COMMENTARY
crossm New Connections: Cell-to-Cell HIV-1 Transmission, Resistance to Broadly Neutralizing Antibodies, and an Envelope Sorting Motif S. Abigail Smith,a,b Cynthia A. Derdeyna,b,c Yerkes National Primate Research Center,a Emory Vaccine Center,b and Department of Pathology and Laboratory Medicine,c Emory University, Atlanta, Georgia, USA
HIV-1 infection from cell-to-cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al. (H. Li, C. Zony, P. Chen, and B. K. Chen, J. Virol. 91:e02425-16, 2017, https://doi.org/ 10.1128/JVI.02425-16) showed that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell-to-cell infection in a context-dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes. ABSTRACT
KEYWORDS broadly neutralizing antibody, human immunodeficiency virus, viral
envelope, virological synapse
Citation Smith SA, Derdeyn CA. 2017. New connections: cell-to-cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif. J Virol 91: e00149-17. https://doi.org/10.1128/ JVI.00149-17. Editor Guido Silvestri, Emory University Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Cynthia A. Derdeyn, [email protected].
he best-understood process by which HIV-1 mediates infection of susceptible CD4⫹ target cells is via cell-free virions. Independent virions attach to the target cell membrane, bind CD4 and the coreceptor, mediate membrane fusion at neutral pH, and deposit the nucleocapsid into the new host cell to initiate de novo replication. However, HIV-1 can also mediate infection via virological synapses, where direct cell-to-cell contact between the envelope (Env) glycoprotein gp120 subunit expressed on the infected CD4⫹ T cell surface interacts with the CD4 receptor on nearby uninfected T cells (reviewed in reference 1). This mode of virus transfer is known as a virological synapse, whereas an immunological synapse mediates transfer from a virus-harboring antigen-presenting cell to an uninfected CD4⫹ T cell. Cell-to-cell HIV-1 infection was described as early as 1989, including one study that also established the relative levels of resistance of this transmission mode to neutralizing antibody (nAb) and the nucleoside reverse transcriptase inhibitor AZT (2, 3). Cell-to-cell HIV-1 infection has also been estimated to be several orders of magnitude more efficient than cell-free infection (3–6). Although it could represent a predominant mode of viral spread in vivo, cell-tocell transmission has not been studied to the same depth as cell-free infection, as almost all in vitro neutralization assays and in vivo broadly neutralizing antibody (bnAb) protection experiments have been performed using cell-free virus. Synaptic transfer of HIV-1 allows the virus to exploit the extensive system of naturally occurring interactions between immune cells. Virological synapses are initiated by Env-CD4 interactions (6) but may be stabilized and regulated by cellular adhesion molecules such as LFA-1 and ICAM-1 (7–11) or the gut-homing receptor ␣47 (12). Cell-to-cell spread through virological synapses could present a number of potential advantages for the virus. Viral gene expression can be detected earlier in cells infected through cell contact than with cell-free virus (13). Cell-to-cell infection can also facilitate transfer of multiple copies of HIV-1 (14–17), increasing the multiplicity of infection. This can lessen the susceptibility of the virus to some classes of antiretroviral
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For the article discussed, see https://doi.org/ 10.1128/JVI.02425-16. The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.
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inhibitors, as well as provide greater genetic diversity and allow persistence of less-fit variants (18, 19). A single cell can also form virological synapses simultaneously with multiple uninfected target cells, further amplifying infectivity (11). The presence of cells that harbor multiple copies of HIV-1 from infected patients supports the concept that this phenomenon occurs during in vivo HIV-1 infection (20–22). Further support for the idea of the importance of cell-to-cell contact in vivo comes from the recent identification of a cell aggregation factor, activated leukocyte cell adhesion molecule (ALCAM), as a host dependency genetic marker of HIV-1 infection (23). Interestingly, that study also demonstrated that knockdown of ALCAM protected cells in vitro from cellassociated infection but not from cell-free infection. HIV-1 has mechanisms in place to regulate formation of syncytia during contact between infected and uninfected cells, such that membrane fusion does not occur prematurely (24–26). Recent evidence demonstrated that HIV-1 Env can function to selectively transmit virus to activated CD4 T cells during a synapse, as well as prolonging the activated state of the acceptor target cells and enhancing the potential for productive infection (27). Cell-to-cell HIV-1 transmission also appears to contribute to pathogenesis by inducing pyroptosis of neighboring lymphoid CD4⫹ T cells, while exposure to cell-free virions does not mimic this process (28). The authors of that study proposed that a cycle of pathogenesis is initiated by a virological synapse, in which the productively infected donor cell dies of apoptosis while the bystander acceptor cell dies through pyroptosis. There is strong evidence that cell-to-cell infection facilitates local spread of virus in lymph node microclusters through T cell motility and tethering interactions (17, 29, 30). All of these studies support the notion that cell-to-cell transfer of HIV-1 through virological synapses is an important component of viral spread and ensuing pathogenesis. Cell-to-cell spread may also be important in promoting resistance to antibody neutralization and persistence of virus in vivo. Cell-to-cell transmission reduces susceptibility to single antiretroviral agents, likely due to increases in the multiplicity of infection and tolerance of antiretroviral-resistant, fitness-impaired variants (31). However, combinations of antiretroviral therapies overcome this and are effective against cell-to-cell transmission, contributing to the success of these multidrug approaches in vivo (32). Recent studies have also provided evidence that cell-to-cell HIV-1 infection has the potential to lessen the effectiveness of neutralizing antibodies in vivo. Even a slight decrease in susceptibility in vivo could result in a substantial effect on virus production in amplification over multiple rounds of replication. Studies have consistently shown that HIV-1 broadly neutralizing antibodies can block cell-to-cell infection but that much higher concentrations or bnAb combinations are frequently required (33–37). All of those studies reported that differences between cell-free neutralization and cell-associated neutralization were seen across virus strains and antibody epitopes. Indeed, substantial variability can also be attributable to whether the assay system utilized cell lines or primary cells, adherent or suspension cells, acutely transfected or chronically infected donor cells, virus transfer or productive infection as an endpoint, and laboratory-adapted or patient-derived viral Envs (1, 4). In fact, chronically infected cells appear to transfer virus more directly, via Env-mediated fusion at the plasma membrane (38), while acutely transfected cells seem to require the endocytosis of immature virions following membrane fusion (39). These findings have important implications for neutralizing antibody-based vaccination and therapeutic strategies (1), as there might be an increased probability of bnAb resistance mutations in cell-cell compared to cell-free virus spread, further highlighting the importance of understanding the decreased susceptibility of cell-to-cell HIV-1 infection to neutralization (37). In this issue of the Journal of Virology, Li et al. provide new insight into the relative resistance of cell-to-cell HIV-1 infection to antibody neutralization (40). The authors investigated a panel of HIV-1 bnAbs that included epitopes in V1/V2, CD4bs, V3-glycan patch, the gp41 membrane-proximal external region (MPER), and the gp120/gp41 interface. Two clade B transmitted/founder (T/F) Envs, along with clade B laboratoryadapted strain NL4.3 and a well-characterized clade B Env from chronic infection (JR-FL), were analyzed. The main findings of the study were that consistent relative May 2017 Volume 91 Issue 9 e00149-17
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resistance of HIV-1 to bnAbs was observed in cell-to-cell infections compared to cell-free infections. This resistance was reflected in decreased antibody potency and diminished maximum neutralization capacity compared to the levels seen with cell-free virus infection. Recent studies have highlighted the importance of the slope of the neutralization curve (41) and the level of incomplete neutralization (42), in addition to the 50% inhibitory concentration (IC50), in predicting the therapeutic potential of bnAbs, but these were not measured in the context of cell-to-cell infection. Li et al. demonstrated that some differences between cell-to-cell infection and cell-free infection were dramatic; for example, gp120/gp41 interface bnAb 35022 was 3,400-fold less effective in neutralizing one T/F Env during cell-to-cell infection. bnAbs PGT126 and 10-1074, which target V3-glycan patch epitopes, were 1,900- and 2,400-fold less effective against cell-to-cell infection of a T/F Env. VRC01 is a potent and wellcharacterized CD4 binding site bnAb that has been passively administered to chronically HIV-1-infected individuals (43) and to those undergoing discontinuation of antiretroviral therapy (44). This bnAb was anywhere from 3.7-fold to 45.7-fold less effective against cell-to-cell infection, depending on the Env variant. Overall, the T/F Envs tended to show greater disparities in cell-to-cell versus cell-free susceptibility to bnAb neutralization than the more commonly used laboratory strains, highlighting the need for caution in interpreting results solely on the basis of laboratory isolates. Furthermore, studies of bnAb cell-to-cell infection have been carried out mainly with clade B HIV-1 Envs, and inclusion of more genetically diverse variants that are representative of other globally significant subtypes will be important (37). As discussed previously, the decreased effectiveness against cell-to-cell infection was consistent, but the magnitude ranged widely depending on the Env variant and epitope targeted by the bnAb. The authors also demonstrated that plasma samples from HIV-1-infected individuals were less able to inhibit cell-to-cell HIV-1 infection, which is consistent with prior studies (2, 6, 39, 45). These polyclonal antibodies may be more reflective of the neutralization activity produced in most HIV-1-infected individuals than bnAbs, which occur only rarely. It is not known whether neutralization of autologous variants would also be less effective against cell-to-cell infection. In the study by Li et al., the disparity between the levels of cell-to-cell and cell-free neutralization susceptibility could not be attributed to differences in the virus producer cell. Along with those from other studies, the results from Li et al. highlight the fact that the disconnection between cell-free and cell-to-cell infection should be considered in designing and evaluating neutralizing antibodybased vaccination and therapeutic strategies. Li et al. also provided novel mechanistic insight into the barriers raised by cell-to-cell infection. After having shown in a previous study that the gp41 cytoplasmic tail was important in regulating neutralizing epitope exposure during cell-to-cell infection (6, 46), the authors homed in on a tyrosine residue that forms the anchor for the YXXL membrane-proximal sorting motif in gp41 as a contributor to neutralization susceptibility during cell-to-cell infection. In that study, the tyrosine was mutated to alanine (Y712A, based on HXB2 numbering), which is not a substitution that occurs naturally in circulating HIV-1 variants. This change was shown to increase susceptibility to neutralization during cell-to-cell infection mediated by a number of bnAbs, in some cases dramatically. In contrast, in the case of cell-free infection, the one Env-bnAb combination that was tested showed that the Y712A mutant Env was more susceptible to neutralization than the wild-type Env. Interestingly, this motif conforms to the defined YXXphi trafficking/sorting motif, where Y represents tyrosine, XX represents any amino acid residues, and phi represents an amino acid with a bulky hydrophobic side chain, such as leucine. A motif of this sort at a location proximal to that of the membranespanning domain of viral glycoproteins is highly conserved across primate lentiviruses (47) and is found commonly in cellular membrane proteins. In vivo studies of alpha herpesviruses have shown that YXXL-dependent endocytosis of certain viral proteins contributes to cell-to-cell spread and virulence (48). The Y712-XXL motif is highly conserved across HIV-1/chimpanzee simian immunodeficiency virus (SIVcpz), HIV-2, and sooty mangabey SIV (SIVsmm) variants (for HIV-1 sequence conservation data, see May 2017 Volume 91 Issue 9 e00149-17
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FIG 1 The gp41 membrane-proximal tyrosine-based endocytosis motif is highly conserved across HIV-1 variants. The AnalyzeAlign tool (https://www.hiv.lanl.gov/content/sequence/ANALYZEALIGN/analyze_ align.html) was used to create a weblogo that included 4,632 HIV-1 sequences from the Los Alamos HIV Database. The amino acid positions correspond to the YXXL motif and are shown on the x axis in reference to HXB2 numbering. The y axis data indicate the abundance of each amino acid occurring at the indicated positions. The amino acids are colored according to hydrophobicity.
Fig. 1). In HIV-1, the tyrosine is virtually invariant, whereas little to no variation is tolerated in the other positions. This motif has a role in the internalization of HIV-1 and SIV Env from the cell surface, as well as in basolateral targeting of Env, through interactions with the clathrin adaptor complex (49–52). Other studies also support the concept that YXXL influences susceptibility to immune responses and facilitates cellto-cell spread of virus (49, 53–55). Studies of the role of YXXL in pathogenesis and disease progression have been carried out using SIVmac239, where the analogous position is Y721. Early studies found that this tyrosine and the surrounding residues were dispensable for SIVmac239 replication in vitro but were critical for in vivo viral fitness and pathogenesis in rhesus macaques (56). Disruption of the YXXL motif in SIVmac239 resulted in an attenuated virus that exhibited a profound reduction in viral load and delayed progression to disease compared to the wild-type strain (56). Furthermore, reversion of the Y721I mutation led to reinstatement of high viral loads and disease progression (56). Morerecent studies of the contributions of this motif to pathogenesis have utilized a G720 Y721 deletion (ΔGY) SIVmac239 virus in the rhesus macaque model (57). This deletion virus, unlike the wild-type virus, did not deplete mucosal CD4 T cells. Nevertheless, ΔGY-infected animals exhibited immune activation and progressed to AIDS-like disease. In a recent study using a different nonhuman primate host, the pig-tailed macaque, infection with the ΔGY virus again resulted in preservation of mucosal CD4 T cells; however, in contrast to the results seen with rhesus macaques, viral replication of the ΔGY virus was controlled to elite levels, and the pig-tailed macaques did not develop immune activation or progress to disease (58). Thus, disruption of this motif appears to alter the course of SIVmac239 pathogenesis in a manner that is specific to the macaque species. The prominence of the tyrosine-based endocytosis motif for HIV-1 was further highlighted in a recent structural study, which placed the YXXL motif within a hydrophilic core of the membrane-spanning domain and demonstrated that this core is critical for maintaining stability of the Env trimer (59). Deletion of the hydrophilic core disrupts the trimer, such that the mutant becomes sensitive to nonneutralizing antibodies and resistant to trimer-specific neutralizing antibodies. This effect was most pronounced in the context of cell-to-cell fusion and was similar but less dramatic in the context of Env pseudoviruses. Thus, in addition to serving as an endocytosis and basolateral targeting signal and playing a significant role in SIV pathogenesis, the membrane-proximal, tyrosine-based sorting motif appears to participate directly in maintaining trimer stability. May 2017 Volume 91 Issue 9 e00149-17
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Why would cell-to-cell HIV-1 infection be less susceptible to neutralizing antibodies? One explanation could be the increased multiplicity of infection observed in cell-to-cell infection; however, this seems to be directly proportional to resistance to antiretroviral inhibitors but not to resistance to neutralizing antibodies (1, 32). It is also possible that Env conformations differ between cell-free and cell-cell infections and that this is regulated by signals in the cytoplasmic tail, as shown previously and in the recent study by Li et al. (6, 46). Furthermore, the virological synapse is dependent on Env binding to CD4 on the cell surface, suggesting that Env is accessible to antibodies at some point during this process. However, the synapse may sterically limit access by some antibodies, and this may affect some epitopes more than others, as well as being subject to variation between genetically diverse HIV-1 Envs. There may also be kinetic barriers as Env transitions through conformational states, as well as differences among the on-off rates of the individual antibodies, which could also behave differently for different HIV-1 Envs, since all combinations are heterologous. Finally, if membrane fusion occurs within endosomal compartments in the target cell, this could also limit the exposure of some neutralization epitopes (39). Additional studies are necessary to understand why some neutralizing antibodies are less efficient against cell-to-cell infection and why this varies with different HIV-1 strains and cell types. In summary, Li et al. have provided new information about why HIV-1 is consistently less susceptible to neutralization by multiple classes of bnAbs, in a manner that is dependent upon both the viral strain and epitope targeted by the antibody. Importantly, they have shown that some bnAbs lose potency and the ability to achieve complete neutralization against cell-to-cell infection, even at high concentrations, and that this should be considered for antibody-based therapeutics and protection strategies. The fact that the neutralization susceptibility of cell-to-cell infection is modulated by a highly conserved motif in gp41 that contributes to Env internalization, basolateral targeting, and trimer stability supports the concept that Env conformation and epitope accessibility may play a role in the relative resistance of cell-to-cell infection which warrants further investigation.
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56. Fultz PN, Vance PJ, Endres MJ, Tao B, Dvorin JD, Davis IC, Lifson JD, Montefiori DC, Marsh M, Malim MH, Hoxie JA. 2001. In vivo attenuation of simian immunodeficiency virus by disruption of a tyrosine-dependent sorting signal in the envelope glycoprotein cytoplasmic tail. J Virol 75:278 –291. https://doi.org/10.1128/JVI.75.1.278-291.2001. 57. Breed MW, Jordan AP, Aye PP, Lichtveld CF, Midkiff CC, Schiro FR, Haggarty BS, Sugimoto C, Alvarez X, Sandler NG, Douek DC, Kuroda MJ, Pahar B, Piatak M, Jr, Lifson JD, Keele BF, Hoxie JA, Lackner AA. 2013. Loss of a tyrosine-dependent trafficking motif in the simian immunodeficiency virus envelope cytoplasmic tail spares mucosal CD4 cells but does not prevent disease progression. J Virol 87:1528 –1543. https://doi.org/ 10.1128/JVI.01928-12. 58. Breed MW, Elser SE, Torben W, Jordan AP, Aye PP, Midkiff C, Schiro F, Sugimoto C, Alvarez-Hernandez X, Blair RV, Somasunderam A, Utay NS, Kuroda MJ, Pahar B, Wiseman RW, O’Connor DH, LaBranche CC, Montefiori DC, Marsh M, Li Y, Piatak M, Jr, Lifson JD, Keele BF, Fultz PN, Lackner AA, Hoxie JA. 2015. Elite control, gut CD4 T cell sparing, and enhanced mucosal T cell responses in Macaca nemestrina infected by a simian immunodeficiency virus lacking a gp41 trafficking motif. J Virol 89: 10156 –10175. https://doi.org/10.1128/JVI.01134-15. 59. Dev J, Park D, Fu Q, Chen J, Ha HJ, Ghantous F, Herrmann T, Chang W, Liu Z, Frey G, Seaman MS, Chen B, Chou JJ. 2016. Structural basis for membrane anchoring of HIV-1 envelope spike. Science 353:172–175. https://doi.org/10.1126/science.aaf7066.
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crossm New Connections: Cell-to-Cell HIV-1 Transmission, Resistance to Broadly Neutralizing Antibodies, and an Envelope Sorting Motif S. Abigail Smith,a,b Cynthia A. Derdeyna,b,c Yerkes National Primate Research Center,a Emory Vaccine Center,b and Department of Pathology and Laboratory Medicine,c Emory University, Atlanta, Georgia, USA
HIV-1 infection from cell-to-cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al. (H. Li, C. Zony, P. Chen, and B. K. Chen, J. Virol. 91:e02425-16, 2017, https://doi.org/ 10.1128/JVI.02425-16) showed that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell-to-cell infection in a context-dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes. ABSTRACT
KEYWORDS broadly neutralizing antibody, human immunodeficiency virus, viral
envelope, virological synapse
Citation Smith SA, Derdeyn CA. 2017. New connections: cell-to-cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif. J Virol 91: e00149-17. https://doi.org/10.1128/ JVI.00149-17. Editor Guido Silvestri, Emory University Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Cynthia A. Derdeyn, [email protected].
he best-understood process by which HIV-1 mediates infection of susceptible CD4⫹ target cells is via cell-free virions. Independent virions attach to the target cell membrane, bind CD4 and the coreceptor, mediate membrane fusion at neutral pH, and deposit the nucleocapsid into the new host cell to initiate de novo replication. However, HIV-1 can also mediate infection via virological synapses, where direct cell-to-cell contact between the envelope (Env) glycoprotein gp120 subunit expressed on the infected CD4⫹ T cell surface interacts with the CD4 receptor on nearby uninfected T cells (reviewed in reference 1). This mode of virus transfer is known as a virological synapse, whereas an immunological synapse mediates transfer from a virus-harboring antigen-presenting cell to an uninfected CD4⫹ T cell. Cell-to-cell HIV-1 infection was described as early as 1989, including one study that also established the relative levels of resistance of this transmission mode to neutralizing antibody (nAb) and the nucleoside reverse transcriptase inhibitor AZT (2, 3). Cell-to-cell HIV-1 infection has also been estimated to be several orders of magnitude more efficient than cell-free infection (3–6). Although it could represent a predominant mode of viral spread in vivo, cell-tocell transmission has not been studied to the same depth as cell-free infection, as almost all in vitro neutralization assays and in vivo broadly neutralizing antibody (bnAb) protection experiments have been performed using cell-free virus. Synaptic transfer of HIV-1 allows the virus to exploit the extensive system of naturally occurring interactions between immune cells. Virological synapses are initiated by Env-CD4 interactions (6) but may be stabilized and regulated by cellular adhesion molecules such as LFA-1 and ICAM-1 (7–11) or the gut-homing receptor ␣47 (12). Cell-to-cell spread through virological synapses could present a number of potential advantages for the virus. Viral gene expression can be detected earlier in cells infected through cell contact than with cell-free virus (13). Cell-to-cell infection can also facilitate transfer of multiple copies of HIV-1 (14–17), increasing the multiplicity of infection. This can lessen the susceptibility of the virus to some classes of antiretroviral
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For the article discussed, see https://doi.org/ 10.1128/JVI.02425-16. The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.
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inhibitors, as well as provide greater genetic diversity and allow persistence of less-fit variants (18, 19). A single cell can also form virological synapses simultaneously with multiple uninfected target cells, further amplifying infectivity (11). The presence of cells that harbor multiple copies of HIV-1 from infected patients supports the concept that this phenomenon occurs during in vivo HIV-1 infection (20–22). Further support for the idea of the importance of cell-to-cell contact in vivo comes from the recent identification of a cell aggregation factor, activated leukocyte cell adhesion molecule (ALCAM), as a host dependency genetic marker of HIV-1 infection (23). Interestingly, that study also demonstrated that knockdown of ALCAM protected cells in vitro from cellassociated infection but not from cell-free infection. HIV-1 has mechanisms in place to regulate formation of syncytia during contact between infected and uninfected cells, such that membrane fusion does not occur prematurely (24–26). Recent evidence demonstrated that HIV-1 Env can function to selectively transmit virus to activated CD4 T cells during a synapse, as well as prolonging the activated state of the acceptor target cells and enhancing the potential for productive infection (27). Cell-to-cell HIV-1 transmission also appears to contribute to pathogenesis by inducing pyroptosis of neighboring lymphoid CD4⫹ T cells, while exposure to cell-free virions does not mimic this process (28). The authors of that study proposed that a cycle of pathogenesis is initiated by a virological synapse, in which the productively infected donor cell dies of apoptosis while the bystander acceptor cell dies through pyroptosis. There is strong evidence that cell-to-cell infection facilitates local spread of virus in lymph node microclusters through T cell motility and tethering interactions (17, 29, 30). All of these studies support the notion that cell-to-cell transfer of HIV-1 through virological synapses is an important component of viral spread and ensuing pathogenesis. Cell-to-cell spread may also be important in promoting resistance to antibody neutralization and persistence of virus in vivo. Cell-to-cell transmission reduces susceptibility to single antiretroviral agents, likely due to increases in the multiplicity of infection and tolerance of antiretroviral-resistant, fitness-impaired variants (31). However, combinations of antiretroviral therapies overcome this and are effective against cell-to-cell transmission, contributing to the success of these multidrug approaches in vivo (32). Recent studies have also provided evidence that cell-to-cell HIV-1 infection has the potential to lessen the effectiveness of neutralizing antibodies in vivo. Even a slight decrease in susceptibility in vivo could result in a substantial effect on virus production in amplification over multiple rounds of replication. Studies have consistently shown that HIV-1 broadly neutralizing antibodies can block cell-to-cell infection but that much higher concentrations or bnAb combinations are frequently required (33–37). All of those studies reported that differences between cell-free neutralization and cell-associated neutralization were seen across virus strains and antibody epitopes. Indeed, substantial variability can also be attributable to whether the assay system utilized cell lines or primary cells, adherent or suspension cells, acutely transfected or chronically infected donor cells, virus transfer or productive infection as an endpoint, and laboratory-adapted or patient-derived viral Envs (1, 4). In fact, chronically infected cells appear to transfer virus more directly, via Env-mediated fusion at the plasma membrane (38), while acutely transfected cells seem to require the endocytosis of immature virions following membrane fusion (39). These findings have important implications for neutralizing antibody-based vaccination and therapeutic strategies (1), as there might be an increased probability of bnAb resistance mutations in cell-cell compared to cell-free virus spread, further highlighting the importance of understanding the decreased susceptibility of cell-to-cell HIV-1 infection to neutralization (37). In this issue of the Journal of Virology, Li et al. provide new insight into the relative resistance of cell-to-cell HIV-1 infection to antibody neutralization (40). The authors investigated a panel of HIV-1 bnAbs that included epitopes in V1/V2, CD4bs, V3-glycan patch, the gp41 membrane-proximal external region (MPER), and the gp120/gp41 interface. Two clade B transmitted/founder (T/F) Envs, along with clade B laboratoryadapted strain NL4.3 and a well-characterized clade B Env from chronic infection (JR-FL), were analyzed. The main findings of the study were that consistent relative May 2017 Volume 91 Issue 9 e00149-17
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resistance of HIV-1 to bnAbs was observed in cell-to-cell infections compared to cell-free infections. This resistance was reflected in decreased antibody potency and diminished maximum neutralization capacity compared to the levels seen with cell-free virus infection. Recent studies have highlighted the importance of the slope of the neutralization curve (41) and the level of incomplete neutralization (42), in addition to the 50% inhibitory concentration (IC50), in predicting the therapeutic potential of bnAbs, but these were not measured in the context of cell-to-cell infection. Li et al. demonstrated that some differences between cell-to-cell infection and cell-free infection were dramatic; for example, gp120/gp41 interface bnAb 35022 was 3,400-fold less effective in neutralizing one T/F Env during cell-to-cell infection. bnAbs PGT126 and 10-1074, which target V3-glycan patch epitopes, were 1,900- and 2,400-fold less effective against cell-to-cell infection of a T/F Env. VRC01 is a potent and wellcharacterized CD4 binding site bnAb that has been passively administered to chronically HIV-1-infected individuals (43) and to those undergoing discontinuation of antiretroviral therapy (44). This bnAb was anywhere from 3.7-fold to 45.7-fold less effective against cell-to-cell infection, depending on the Env variant. Overall, the T/F Envs tended to show greater disparities in cell-to-cell versus cell-free susceptibility to bnAb neutralization than the more commonly used laboratory strains, highlighting the need for caution in interpreting results solely on the basis of laboratory isolates. Furthermore, studies of bnAb cell-to-cell infection have been carried out mainly with clade B HIV-1 Envs, and inclusion of more genetically diverse variants that are representative of other globally significant subtypes will be important (37). As discussed previously, the decreased effectiveness against cell-to-cell infection was consistent, but the magnitude ranged widely depending on the Env variant and epitope targeted by the bnAb. The authors also demonstrated that plasma samples from HIV-1-infected individuals were less able to inhibit cell-to-cell HIV-1 infection, which is consistent with prior studies (2, 6, 39, 45). These polyclonal antibodies may be more reflective of the neutralization activity produced in most HIV-1-infected individuals than bnAbs, which occur only rarely. It is not known whether neutralization of autologous variants would also be less effective against cell-to-cell infection. In the study by Li et al., the disparity between the levels of cell-to-cell and cell-free neutralization susceptibility could not be attributed to differences in the virus producer cell. Along with those from other studies, the results from Li et al. highlight the fact that the disconnection between cell-free and cell-to-cell infection should be considered in designing and evaluating neutralizing antibodybased vaccination and therapeutic strategies. Li et al. also provided novel mechanistic insight into the barriers raised by cell-to-cell infection. After having shown in a previous study that the gp41 cytoplasmic tail was important in regulating neutralizing epitope exposure during cell-to-cell infection (6, 46), the authors homed in on a tyrosine residue that forms the anchor for the YXXL membrane-proximal sorting motif in gp41 as a contributor to neutralization susceptibility during cell-to-cell infection. In that study, the tyrosine was mutated to alanine (Y712A, based on HXB2 numbering), which is not a substitution that occurs naturally in circulating HIV-1 variants. This change was shown to increase susceptibility to neutralization during cell-to-cell infection mediated by a number of bnAbs, in some cases dramatically. In contrast, in the case of cell-free infection, the one Env-bnAb combination that was tested showed that the Y712A mutant Env was more susceptible to neutralization than the wild-type Env. Interestingly, this motif conforms to the defined YXXphi trafficking/sorting motif, where Y represents tyrosine, XX represents any amino acid residues, and phi represents an amino acid with a bulky hydrophobic side chain, such as leucine. A motif of this sort at a location proximal to that of the membranespanning domain of viral glycoproteins is highly conserved across primate lentiviruses (47) and is found commonly in cellular membrane proteins. In vivo studies of alpha herpesviruses have shown that YXXL-dependent endocytosis of certain viral proteins contributes to cell-to-cell spread and virulence (48). The Y712-XXL motif is highly conserved across HIV-1/chimpanzee simian immunodeficiency virus (SIVcpz), HIV-2, and sooty mangabey SIV (SIVsmm) variants (for HIV-1 sequence conservation data, see May 2017 Volume 91 Issue 9 e00149-17
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FIG 1 The gp41 membrane-proximal tyrosine-based endocytosis motif is highly conserved across HIV-1 variants. The AnalyzeAlign tool (https://www.hiv.lanl.gov/content/sequence/ANALYZEALIGN/analyze_ align.html) was used to create a weblogo that included 4,632 HIV-1 sequences from the Los Alamos HIV Database. The amino acid positions correspond to the YXXL motif and are shown on the x axis in reference to HXB2 numbering. The y axis data indicate the abundance of each amino acid occurring at the indicated positions. The amino acids are colored according to hydrophobicity.
Fig. 1). In HIV-1, the tyrosine is virtually invariant, whereas little to no variation is tolerated in the other positions. This motif has a role in the internalization of HIV-1 and SIV Env from the cell surface, as well as in basolateral targeting of Env, through interactions with the clathrin adaptor complex (49–52). Other studies also support the concept that YXXL influences susceptibility to immune responses and facilitates cellto-cell spread of virus (49, 53–55). Studies of the role of YXXL in pathogenesis and disease progression have been carried out using SIVmac239, where the analogous position is Y721. Early studies found that this tyrosine and the surrounding residues were dispensable for SIVmac239 replication in vitro but were critical for in vivo viral fitness and pathogenesis in rhesus macaques (56). Disruption of the YXXL motif in SIVmac239 resulted in an attenuated virus that exhibited a profound reduction in viral load and delayed progression to disease compared to the wild-type strain (56). Furthermore, reversion of the Y721I mutation led to reinstatement of high viral loads and disease progression (56). Morerecent studies of the contributions of this motif to pathogenesis have utilized a G720 Y721 deletion (ΔGY) SIVmac239 virus in the rhesus macaque model (57). This deletion virus, unlike the wild-type virus, did not deplete mucosal CD4 T cells. Nevertheless, ΔGY-infected animals exhibited immune activation and progressed to AIDS-like disease. In a recent study using a different nonhuman primate host, the pig-tailed macaque, infection with the ΔGY virus again resulted in preservation of mucosal CD4 T cells; however, in contrast to the results seen with rhesus macaques, viral replication of the ΔGY virus was controlled to elite levels, and the pig-tailed macaques did not develop immune activation or progress to disease (58). Thus, disruption of this motif appears to alter the course of SIVmac239 pathogenesis in a manner that is specific to the macaque species. The prominence of the tyrosine-based endocytosis motif for HIV-1 was further highlighted in a recent structural study, which placed the YXXL motif within a hydrophilic core of the membrane-spanning domain and demonstrated that this core is critical for maintaining stability of the Env trimer (59). Deletion of the hydrophilic core disrupts the trimer, such that the mutant becomes sensitive to nonneutralizing antibodies and resistant to trimer-specific neutralizing antibodies. This effect was most pronounced in the context of cell-to-cell fusion and was similar but less dramatic in the context of Env pseudoviruses. Thus, in addition to serving as an endocytosis and basolateral targeting signal and playing a significant role in SIV pathogenesis, the membrane-proximal, tyrosine-based sorting motif appears to participate directly in maintaining trimer stability. May 2017 Volume 91 Issue 9 e00149-17
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Why would cell-to-cell HIV-1 infection be less susceptible to neutralizing antibodies? One explanation could be the increased multiplicity of infection observed in cell-to-cell infection; however, this seems to be directly proportional to resistance to antiretroviral inhibitors but not to resistance to neutralizing antibodies (1, 32). It is also possible that Env conformations differ between cell-free and cell-cell infections and that this is regulated by signals in the cytoplasmic tail, as shown previously and in the recent study by Li et al. (6, 46). Furthermore, the virological synapse is dependent on Env binding to CD4 on the cell surface, suggesting that Env is accessible to antibodies at some point during this process. However, the synapse may sterically limit access by some antibodies, and this may affect some epitopes more than others, as well as being subject to variation between genetically diverse HIV-1 Envs. There may also be kinetic barriers as Env transitions through conformational states, as well as differences among the on-off rates of the individual antibodies, which could also behave differently for different HIV-1 Envs, since all combinations are heterologous. Finally, if membrane fusion occurs within endosomal compartments in the target cell, this could also limit the exposure of some neutralization epitopes (39). Additional studies are necessary to understand why some neutralizing antibodies are less efficient against cell-to-cell infection and why this varies with different HIV-1 strains and cell types. In summary, Li et al. have provided new information about why HIV-1 is consistently less susceptible to neutralization by multiple classes of bnAbs, in a manner that is dependent upon both the viral strain and epitope targeted by the antibody. Importantly, they have shown that some bnAbs lose potency and the ability to achieve complete neutralization against cell-to-cell infection, even at high concentrations, and that this should be considered for antibody-based therapeutics and protection strategies. The fact that the neutralization susceptibility of cell-to-cell infection is modulated by a highly conserved motif in gp41 that contributes to Env internalization, basolateral targeting, and trimer stability supports the concept that Env conformation and epitope accessibility may play a role in the relative resistance of cell-to-cell infection which warrants further investigation.
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