HIV-1 envelope (Env), composed of the glycoproteins gp120 and gp41, plays a crucial role in virus entry. In most instances, to enter a target cell, HIV-1 Env gp120, which is expressed at the surface of free virions and infected cells, must bind to CD4. The interaction between gp120 and CD4 triggers conformational changes leading to increased exposure of gp120 regions (including the V3 loop) that can bind to CCR5 or CXCR4 (Pierson and Doms, 2003). Finally, interaction of gp120 with the coreceptor induces a structural rearrangement of the transmembrane Env subunit gp41 and insertion of the fusion domain at the N-terminus of gp41 into the target cell membrane. At this stage, gp41 adopts a trimeric extended pre-hairpin intermediate conformation before the formation of a stable six-helix bundle structure and virus/cell-to-cell fusion.

Infection by cell-free viruses or through contact between infected and uninfected cells leads to HIV-1 spread (Sattentau, 2008), but the efficiency of HIV-1 infection is higher when the virus is delivered through cell-to-cell contacts. DCs and macrophages, which are among the first cells encountering HIV-1, efficiently transmit HIV-1 to CD4+ T cells through virological synapses (McDonald et al., 2003; Arrighi et al., 2004; Groot et al., 2008; Felts et al., 2010). This way of spread also occurs between HIV-1-infected and uninfected CD4+ T cells (Jolly et al., 2004; Rudnicka et al., 2009). In vitro, cell-to-cell fusion leads to the formation of giant, multinucleated cells called syncytia.

Coreceptor (CCR5 or CXCR4) use is correlated, at least in part, with the different phases of the disease. R5 viruses, which utilize CCR5, are predominantly isolated during the early stages of HIV-1 infection. The emergence in a patient of X4 variants, which use CXCR4, is almost invariably associated with faster decline of circulating CD4+ T cells, accelerated disease progression and poor prognosis for survival (Koot et al., 1993; Richman and Bozzette, 1994). However, the presence of X4 viruses is not an obligatory prerequisite for disease progression and a significant proportion of individuals who progress to AIDS harbor exclusively R5 variants. The selective transmission of R5 viruses is not fully understood, but it may depend on the superimposition of multiple imperfect gatekeepers that restrict HIV-1 X4 transmission at different steps of the infection process (Margolis and Shattock, 2006).

Only about 15–30% of CD4+ T lymphocytes express detectable levels of CCR5 at the cell surface, whereas CXCR4 is expressed on nearly all of these T cells (Bleul et al., 1997; Grivel and Margolis, 1999). High levels of CCR5 are observed in activated/memory CD4+ T cells, which are the CD4+ T cells that can be productively infected. In addition, the first CD4+ T cells to undergo intense HIV-1 replication after infection are the resting memory CD4+ T cells present in the gut-associated lymphoid tissue (GALT), which also express CCR5 (Li et al., 2005).

Macrophages are also infected by X4 and R5 strains through its receptors, CD4, CXCR4, and CCR5. However, they are more frequently infected by HIV-1 R5 strains. This phenomenon is not fully understood, but one explanation is that R5 strains can exploit low levels of CD4 and/or CCR5 to enter macrophages (Peters et al., 2004, 2006).

Besides this route of entry, endocytosis of HIV-1 has been described, especially in macrophages. Most virions are subsequently degraded, but productive infection may nevertheless occur through this CD4-independent mechanism (Marechal et al., 2001). Entry by endocytosis has also been described in CD4+ T cells, but in such cells it requires the presence of CD4 and leads to productive infection (Pauza and Price, 1988). Recently, Miyauchi et al. (2009) have demonstrated that HIV-1 likely enters lymphoid cells via an endocytic pathway, and that fusion between Env and its receptors occur in intracellular compartments.

Additional membrane components may support HIV-1 entry in macrophages, including syndecan, a heparan sulfate proteoglycan (Saphire et al., 2001; de Parseval et al., 2005); gp340, a cysteine-rich scavenger receptor (Wu et al., 2004); the macrophage mannose receptor (Larkin et al., 1989; Chehimi et al., 2003; Nguyen and Hildreth, 2003); elastase (Bristow et al., 2003); and α-v-integrin (Bosch et al., 2006). These constituents may facilitate virus attachment, binding, entry and/or fusion. Another potential membrane ligand of HIV-1 is annexin II, which is expressed on the membrane of macrophages, but not of T cells. Annexin II, which binds to phosphatidyl serine (PS), an anionic phospholipid captured during HIV-1 budding, contributes to the early events of macrophage HIV-1 infection (Ma et al., 2004). Other candidate host cell surface proteins that are incorporated in HIV-1 membranes and are potentially needed for HIV-1 entry might include CD28, CD44, and CD62L (Herzberg et al., 2006).

After reaching the cytoplasm of the infected cell, HIV-1 reverse transcription takes place in the reverse transcription complex (RTC) that is constituted by virion core proteins, cellular proteins, and the RNA genome (McDonald et al., 2002; Nermut and Fassati, 2003). Double-stranded DNA is synthesized within a few hours in CD4+ T cells, whereas this process is slower in macrophages (Collin and Gordon, 1994; O’Brien et al., 1994). The RTC moves along the microtubule network to reach the microtubule organizing center (MTOC) and the nucleus. The viral DNA is then imported into the nucleus and integrated in the cell genome through a process that requires mitosis in CD4+ T cells but not in macrophages. This suggests that cellular rather than viral components of the preintegration complex (PIC), in which the viral DNA is associated with many proteins, may play a major role in viral nuclear import and integration in macrophages (Yamashita and Emerman, 2005). After integration of the viral genome, most of the assembling HIV-1 particles localize at the plasma membrane in infected CD4+ T cells, whereas in macrophages viral particles are mostly concentrated in apparently internal compartments that possess the characteristics of late endosomes/multivesicular bodies (LEs/MVBs). However, a fraction of the intracellular virus-containing compartments in macrophages could be large and complex invaginations of the plasma membrane (Deneka et al., 2007; Jouve et al., 2007; Welsch et al., 2007).

Several microarray studies also indicate that HIV-1 infection leads to changes in host gene expression, depending on the target cell-type. Specifically, modulation of genes associated with the host defense, signal transduction, cell cycle transcription, and arrest occurs predominantly in HIV-1-infected macrophages (Coberley et al., 2004; Vazquez et al., 2005). This differential modulation of gene expression contributes to the greater survival of macrophages in comparison to CD4+ T cells following HIV-1 infection (Giri et al., 2006). Transcription of the HIV-1 DNA is also a highly regulated process that exploits the specific environment of the cell host and involves multiple interplays between cell and viral factors (Rohr et al., 2003).

Acute HIV-1 infection leads to a dramatic decrease in the number of CD4+ T cells, which then return to normal level in the majority of patients. Afterward, the asymptomatic phase is characterized by a progressive and continuous decline in the level of circulating CD4+ T cells. In contrast, macrophages are more resistant to HIV-1 cytopathic effects and their number is stable over years, although several macrophage functions are affected by HIV-1 infection (Biggs et al., 1995; Yoo et al., 1996; Polyak et al., 1997; Kumar et al., 1999). CD4+ T lymphocyte depletion is due to continued and accelerated apoptosis, probably triggered by multifactorial mechanisms (Espert et al., 2006; Cummins and Badley, 2010). HIV-1-induced apoptosis in bystander, uninfected immune cells is likely to be a key factor in the gradual depletion of T lymphocytes observed in HIV-1-infected patients, since the degree of cell loss largely exceeds the number of infected cells. Furthermore, the vast majority of CD4+ T cells undergoing apoptosis in peripheral blood and lymph nodes of HIV-1 patients are uninfected (Krammer et al., 1994; Finkel et al., 1995; Doitsh et al., 2010).

Among the HIV-1 proteins known to induce apoptosis of CD4+ T cells, cumulative data have demonstrated a major role of Env in apoptosis of uninfected, bystander lymphocytes (Laurent-Crawford et al., 1993; Heinkelein et al., 1995; Ohnimus et al., 1997; Blanco et al., 1999, 2000; Roggero et al., 2001; Roshal et al., 2001; Ahr et al., 2004). Indeed, binding of Env to its receptors constitutes the primary interface between HIV-1 and its target cells, and both HIV-1 X4 and R5 Env can induce CD4+ T cell death (Joshi et al., 2011). Although interaction of gp120 with CD4 and the coreceptor is required for apoptosis induction, Env-mediated apoptosis of target CD4+ T cells is induced through the fusogenic function of gp41. Interestingly, hemifusion, an intermediate step in the fusion process characterized by the merger of the outer membrane leaflets of two biological membranes without the formation of a fusion pore or mixing of the inner leaflets, is sufficient to trigger Env-mediated apoptosis of bystander CD4+ T cells (Garg and Blumenthal, 2006, 2008; Garg et al., 2007).

Many fundamental questions remain unsolved, such as how host factors can influence the susceptibility to HIV-1 infection and resistance to death and what are the mechanisms leading to the establishment and maintenance of HIV-1 latency. This latter point is of major importance because the latent viral reservoirs, which are constituted mainly of resting CD4+ T cells (Chun et al., 1995, 1997; Finzi et al., 1997; Wong et al., 1997; Siliciano et al., 2003) and cells of the monocyte–macrophage lineage (Folks et al., 1988; Biswas et al., 1992, 1994; Coleman and Wu, 2009), are unaffected by highly active anti-retroviral therapies (HAART; Finzi et al., 1999) and can reinitiate systemic infection upon interruption of HAART, or following the development of resistance (Marcello, 2006; Suyama et al., 2009).

Autophagy of uninfected CD4+ T cells, when co-cultured with lymphocytes that express Env, was described for the first time in 2006 (Espert et al., 2006). It is triggered by binding of Env to its receptors CD4 and CXCR4, but is independent of CD4 or CXCR4 signaling pathways. Autophagy can be induced in single cells (hemifusion) and syncytia (complete fusion) and depends on the fusogenic function of gp41. These results indicate that the entire process leading to HIV-1 entry into target cells through binding of Env to its receptors CD4 and CXCR4 is responsible for autophagy in CD4+ T lymphocytes (Denizot et al., 2008). As autophagy is gp41-dependent, it can be induced whatever the coreceptor used for HIV-1 entry (CCR5 or CXCR4; Espert et al., 2009). The mechanisms by which gp41 induces autophagy are unknown, but it could be a consequence of the cellular stress induced after hemifusion of the membranes. A proteomics study has already demonstrated that rapid and sustained accumulation of ROS is induced in CD4+ T cells after contact with Env-expressing cells (Molina et al., 2007), and the production of ROS is known to be directly involved in autophagy (Scherz-Shouval et al., 2007).

Importantly, Env-mediated autophagy is required to trigger CD4+ T cell apoptosis. Indeed, blockade of autophagy at different steps by drugs (3-methyladenine or Bafilomycin A1) or short interfering RNAs against Beclin 1 and Atg7 fully inhibits Env-mediated apoptosis. At present, nothing is known about the mechanisms that link autophagy to apoptosis, but Beclin 1 might play a key role since this Atg also binds to anti-apoptotic Bcl-2 proteins (Bcl-2 and Bcl-xL). Interaction of Beclin 1 with Bcl-2 or Bcl-xL inhibits Beclin 1-dependent autophagy, demonstrating a direct crosstalk between the core machineries regulating autophagy and apoptosis (Pattingre et al., 2005).

Interestingly, Beclin 1 is accumulated during the early steps of the Env-induced signaling cascade, and this phenomenon precedes autophagic vacuolization. Accumulation of Beclin 1 has also been reported in response to drugs that trigger cell death with autophagic features (Scarlatti et al., 2004; Shimizu et al., 2004; Furuya et al., 2005). Deciphering the molecular mechanism of Beclin 1 accumulation will be a crucial step for understanding the role of autophagy in HIV-1 pathogenicity.

Conversely, when cells that express HIV-1 R5 or X4 Env are co-cultured with cells from the monocyte/macrophage lineage, autophagy is not observed in uninfected cells, although they are susceptible to autophagy induced by different agents. Env-mediated autophagy is thus a cell-type dependent process. The state of differentiation is not responsible for their intrinsic resistance to Env-mediated autophagy because cells at different stage of differentiation (from promonocytic cells to monocyte-differentiated macrophages) are all equally resistant. Macrophages also do not undergo Env-mediated apoptosis and they are resistant to cell depletion during HIV-1 infection.

These major differences between CD4+ T cells and macrophages raise unsolved questions: what are the mechanisms that lead to Env-mediated autophagy, how autophagy is regulated in the different cell-types, and how autophagy triggers apoptosis in CD4+ T lymphocytes.

The fact that the fusogenic function of gp41 induces Env-mediated autophagy only in CD4+ T cells is surprising since fusion also occurs in uninfected macrophages co-cultured with HIV-1-infected cells. An important and still unexplained difference between these two target cells is the ability to form syncytia. This has been observed since the discovery of the disease and HIV strains were first classified based on this observation. Indeed “syncytia-inducing” viruses correspond to HIV-1 strains capable of infecting CD4+ T cells through CXCR4, while “non-syncytia-inducing” viruses are HIV-1 strains that infect macrophages through CCR5 (Goodenow and Collman, 2006). One hypothesis to explain why Env-mediated autophagy is a cell-specific mechanism is that the gp41-induced perturbations, triggered at the membrane of both macrophages and CD4+ T cells, could be different in the two cell-types or differentially regulated. Indeed, HIV-1 entry in macrophages is supported by additional specific interactions with host membrane molecules following Env binding to the receptor/coreceptor (see HIV-1 Entry in CD4+ T Cells and Macrophages). HIV-1 can also enter macrophages by endocytosis. Furthermore, recent data have demonstrated that ceramides, which are known inducers of autophagy (Pattingre et al., 2009), play an important role in the reorganization of membrane proteins (Chiantia et al., 2008) that is necessary for HIV-1 entry into cells. Another hypothesis is that gp120 binding to CD4 and to the coreceptor, two steps that precede gp41 insertion into the target membrane, transduces signals that counteract Env-mediated autophagy in a cell-type dependent manner. We also cannot exclude the involvement of secreted chemokines and/or cytokines in the regulation of gp41-induced autophagy. Further investigation is needed to elucidate this point.

HIV-1 X4 or R5 Env expression at the cell surface (Env-transfected or HIV-1-infected cells) triggers autophagy in uninfected CD4+ T cells, leading to apoptosis. Surprisingly, when this contact leads to a productive infection, Env-mediated autophagy is inhibited in CD4+ T cells (Zhou and Spector, 2008; Espert et al., 2009). Moreover, the levels of the autophagy factor LC3-II (Zhou and Spector, 2008; Espert et al., 2009) and of Beclin 1 (Zhou and Spector, 2008) are dramatically decreased in these newly infected CD4+ T cells, suggesting that HIV-1 can actively down-regulate autophagy. Interestingly, when autophagy is triggered by different inducers in HIV-1-infected CD4+ T cells, production of HIV-1 is decreased, suggesting that autophagy can limit HIV-1 replication (unpublished data from our group).

Thus, HIV-1, as many viruses, can counteract the anti-viral function of autophagy, but the mechanism(s) by which HIV-1 can interfere with this essential cellular pathway is (are) still unknown. One or several viral proteins might block autophagy to avoid HIV-1 destruction.

Conversely, in cells from the monocyte/macrophage lineage, autophagy is induced following productive infection through contact with HIV-1 X4 or R5-infected effector cells (Espert et al., 2009). However, the observation that viruses are present only in moderately autophagic cells (Espert et al., 2009), suggests that autophagy is still controlled by HIV-1 in these cells to avoid degradation. Interestingly, early, non-degradative steps of autophagy promote HIV-1 production since blockade of this process dramatically decreases the quantity of p24 Gag (Espert et al., 2009; Kyei et al., 2009). In addition, the HIV-1 precursor Gag is found in complexes with LC3 and is present at LC3-II-enriched membranes, suggesting that autophagy could favor Gag processing and thus production of viral particles (Kyei et al., 2009). In contrast, the degradative step of autophagy has an anti-HIV-1 activity that must be controlled by the virus to prevent its degradation. Indeed, blockage of the degradative step of autophagy increases HIV-1 production (Espert et al., 2009; Kyei et al., 2009). Interestingly, the auxiliary HIV-1 protein Nef plays a major role in the inhibition of the degradative stage of autophagy by binding to Beclin 1 (Kyei et al., 2009). Nef also interacts with immunity-associated GTPase family M (IRGM), a protein known to play an autophagy-dependent anti-bacterial function (Deretic et al., 2006; McCarroll et al., 2008; Deretic, 2010a; Singh et al., 2010) and to bind to several key proteins of the autophagy process such as Atg5 and Atg10 (Pombo-Grégoire et al., 2011). Nef/IRGM interaction promotes autophagosome accumulation and improves HIV-1 replication (Pombo-Grégoire et al., 2011). In contrast, its absence is detrimental for the viral production. IRGM also triggers autophagy in cells infected by other RNA viruses, such as hepatitis C virus (HCV) and measles virus (MeV), suggesting that different RNA virus families use similar strategies, involving IRGM, to fine-tune autophagy to their own benefit.

Taken together, all these results (summarized in Table 2) suggest a complex, cell-type specific relationship between HIV-1 and the autophagic response and highlight the complexity of HIV-1 pathogenesis.