AMD3100 (catalog number 8128) and maraviroc (catalog number 11580) were obtained from the NIH AIDS Reagent Program.

 

HIV-1 Env stability assay.

 

HEK293T cells were transfected with proviral DNA (67). Twenty-four hours after transfection, the cells were metabolically labeled with 100 μCi/ml of [35S]methionine-cysteine (catalog number NEG772007MC; PerkinElmer) dissolved in 5% dialyzed FBS-supplemented methionine- and cysteine-negative DMEM for 16 h. The cells were then lysed in radioimmunoprecipitation assay (RIPA) buffer containing 140 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 1% NP-40, and 0.05% SDS. The radiolabeled Env proteins in the cell lysates or supernatants were precipitated by using serum from HIV-1 patients for 1 h at 37°C. The immunoprecipitated samples were separated by electrophoresis in polyacrylamide gels and analyzed by using a PhosphorImager (Molecular Dynamics). The association index measures the relative ability of the gp120 domain to stay on the Env trimer on virus-producing cells. The association index values thus describe the intrinsic stability of Env (i.e., the ability of gp120 to remain associated with gp41). A low association index is a good indicator of decreased levels of gp120 in virus particles (68, 69). The association index of NL4-3 was arbitrarily set to a value of 1, and the values for other viruses were calculated relative to the value for NL4-3 {association index = [(target gp120)cell lysate × (NL4-3 gp120)supernatant]/[(target gp120)supernatant × (NL4-3 gp120)cell lysate]}. The processing index measures the relative efficiency of Env maturation from the gp160 precursor to gp120. The processing index of NL4-3 was arbitrarily set to a value of 1, and the values for other viruses were calculated relative to the value for NL4-3 {processing index = [(total gp120)target × (gp160)NL4-3]/[(gp160)target × (total gp120)NL4-3]}.

 

Statistical analysis.

The P values were calculated based on the unpaired two-tailed t test. A P value of <0.05 was deemed statistically significant. The R2 values and the P values in the correlation graphs were calculated based on the linear regression module implemented in the Excel program.

ACKNOWLEDGMENTS

We thank Eric O. Freed for providing the pAD8-1 and pNL(AD8) proviral DNA clones.

This work was supported by funding from the Canadian Institutes of Health Research to C.L. and from the National Institutes of Health to S.-L.L. (R01 AI112381). A.F. is the recipient of a Canada research chair on retroviral entry. Y.W. is a recipient of a Canada graduate scholarship (master's) from the Natural Sciences and Engineering Research Council of Canada. S.D. is a recipient of a CRCHUM postdoctoral fellowship.

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Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

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