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J Virol. 2017 April 1; 91(7): e02441-16.
Published online 2017 March 13. Prepublished online 2017 January 18. doi:  10.1128/JVI.02441-16
PMCID: PMC5355610

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

Susan R. Ross, Editor
Susan R. Ross, University of Illinois at Chicago;

ABSTRACT

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

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.

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).

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 ...
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, ...

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).

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 ...

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 ...

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 ...

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 ...

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).

FIG 7
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), ...

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.

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, ...

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 and8).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.

REFERENCES

1. Brass AL, Huang IC, Benita Y, John SP, Krishnan MN, Feeley EM, Ryan BJ, Weyer JL, van der Weyden L, Fikrig E, Adams DJ, Xavier RJ, Farzan M, Elledge SJ 2009. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 139:1243–1254. doi:.10.1016/j.cell.2009.12.017 [PMC free article] [PubMed] [Cross Ref]
2. Huang IC, Bailey CC, Weyer JL, Radoshitzky SR, Becker MM, Chiang JJ, Brass AL, Ahmed AA, Chi X, Dong L, Longobardi LE, Boltz D, Kuhn JH, Elledge SJ, Bavari S, Denison MR, Choe H, Farzan M 2011. Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus. PLoS Pathog 7:e1001258. doi:.10.1371/journal.ppat.1001258 [PMC free article] [PubMed] [Cross Ref]
3. Lu J, Pan Q, Rong L, He W, Liu SL, Liang C 2011. The IFITM proteins inhibit HIV-1 infection. J Virol 85:2126–2137. doi:.10.1128/JVI.01531-10 [PMC free article] [PubMed] [Cross Ref]
4. Weidner JM, Jiang D, Pan XB, Chang J, Block TM, Guo JT 2010. Interferon-induced cell membrane proteins, IFITM3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms. J Virol 84:12646–12657. doi:.10.1128/JVI.01328-10 [PMC free article] [PubMed] [Cross Ref]
5. Bailey CC, Zhong G, Huang IC, Farzan M 2014. IFITM-family proteins: the cell's first line of antiviral defense. Annu Rev Virol 1:261–283. doi:.10.1146/annurev-virology-031413-085537 [PMC free article] [PubMed] [Cross Ref]
6. Bailey CC, Huang IC, Kam C, Farzan M 2012. Ifitm3 limits the severity of acute influenza in mice. PLoS Pathog 8:e1002909. doi:.10.1371/journal.ppat.1002909 [PMC free article] [PubMed] [Cross Ref]
7. Everitt AR, Clare S, Pertel T, John SP, Wash RS, Smith SE, Chin CR, Feeley EM, Sims JS, Adams DJ, Wise HM, Kane L, Goulding D, Digard P, Anttila V, Baillie JK, Walsh TS, Hume DA, Palotie A, Xue Y, Colonna V, Tyler-Smith C, Dunning J, Gordon SB, GenISIS Investigators, MOSAIC Investigators, Smyth RL, Openshaw PJ, Dougan G, Brass AL, Kellam P 2012. IFITM3 restricts the morbidity and mortality associated with influenza. Nature 484:519–523. doi:.10.1038/nature10921 [PMC free article] [PubMed] [Cross Ref]
8. Zhang Y, Makvandi-Nejad S, Qin L, Zhao Y, Zhang T, Wang L, Repapi E, Taylor S, McMichael A, Li N 2015. Interferon-induced transmembrane protein-3 rs12252-C is associated with rapid progression of acute HIV-1 infection in Chinese MSM cohort. AIDS 29:889–894. doi:.10.1097/QAD.0000000000000632 [PMC free article] [PubMed] [Cross Ref]
9. Zhang Y-H, Zhao Y, Li N, Peng Y-C, Giannoulatou E, Jin R-H, Yan H-P, Wu H, Liu J-H, Liu N 2013. Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals. Nat Commun 4:1418. doi:.10.1038/ncomms2433 [PMC free article] [PubMed] [Cross Ref]
10. Diamond MS, Farzan M 2013. The broad-spectrum antiviral functions of IFIT and IFITM proteins. Nat Rev Immunol 13:46–57. doi:.10.1038/nri3344 [PMC free article] [PubMed] [Cross Ref]
11. Perreira JM, Chin CR, Feeley EM, Brass AL 2013. IFITMs restrict the replication of multiple pathogenic viruses. J Mol Biol 425:4937–4955. doi:.10.1016/j.jmb.2013.09.024 [PMC free article] [PubMed] [Cross Ref]
12. Smith S, Weston S, Kellam P, Marsh M 2014. IFITM proteins—cellular inhibitors of viral entry. Curr Opin Virol 4:71–77. doi:.10.1016/j.coviro.2013.11.004 [PubMed] [Cross Ref]
13. Feeley EM, Sims JS, John SP, Chin CR, Pertel T, Chen L-M, Gaiha GD, Ryan BJ, Donis RO, Elledge SJ 2011. IFITM3 inhibits influenza A virus infection by preventing cytosolic entry. PLoS Pathog 7:e1002337. doi:.10.1371/journal.ppat.1002337 [PMC free article] [PubMed] [Cross Ref]
14. Desai TM, Marin M, Chin CR, Savidis G, Brass AL, Melikyan GB 2014. IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion. PLoS Pathog 10:e1004048. doi:.10.1371/journal.ppat.1004048 [PMC free article] [PubMed] [Cross Ref]
15. Li K, Markosyan RM, Zheng YM, Golfetto O, Bungart B, Li M, Ding S, He Y, Liang C, Lee JC, Gratton E, Cohen FS, Liu SL 2013. IFITM proteins restrict viral membrane hemifusion. PLoS Pathog 9:e1003124. doi:.10.1371/journal.ppat.1003124 [PMC free article] [PubMed] [Cross Ref]
16. Amini-Bavil-Olyaee S, Choi YJ, Lee JH, Shi M, Huang IC, Farzan M, Jung JU 2013. The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry. Cell Host Microbe 13:452–464. doi:.10.1016/j.chom.2013.03.006 [PMC free article] [PubMed] [Cross Ref]
17. Bailey CC, Kondur HR, Huang I-C, Farzan M 2013. Interferon-induced transmembrane protein 3 is a type II transmembrane protein. J Biol Chem 288:32184–32193. doi:.10.1074/jbc.M113.514356 [PMC free article] [PubMed] [Cross Ref]
18. Jia R, Pan Q, Ding S, Rong L, Liu S-L, Geng Y, Qiao W, Liang C 2012. The N-terminal region of IFITM3 modulates its antiviral activity by regulating IFITM3 cellular localization. J Virol 86:13697–13707. doi:.10.1128/JVI.01828-12 [PMC free article] [PubMed] [Cross Ref]
19. Jia R, Xu F, Qian J, Yao Y, Miao C, Zheng YM, Liu SL, Guo F, Geng Y, Qiao W 2014. Identification of an endocytic signal essential for the antiviral action of IFITM3. Cell Microbiol 16:1080–1093. doi:.10.1111/cmi.12262 [PMC free article] [PubMed] [Cross Ref]
20. John SP, Chin CR, Perreira JM, Feeley EM, Aker AM, Savidis G, Smith SE, Elia AE, Everitt AR, Vora M 2013. The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication. J Virol 87:7837–7852. doi:.10.1128/JVI.00481-13 [PMC free article] [PubMed] [Cross Ref]
21. Ling S, Zhang C, Wang W, Cai X, Yu L, Wu F, Zhang L, Tian C 2016. Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3. Sci Rep 6:24029. doi:.10.1038/srep24029 [PMC free article] [PubMed] [Cross Ref]
22. Weston S, Czieso S, White IJ, Smith SE, Kellam P, Marsh M 2014. A membrane topology model for human interferon inducible transmembrane protein 1. PLoS One 9:e104341. doi:.10.1371/journal.pone.0104341 [PMC free article] [PubMed] [Cross Ref]
23. Compton AA, Bruel T, Porrot F, Mallet A, Sachse M, Euvrard M, Liang C, Casartelli N, Schwartz O 2014. IFITM proteins incorporated into HIV-1 virions impair viral fusion and spread. Cell Host Microbe 16:736–747. doi:.10.1016/j.chom.2014.11.001 [PubMed] [Cross Ref]
24. Tartour K, Appourchaux R, Gaillard J, Nguyen XN, Durand S, Turpin J, Beaumont E, Roch E, Berger G, Mahieux R, Brand D, Roingeard P, Cimarelli A 2014. IFITM proteins are incorporated onto HIV-1 virion particles and negatively imprint their infectivity. Retrovirology 11:103. doi:.10.1186/s12977-014-0103-y [PMC free article] [PubMed] [Cross Ref]
25. Yu J, Li M, Wilkins J, Ding S, Swartz TH, Esposito AM, Zheng YM, Freed EO, Liang C, Chen BK, Liu SL 2015. IFITM proteins restrict HIV-1 infection by antagonizing the envelope glycoprotein. Cell Rep 13:145–156. doi:.10.1016/j.celrep.2015.08.055 [PMC free article] [PubMed] [Cross Ref]
26. Qian J, Le Duff Y, Wang Y, Pan Q, Ding S, Zheng YM, Liu SL, Liang C 2015. Primate lentiviruses are differentially inhibited by interferon-induced transmembrane proteins. Virology 474:10–18. doi:.10.1016/j.virol.2014.10.015 [PMC free article] [PubMed] [Cross Ref]
27. Wilkins J, Zheng YM, Yu J, Liang C, Liu SL 2016. Nonhuman primate IFITM proteins are potent inhibitors of HIV and SIV. PLoS One 11:e0156739. doi:.10.1371/journal.pone.0156739 [PMC free article] [PubMed] [Cross Ref]
28. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59:284–291. [PMC free article] [PubMed]
29. Collman R, Balliet J, Gregory S, Friedman H, Kolson D, Nathanson N, Srinivasan A 1992. An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J Virol 66:7517–7521. [PMC free article] [PubMed]
30. Koup R, Safrit JT, Cao Y, Andrews CA, McLeod G, Borkowsky W, Farthing C, Ho DD 1994. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J Virol 68:4650–4655. [PMC free article] [PubMed]
31. Li Y, Hui H, Burgess CJ, Price RW, Sharp PM, Hahn BH, Shaw GM 1992. Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation. J Virol 66:6587–6600. [PMC free article] [PubMed]
32. Li Y, Kappes JC, Conway JA, Price RW, Shaw GM, Hahn BH 1991. Molecular characterization of human immunodeficiency virus type 1 cloned directly from uncultured human brain tissue: identification of replication-competent and -defective viral genomes. J Virol 65:3973–3985. [PMC free article] [PubMed]
33. Foster TL, Wilson H, Iyer SS, Coss K, Doores K, Smith S, Kellam P, Finzi A, Borrow P, Hahn BH, Neil SJ 2016. Resistance of transmitted founder HIV-1 to IFITM-mediated restriction. Cell Host Microbe 20:429–442. doi:.10.1016/j.chom.2016.08.006 [PMC free article] [PubMed] [Cross Ref]
34. Freed EO, Englund G, Martin MA 1995. Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection. J Virol 69:3949–3954. [PMC free article] [PubMed]
35. Hamoudi M, Simon-Loriere E, Gasser R, Negroni M 2013. Genetic diversity of the highly variable V1 region interferes with human immunodeficiency virus type 1 envelope functionality. Retrovirology 10:114. doi:.10.1186/1742-4690-10-114 [PMC free article] [PubMed] [Cross Ref]
36. Cordonnier A, Montagnier L, Emerman M 1989. Single amino-acid changes in HIV envelope affect viral tropism and receptor binding. Nature 340:571–574. doi:.10.1038/340571a0 [PubMed] [Cross Ref]
37. Hwang SS, Boyle TJ, Lyerly HK, Cullen BR 1991. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 253:71–74. doi:.10.1126/science.1905842 [PubMed] [Cross Ref]
38. Fatkenheuer G, Pozniak AL, Johnson MA, Plettenberg A, Staszewski S, Hoepelman AI, Saag MS, Goebel FD, Rockstroh JK, Dezube BJ, Jenkins TM, Medhurst C, Sullivan JF, Ridgway C, Abel S, James IT, Youle M, van der Ryst E 2005. Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1. Nat Med 11:1170–1172. doi:.10.1038/nm1319 [PubMed] [Cross Ref]
39. Hendrix CW, Flexner C, MacFarland RT, Giandomenico C, Fuchs EJ, Redpath E, Bridger G, Henson GW 2000. Pharmacokinetics and safety of AMD-3100, a novel antagonist of the CXCR-4 chemokine receptor, in human volunteers. Antimicrob Agents Chemother 44:1667–1673. doi:.10.1128/AAC.44.6.1667-1673.2000 [PMC free article] [PubMed] [Cross Ref]
40. Hoffman TL, LaBranche CC, Zhang W, Canziani G, Robinson J, Chaiken I, Hoxie JA, Doms RW 1999. Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein. Proc Natl Acad Sci U S A 96:6359–6364. doi:.10.1073/pnas.96.11.6359 [PubMed] [Cross Ref]
41. Thali M, Moore J, Furman C, Charles M, Ho D, Robinson J, Sodroski J 1993. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120-CD4 binding. J Virol 67:3978–3988. [PMC free article] [PubMed]
42. Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, Zhou T, Schmidt SD, Wu L, Xu L, Longo NS, McKee K, O'Dell S, Louder MK, Wycuff DL, Feng Y, Nason M, Doria-Rose N, Connors M, Kwong PD, Roederer M, Wyatt RT, Nabel GJ, Mascola JR 2010. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329:856–861. doi:.10.1126/science.1187659 [PMC free article] [PubMed] [Cross Ref]
43. Sheehy AM, Gaddis NC, Choi JD, Malim MH 2002. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646–650. doi:.10.1038/nature00939 [PubMed] [Cross Ref]
44. Yu X, Yu Y, Liu B, Luo K, Kong W, Mao P, Yu XF 2003. Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302:1056–1060. doi:.10.1126/science.1089591 [PubMed] [Cross Ref]
45. Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J 2004. The cytoplasmic body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848–853. doi:.10.1038/nature02343 [PubMed] [Cross Ref]
46. Mudhasani R, Tran JP, Retterer C, Radoshitzky SR, Kota KP, Altamura LA, Smith JM, Packard BZ, Kuhn JH, Costantino J 2013. IFITM-2 and IFITM-3 but not IFITM-1 restrict Rift Valley fever virus. J Virol 87:8451–8464. doi:.10.1128/JVI.03382-12 [PMC free article] [PubMed] [Cross Ref]
47. Warren CJ, Griffin LM, Little AS, Huang I-C, Farzan M, Pyeon D 2014. The antiviral restriction factors IFITM1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus. PLoS One 9:e96579. doi:.10.1371/journal.pone.0096579 [PMC free article] [PubMed] [Cross Ref]
48. Ding S, Pan Q, Liu SL, Liang C 2014. HIV-1 mutates to evade IFITM1 restriction. Virology 454–455:11–24. doi:.10.1016/j.virol.2014.01.020 [PMC free article] [PubMed] [Cross Ref]
49. Jia R, Ding S, Pan Q, Liu SL, Qiao W, Liang C 2015. The C-terminal sequence of IFITM1 regulates its anti-HIV-1 activity. PLoS One 10:e0118794. doi:.10.1371/journal.pone.0118794 [PMC free article] [PubMed] [Cross Ref]
50. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C, Sodroski J 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135–1148. doi:.10.1016/S0092-8674(00)81313-6 [PubMed] [Cross Ref]
51. Cocchi F, DeVico AL, Garzino-Demo A, Cara A, Gallo RC, Lusso P 1996. The V3 domain of the HIV-1 gp120 envelope glycoprotein is critical for chemokine-mediated blockade of infection. Nat Med 2:1244–1247. doi:.10.1038/nm1196-1244 [PubMed] [Cross Ref]
52. Trkola A, Dragic T, Arthos J, Binley JM, Olson WC, Allaway GP, Cheng-Mayer C, Robinson J, Maddon PJ, Moore JP 1996. CD4-dependent, antibody-sensitive interactions between HIV-1 and its co-receptor CCR-5. Nature 384:184–187. doi:.10.1038/384184a0 [PubMed] [Cross Ref]
53. Pancera M, Zhou T, Druz A, Georgiev IS, Soto C, Gorman J, Huang J, Acharya P, Chuang GY, Ofek G, Stewart-Jones GB, Stuckey J, Bailer RT, Joyce MG, Louder MK, Tumba N, Yang Y, Zhang B, Cohen MS, Haynes BF, Mascola JR, Morris L, Munro JB, Blanchard SC, Mothes W, Connors M, Kwong PD 2014. Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature 514:455–461. doi:.10.1038/nature13808 [PMC free article] [PubMed] [Cross Ref]
54. Davis KL, Gray ES, Moore PL, Decker JM, Salomon A, Montefiori DC, Graham BS, Keefer MC, Pinter A, Morris L, Hahn BH, Shaw GM 2009. High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination. Virology 387:414–426. doi:.10.1016/j.virol.2009.02.022 [PMC free article] [PubMed] [Cross Ref]
55. Letvin NL, Robinson S, Rohne D, Axthelm MK, Fanton JW, Bilska M, Palker TJ, Liao HX, Haynes BF, Montefiori DC 2001. Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope. J Virol 75:4165–4175. doi:.10.1128/JVI.75.9.4165-4175.2001 [PMC free article] [PubMed] [Cross Ref]
56. Zolla-Pazner S. 2005. Improving on nature: focusing the immune response on the V3 loop. Hum Antibodies 14:69–72. [PubMed]
57. Munro JB, Gorman J, Ma X, Zhou Z, Arthos J, Burton DR, Koff WC, Courter JR, Smith AB III, Kwong PD, Blanchard SC, Mothes W 2014. Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science 346:759–763. doi:.10.1126/science.1254426 [PMC free article] [PubMed] [Cross Ref]
58. Zhao X, Guo F, Liu F, Cuconati A, Chang J, Block TM, Guo J-T 2014. Interferon induction of IFITM proteins promotes infection by human coronavirus OC43. Proc Natl Acad Sci U S A 111:6756–6761. doi:.10.1073/pnas.1320856111 [PubMed] [Cross Ref]
59. Krapp C, Hotter D, Gawanbacht A, McLaren PJ, Kluge SF, Sturzel CM, Mack K, Reith E, Engelhart S, Ciuffi A, Hornung V, Sauter D, Telenti A, Kirchhoff F 2016. Guanylate binding protein (GBP) 5 is an interferon-inducible inhibitor of HIV-1 infectivity. Cell Host Microbe 19:504–514. doi:.10.1016/j.chom.2016.02.019 [PubMed] [Cross Ref]
60. Tada T, Zhang Y, Koyama T, Tobiume M, Tsunetsugu-Yokota Y, Yamaoka S, Fujita H, Tokunaga K 2015. MARCH8 inhibits HIV-1 infection by reducing virion incorporation of envelope glycoproteins. Nat Med 21:1502–1507. doi:.10.1038/nm.3956 [PubMed] [Cross Ref]
61. Rosa A, Chande A, Ziglio S, De Sanctis V, Bertorelli R, Goh SL, McCauley SM, Nowosielska A, Antonarakis SE, Luban J 2015. HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation. Nature 526:212–217. doi:.10.1038/nature15399 [PMC free article] [PubMed] [Cross Ref]
62. Usami Y, Wu Y, Gottlinger HG 2015. SERINC3 and SERINC5 restrict HIV-1 infectivity and are counteracted by Nef. Nature 526:218–223. doi:.10.1038/nature15400 [PMC free article] [PubMed] [Cross Ref]
63. Krowicka H, Robinson JE, Clark R, Hager S, Broyles S, Pincus SH 2008. Use of tissue culture cell lines to evaluate HIV antiviral resistance. AIDS Res Hum Retroviruses 24:957–967. doi:.10.1089/aid.2007.0242 [PMC free article] [PubMed] [Cross Ref]
64. Harada S, Koyanagi Y, Yamamoto N 1985. Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay. Science 229:563–566. doi:.10.1126/science.2992081 [PubMed] [Cross Ref]
65. Cavrois M, de Noronha C, Greene WC 2002. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat Biotechnol 20:1151–1154. doi:.10.1038/nbt745 [PubMed] [Cross Ref]
66. Cavrois M, Neidleman J, Bigos M, Greene WC 2004. Fluorescence resonance energy transfer-based HIV-1 virion fusion assay. Methods Mol Biol 263:333–343. [PubMed]
67. Ding S, Medjahed H, Prévost J, Coutu M, Xiang SH, Finzi A 2016. Lineage-specific differences between the gp120 inner domain layer 3 of human immunodeficiency virus and that of simian immunodeficiency virus. J Virol 90:10065–10073. doi:.10.1128/JVI.01215-16 [PMC free article] [PubMed] [Cross Ref]
68. Desormeaux A, Coutu M, Medjahed H, Pacheco B, Herschhorn A, Gu C, Xiang SH, Mao Y, Sodroski J, Finzi A 2013. The highly conserved layer-3 component of the HIV-1 gp120 inner domain is critical for CD4-required conformational transitions. J Virol 87:2549–2562. doi:.10.1128/JVI.03104-12 [PMC free article] [PubMed] [Cross Ref]
69. Finzi A, Xiang SH, Pacheco B, Wang L, Haight J, Kassa A, Danek B, Pancera M, Kwong PD, Sodroski J 2010. Topological layers in the HIV-1 gp120 inner domain regulate gp41 interaction and CD4-triggered conformational transitions. Mol Cell 37:656–667. doi:.10.1016/j.molcel.2010.02.012 [PMC free article] [PubMed] [Cross Ref]

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