Molecular Basis for Cell Tropism of CXCR4-Dependent Human Immunodeficiency Virus Type 1 Isolates

Laboratory isolates of human immunodeficiency virus type 1 (HIV-1) that utilize CXCR4 as a coreceptor infect primary human macrophages inefficiently even though these express a low but detectable level of cell surface CXCR4. In contrast, infection of primary macrophages by primary CXCR4-tropic HIV-1 isolates is readily detectable. Here, we provide evidence suggesting that this difference in cell tropism results from a higher requirement for cell surface CXCR4 for infection by laboratory HIV-1 isolates. Transfected COS7 cells that express a high level of CD4 but a low level of CXCR4 were infected significantly more efficiently by two primary CXCR4-tropic HIV-1 isolates compared to the prototypic laboratory HIV-1 isolate IIIB. More importantly, overexpression of either wild-type or signaling-defective CXCR4 on primary macrophages dramatically enhanced the efficiency of infection by the laboratory HIV-1 isolate yet only modestly enhanced infection by either primary CXCR4-tropic virus. Overexpression of CD4 had, in contrast, only a limited effect on macrophage infection by the laboratory HIV-1, although infection by the primary isolates was markedly enhanced. We therefore conclude that the laboratory CXCR4-tropic HIV-1 isolate exhibits a significantly higher CXCR4 requirement for efficient infection than do the primary CXCR4-tropic isolates and that this difference can explain the poor ability of the laboratory HIV-1 isolate to replicate in primary macrophages. More generally, we propose that the cell tropisms displayed by different strains of HIV-1 in culture can largely be explained on the basis of differential requirements for cell surface CD4 and/or coreceptor expression levels.     

Determinants Flanking the CD4 Binding Loop Modulate Macrophage Tropism of Human Immunodeficiency Virus Type 1 R5 Envelopes

Human immunodeficiency virus type 1 R5 viruses vary extensively in phenotype. Thus, R5 envelopes (env) in the brain tissue of individuals with neurological complications are frequently highly macrophage-tropic. Macrophage tropism correlates with the capacity of the envelope to exploit low CD4 levels for infection. In addition, the presence of an asparagine at residue 283 within the CD4 binding site has been associated with brain-derived envelopes, increased env-CD4 affinity, and enhanced macrophage tropism. Here, we identify additional envelope determinants of R5 macrophage tropism. We compared highly macrophage-tropic (B33) and non-macrophage-tropic (LN40) envelopes from brain and lymph node specimens of one individual. We first examined the role of residue 283 in macrophage tropism. Introduction of N283 into LN40 (T283N) conferred efficient macrophage infectivity. In contrast, substitution of N283 for the more conserved threonine in B33 had little effect on macrophage infection. Thus, B33 carried determinants for macrophage tropism that were independent of N283. We prepared chimeric B33/LN40 envelopes and used site-directed mutagenesis to identify additional determinants. The determinants of macrophage tropism that were identified included residues on the CD4 binding loop flanks that were proximal to CD4 contact residues and residues in the V3 loop. The same residues affected sensitivity to CD4-immunoglobulin G inhibition, consistent with an altered env-CD4 affinity. We predict that these determinants alter exposure of CD4 contact residues. Moreover, the CD4 binding loop flanks are variable and may contribute to a general mechanism for protecting proximal CD4 contact residues from neutralizing antibodies. Our results have relevance for env-based vaccines that will need to expose critical CD4 contact residues to the immune system.Human immunodeficiency virus type 1 (HIV-1) requires interactions between viral envelope glycoproteins and cell surface CD4 and coreceptors to trigger fusion and entry into cells. HIV-1 R5 viruses that specifically use CCR5 as a coreceptor are those predominantly transmitted (3). Yet, our knowledge of R5 virus variation in different biological properties is still limited. In vivo, HIV-1 infection is limited mostly to cells that express CD4 and appropriate coreceptors. Thus, HIV-1 infects CD4⁺ T cells, monocyte/macrophage lineage cells, and dendritic cells. CCR5 is expressed on each of these cell lineages, although on T cells, CCR5 is restricted mainly to RO45⁺ memory cells (1, 16). Early in infection, R5 viruses target and decimate mucosal CD4⁺ memory T cells (2, 18, 26). R5 viruses are also predominant in tissues in which monocyte/macrophage lineage cells are prevalent, and several reports have described the presence of highly macrophage-tropic R5 viruses in brain tissue (11, 12, 20, 22). Previously, we used PCR to amplify HIV-1 envelope genes directly from patient tissues. We found that R5 virus envelopes amplified from brain tissue frequently conferred highly efficient infection of macrophages, while the majority of those from lymph nodes, blood, and semen infected macrophages inefficiently (20, 22). Although those studies examined relatively few infected individuals, they demonstrated over 1,000-fold variation in macrophage-tropic HIV-1 R5 viruses. Such variation is likely to have a significant impact on transmission and pathogenesis.The envelope (env) determinants of R5 macrophage-tropic strains are poorly understood. Several studies have shown that highly macrophage-tropic brain envelopes are able to exploit low levels of CD4 on macrophages for infection, consistent with an enhanced interaction between gp120 and CD4. Dunfee et al. reported that an asparagine residue at position 283 in the C2 part of the CD4 binding site was present in 41% of envelope sequences from brain tissue specimens of patients with HIV-associated dementia and in only 8% of envelopes from non-HIV-associated dementia brain tissue (8). The same study showed that the presence of N283 (rather than the more conserved T283) led to an increased affinity of gp120 for CD4, probably because the side chain of asparagine could more readily form a hydrogen bond with Q40 on CD4. However, our previous data showed that N283 is not the only determinant of macrophage infectivity, since several macrophage-tropic R5 envelopes from brain and semen specimens lacked N283, while non-macrophage-tropic envelopes from lymph node specimens carrying N283 were identified (22). Dunfee et al. also reported that a glycosylation site at residue 386, close to the CD4 binding loop, influenced exposure of the CD4 binding site and had an impact on macrophage tropism and sensitivity to the CD4 binding site antibody b12 (9). We have recently confirmed a role for N386 in resistance to the CD4 binding site monoclonal antibody (MAb) b12. However, resistance was dependent on the presence of a proximal residue, R373, which acted together with N386 to block b12 (7).Here, we have further investigated envelope determinants of macrophage tropism by preparing chimeric envelopes from highly macrophage-tropic and non-macrophage-tropic R5 envelopes from brain and lymph node specimens from the same subject. We show that R5 macrophage tropism is controlled by several determinants in gp120 that are focused on amino acids flanking the CD4 binding loop, with a contribution from residues in the V3 loop.     

Strong Ability of Nef-Specific CD4+ Cytotoxic T Cells To Suppress Human Immunodeficiency Virus Type 1 (HIV-1) Replication in HIV-1-Infected CD4+ T Cells and Macrophages

A restricted number of studies have shown that human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic CD4⁺ T cells are present in HIV-1-infected individuals. However, the roles of this type of CD4⁺ T cell in the immune responses against an HIV-1 infection remain unclear. In this study, we identified novel Nef epitope-specific HLA-DRB1*0803-restricted cytotoxic CD4⁺ T cells. The CD4⁺ T-cell clones specific for Nef187-203 showed strong gamma interferon production after having been stimulated with autologous B-lymphoblastoid cells infected with recombinant vaccinia virus expressing Nef or pulsed with heat-inactivated virus particles, indicating the presentation of the epitope antigen through both exogenous and endogenous major histocompatibility complex class II processing pathways. Nef187-203-specific CD4⁺ T-cell clones exhibited strong cytotoxic activity against both HIV-1-infected macrophages and CD4⁺ T cells from an HLA-DRB1*0803⁺ donor. In addition, these Nef-specific cytotoxic CD4⁺ T-cell clones exhibited strong ability to suppress HIV-1 replication in both macrophages and CD4⁺ T cells in vitro. Nef187-203-specific cytotoxic CD4⁺ T cells were detected in cultures of peptide-stimulated peripheral blood mononuclear cells (PBMCs) and in ex vivo PBMCs from 40% and 20% of DRB1*0803⁺ donors, respectively. These results suggest that HIV-1-specific CD4⁺ T cells may directly control HIV-1 infection in vivo by suppressing virus replication in HIV-1 natural host cells.Human immunodeficiency virus (HIV)-specific CD8⁺ cytotoxic T cells (CTLs) play a central role in the control of HIV type 1 (HIV-1) during acute and chronic phases of an HIV-1 infection (5, 29, 34). However, HIV-1 escapes from the immune surveillance of CD8⁺ CTLs by mechanisms such as mutations of immunodominant CTL epitopes and downregulation of major histocompatibility complex class I (MHC-I) molecules on the infected cells (9, 11, 12, 49). Therefore, most HIV-1-infected patients without highly active antiretroviral therapy (HAART) develop AIDS eventually.HIV-1-specific CD4⁺ T cells also play an important role in host immune responses against HIV-1 infections. An inverse association of CD4⁺ T-cell responses with viral load in chronically HIV-1-infected patients was documented in a series of earlier studies (8, 36, 39, 41, 48), although the causal relationship between them still remains unclear (23). Classically, CD4⁺ T cells help the expansion of CD8⁺ CTLs by producing growth factors such as interleukin-2 (IL-2) or by their CD40 ligand interaction with antigen-processing cells and CD8⁺ CTLs. In addition, CD4⁺ T cells provide activation of macrophages, which can professionally maintain CD8⁺ T-cell memory (17). On the other hand, the direct ability of virus-specific cytotoxic CD4⁺ T cells (CD4⁺ CTLs) to kill target cells has been widely observed in human virus infections such as those by human cytomegalovirus, Epstein-Barr virus (EBV), hepatitis B virus, Dengue virus, and HIV-1 (2, 4, 10, 19, 30, 31, 38, 50). Furthermore, one study showed that mouse CD4⁺ T cells specific for lymphocytic choriomeningitis virus have cytotoxic activity in vivo (25). These results, taken together, indicate that a subset of effector CD4⁺ T cells develops cytolytic activity in response to virus infections.HIV-1-specific CD4⁺ CTLs were found to be prevalent in HIV-1 infections, as Gag-specific cytotoxic CD4⁺ T cells were detected directly ex vivo among peripheral blood mononuclear cells (PBMCs) from an HIV-1-infected long-term nonprogressor (31). Other studies showed that up to 50% of the CD4⁺ T cells in some HIV-1-infected donors can exhibit a clear cytolytic potential, in contrast to the fact that healthy individuals display few of these cells (3, 4). These studies indicate the real existence of CD4⁺ CTLs in HIV-1 infections.The roles of CD4⁺ CTLs in the control of an HIV-1 infection have not been widely explored. It is known that Gag-specific CD4⁺ CTLs can suppress HIV-1 replication in a human T-cell leukemia virus type 1-immortalized CD4⁺ T-cell line (31). However, the functions of CD4⁺ T cells specific for other HIV-1 antigens remain unclear. On the other hand, the abilities of CD4⁺ CTLs to suppress HIV-1 replication in infected macrophages and CD4⁺ T cells may be different, as in the case of CD8⁺ CTLs for HIV-1-infected macrophages (17). In this study, we identified Nef-specific CD4⁺ T cells and investigated their ability to kill HIV-1 R5 virus-infected macrophages and HIV-1 X4 virus-infected CD4⁺ T cells and to suppress HIV-1 replication in the infected macrophages and CD4⁺ T cells. The results obtained in the present study show for the first time the ability of HIV-1-specific CD4⁺ CTLs to suppress HIV-1 replication in natural host cells, i.e., macrophages and CD4⁺ T cells.     

Annexin 2 Is Not Required for Human Immunodeficiency Virus Type 1 Particle Production but Plays a Cell Type-Dependent Role in Regulating Infectivity

During assembly and budding of retroviruses, host cell proteins are incorporated into viral particles. Identification of virion-associated proteins may help pinpoint key cellular components required for virus production and function. The cellular protein annexin 2 (Anx2) is incorporated into HIV-1 particles, and knockdown of Anx2 has been reported to cause defects in Gag processing and infectivity of HIV-1 particles in macrophages. Here, we tested whether Anx2 was required for HIV-1 production in other cell types capable of producing HIV-1 virions. Endogenous Anx2 levels were knocked down by ∼98% using lentivirus encoding short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) targeting Anx2. Under these conditions, there was no reduction in HIV-1 virus-like particle (VLP) production in either COS-1, 293T, or Jurkat T cells or primary human monocyte-derived macrophages (MDMs). Murine embryonic fibroblasts derived from Anx2^−/− mice produced the same levels of VLPs as matched cells from wild-type mice. The calcium-mediated spike in VLP production still occurred in Anx2-depleted COS-1 cells, and there was no apparent alteration in the intracellular Gag localization. Overexpression of Anx2 in trans had no effect on Gag processing or VLP production. Neither Anx2 depletion nor Anx2 overexpression altered the infectivity of HIV-1 particles produced by COS-1 or 293T cells. However, supernatants containing virus from Anx2 siRNA-treated primary human MDMs exhibited decreased infectivity. These data indicate that Anx2 is not required for HIV-1 assembly or Gag processing but rather plays a cell type-dependent role in regulating production of infectious HIV-1 by macrophages.The Gag polyprotein generates the key structural proteins for all retroviruses. Gag is necessary and sufficient for the formation of virus-like particles (VLPs), which are morphologically similar to immature virions. Following its synthesis in the cytoplasm, HIV-1 Gag is trafficked to sites of particle production on membranes. Viral particle production depends on Gag-membrane interactions mediated by the myristoylated MA domain of Gag (18, 22, 31) and Gag-Gag interactions mediated by the CA and NC domains. Budding and release of the new virion are mediated by the Gag p6 domain. For successful particle production to occur, HIV-1 Gag must also interact with numerous host cell proteins and protein complexes. Identification of these interactions provides a crucial window into determining Gag trafficking intermediates as well as clues to the mechanism of virion production.The host cell protein annexin 2 (Anx2) has recently attracted attention for its potential to regulate key processes in both cells and viruses (9, 14, 17, 24). Anx2 belongs to a family of conserved calcium-regulated proteins and interacts with actin, membranes, and negatively charged phospholipids. The major protein binding partner for Anx2 is p11, also known as S100A10. Two populations of Anx2 have been identified: a heterotetrameric complex with two molecules of Anx2 and two molecules of p11 (found predominantly at the plasma membrane) and a monomeric form found mainly in the cytoplasm. Anx2 performs multiple functions in the cell, including regulation of actin-based dynamics, fibrinolysis, calcium-mediated exocytosis, and transport of intermediates from early to late endosomes (10, 14-16) Anx2 also enhances binding and fusion of cytomegalovirus with phospholipid membranes (21). In addition, Anx2 can be detected within influenza virus particles (28), where it has been shown to aid in virus replication (9).Several lines of evidence suggest that Anx2 may play a role in HIV-1 biogenesis. Both Anx2 and its binding partner p11 are incorporated in HIV-1 particles produced by macrophages (2). Anx2 interacts with Gag in macrophages, and annexin 2 knockdown has been reported to cause defective Gag processing and reduced infectivity of the released particles (24). Blockade of Anx2 function, with either anti-Anx2 antibody or small interfering RNA (siRNA)-mediated knockdown, results in suppression of HIV-1 infection in macrophages (11). Anx2 also binds to Gag in 293T cells, and expression of Anx2 in trans in these cells has been reported to lead to increased Gag processing and HIV-1 production (7). Taken together, these findings suggest that Anx2 might play a universal role in Gag trafficking and particle production. To test this hypothesis, we exploited methods to efficiently knock down Anx2 expression and determined the effect of Anx2 knockdown in a variety of cell lines capable of producing HIV-1 virions. Here we show that, in the absence of Anx2 expression, HIV-1 Gag is expressed, trafficked, and capable of mediating viral particle formation in a manner similar to that of control cells expressing Anx2. However, a cell type-dependent effect of Anx2 depletion on HIV-1 infectivity was detected in primary human monocyte-derived macrophages (MDMs). These findings suggest that Anx2 might be a macrophage-specific host cell factor that regulates HIV-1 infectivity.     

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

The role of CD4⁺ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4⁺ T cells are needed for the generation of the protective HSV-1-specific CD8⁺-T-cell response. This study examined the contribution of CD4⁺ T cells in the generation of the primary CD8⁺-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8⁺-T-cell response generated in the draining lymph nodes of CD4⁺-T-cell-depleted C57BL/6 mice and B6-MHC-II^−/− mice is quantitatively and qualitatively distinct from the CD8⁺ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8⁺ T cells express comparable levels of the activation marker CD44 in mice lacking CD4⁺ T cells and normal mice. In contrast, CD8⁺ T cells generated in the absence of CD4⁺ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8⁺ T cells in the CD4⁺-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8⁺ T cells is diminished in the absence of CD4⁺-T-cell help. These results suggest that CD4⁺-T-cell help is essential for the generation of fully functional CD8⁺ T cells during the primary response to HSV-1 infection.Infection due to herpes simplex virus type 1 (HSV-1) results in a wide spectrum of clinical presentations depending on the host''s age, the host''s immune status, and the route of inoculation (47). HSV-1 typically causes mild and self-limited lesions on the orofacial areas or genital sites. However, the disease can be life-threatening, as in the case of neonatal and central nervous system infections (18). The host''s immune responses, particularly CD8⁺ T cells, play an important role in determining the outcome of HSV infections in both the natural human host (18, 19, 28) and experimental murine models (11, 43). Immunodepletion and adoptive transfer studies have demonstrated the role of CD8⁺ T cells in reducing viral replication, resolving cutaneous disease, and providing overall protection upon rechallenge (6, 25, 26). CD8⁺ T cells play a particularly important role in preventing infection of the peripheral nervous system (PNS) and the reactivation of latent virus from neurons in the sensory ganglia of infected mice (21, 24, 36). The mechanisms that CD8⁺ T cells employ include gamma interferon (IFN-γ) production and functions associated with cytolytic granule content at the sites of primary infection (23, 31, 38). In the PNS of infected mice, the mechanisms primarily involve IFN-γ secretion (16, 20, 29), particularly against infected neurons expressing surface Qa-1 (41). Histopathological evidence from HSV-1-infected human ganglion sections show a large CD8⁺-T-cell infiltrate and the presence of inflammatory cytokines, suggesting that the presence of activated, effector memory cells within the PNS is important for maintaining HSV-1 latency in the natural human host (10, 42).The generation of a robust CD8⁺-T-cell response is essential for the control of various infectious pathogens. Some studies suggest that a brief interaction with antigen-presenting cells (APCs) is sufficient for CD8⁺-T-cell activation and expansion into functional effectors (44). However, the magnitude and quality of the overall CD8⁺-T-cell response generated may be dependent on additional factors (49). Recent evidence suggests that CD4⁺ T cells facilitate the activation and development of CD8⁺-T-cell responses either directly through the provision of cytokines or indirectly by the conditioning of dendritic cells (DC) (8, 48, 51). Those studies suggested that the latter mechanism is the dominant pathway, wherein CD4⁺ T cells assist CD8⁺-T-cell priming via the engagement of CD40 ligand (CD154) on CD4⁺ T cells and CD40 expressed on DC (4, 30, 33). This interaction results in the activation and maturation of DC, making them competent to stimulate antigen-specific CD8⁺-T-cell responses (35, 37).The requirement for CD4⁺-T-cell help in the generation of primary and secondary CD8⁺-T-cell responses to antigen varies. Primary CD8⁺-T-cell responses to infectious pathogens, such as Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), influenza virus, and vaccinia virus, can be mounted effectively independently of CD4⁺-T-cell help (3, 12, 22, 34). In contrast, primary CD8⁺-T-cell responses to nonmicrobial antigens display an absolute dependence on CD4⁺-T-cell help (4, 5, 30, 33, 46). This observed difference in the requirement for CD4⁺-T-cell help may ultimately be a product of the initial inflammatory stimulus generated following immunization (49). Microbial antigens trigger an inflammatory response that can lead to the direct activation and priming of APCs, such as DC, thereby bypassing the need for CD4⁺-T-cell help. Nonmicrobial antigens, however, trigger an attenuated inflammatory response that does not directly activate and prime DCs. In the absence of this inflammation, CD4⁺ T cells are thought to condition and license DC functions through CD154/CD40 interactions, which leads to the subsequent activation of antigen-specific CD8⁺-T-cell responses (5, 49). Even in the case of pathogens where primary CD8⁺-T-cell responses were independent of CD4⁺-T-cell help, the secondary responses to these pathogens were found to be defective in the absence of CD4⁺-T-cell help (3, 12, 34, 40).The requirement for CD4⁺-T-cell help in priming CD8⁺-T-cell responses against HSV-1 infection is not well defined. Earlier studies with HSV-1 suggested that CD4⁺ T cells play an important role in the generation of primary CD8⁺-T-cell responses, detected in vitro, to acute infection with HSV-1 (14), principally through the provision of interleukin 2 (IL-2) for optimal CD8⁺-T-cell differentiation and proliferation. Subsequent studies, utilizing an in vivo approach, indicated that CD4⁺ T cells were not required for CD8⁺-T-cell-mediated cytolytic function (23). CD4⁺ T cells are thought to provide help by conditioning DC in a cognate, antigen-specific manner, thereby making them competent to stimulate HSV-1-specific CD8⁺-T-cell responses (37). By contrast, findings from other studies show that CD4⁺-T-cell-depleted mice were able to fully recover from acute infection with HSV-1 (38). These studies imply that the absence of CD4⁺ T cells does not prevent priming of CD8⁺ T cells in vivo.Studies from this laboratory have identified two distinct HSV-1-specific CD8⁺-T-cell subpopulations generated during the primary response, based upon the ability to synthesize IFN-γ following antigenic stimulation in vitro (1). To better understand the need for CD4⁺-T-cell help, we examined the functional characteristics and phenotypes of these CD8⁺-T-cell populations generated during a primary response to acute infection with HSV-1 in mice lacking CD4⁺ T cells. Our findings show that primary CD8⁺-T-cell responses to HSV-1 are compromised in the absence of CD4⁺-T-cell help. Specifically, the HSV-1 gB-specific CD8⁺ T cells produced in the absence of CD4⁺ T cells were found to be active with regard to cytolysis in vivo but were functionally impaired in the production of IFN-γ and TNF-α compared with intact C57BL/6 mice. Virus-specific CD8⁺ T cells were also reduced in number in CD4-depleted mice and in B6 mice lacking major histocompatibility complex (MHC) class II expression (B6-MHC-II^−/−) compared to wild-type (WT) mice. In addition, our data showed higher virus burdens in the infectious tissues obtained from mice lacking CD4⁺ T cells than in those from intact mice. Collectively, these findings demonstrate that CD4⁺-T-cell help is essential for the generation of primary CD8⁺-T-cell responses following acute cutaneous infection with HSV-1.     

A Variant Macaque-Tropic Human Immunodeficiency Virus Type 1 Is Resistant to Alpha Interferon-Induced Restriction in Pig-Tailed Macaque CD4+ T Cells

Human immunodeficiency virus type 1 (HIV-1) antagonizes innate restriction factors in order to infect and persistently replicate in a host. In a previous study, we demonstrated that HIV-1 NL4-3 with a simian immunodeficiency virus mne (SIVmne) vif gene substitution (HSIV-vif-NL4-3) could infect and replicate in pig-tailed macaques (PTM), indicating that APOBEC3 proteins are primary barriers to transmission. Because viral replication was persistent but low, we hypothesized that HSIV-vif-NL4-3 may be suppressed by type I interferons (IFN-I), which are known to upregulate the expression of innate restriction factors. Here, we demonstrate that IFN-α more potently suppresses HSIV-vif-NL4-3 in PTM CD4⁺ T cells than it does pathogenic SIVmne027. Importantly, we identify a variant (HSIV-vif-Yu2) that is resistant to IFN-α, indicating that the IFN-α-induced barrier can be overcome by HSIV-vif chimeras in PTM CD4⁺ T cells. Interestingly, HSIV-vif-Yu2 and HSIV-vif-NL4-3 are similarly restricted by PTM BST2/Tetherin, and neither virus downregulates it from the surface of infected PTM CD4⁺ T cells. Resistance to IFN-α-induced restriction appears to be conferred by a determinant in HSIV-vif-Yu2 that includes env su. Finally, we show that the Yu-2 env su allele may overcome an IFN-α-induced barrier to entry. Together, our data demonstrate that the prototype macaque-tropic HIV-1 clones based on NL4-3 may not sufficiently antagonize innate restriction in PTM cells. However, variants with resistance to IFN-α-induced restriction factors in PTM CD4⁺ T cells may enhance viral replication by overcoming a barrier early in the viral replication cycle.     

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection

Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1(LAI) infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition.     

Necroptosis Takes Place in Human Immunodeficiency Virus Type-1 (HIV-1)-Infected CD4+ T Lymphocytes

Human immunodeficiency virus type 1 (HIV-1) infection is characterized by progressive depletion of CD4⁺ T lymphocytes and dysfunction of the immune system. The numbers of CD4⁺ T lymphocytes in the human body are maintained constantly by homeostatic mechanisms that failed during HIV-1 infection, resulting in progressive loss of CD4⁺ T cells mainly via apoptosis. Recently, a non-apoptotic form of necrotic programmed cell death, named necroptosis, has been investigated in many biological and pathological processes. We then determine whether HIV-1-infected cells also undergo necroptosis. In this report, we demonstrate that HIV-1 not only induces apoptosis, but also mediates necroptosis in the infected primary CD4⁺ T lymphocytes and CD4⁺ T-cell lines. Necroptosis-dependent cytopathic effects are significantly increased in HIV-1-infected Jurkat cells that is lack of Fas-associated protein-containing death domain (FADD), indicating that necroptosis occurs as an alternative cell death mechanism in the absence of apoptosis. Unlike apoptosis, necroptosis mainly occurs in HIV-infected cells and spares bystander damage. Treatment with necrostatin-1(Nec-1), a RIP1 inhibitor that specifically blocks the necroptosis pathway, potently restrains HIV-1-induced cytopathic effect and interestingly, inhibits the formation of HIV-induced syncytia in CD4⁺ T-cell lines. This suggests that syncytia formation is mediated, at least partially, by necroptosis-related processes. Furthermore, we also found that the HIV-1 infection-augmented tumor necrosis factor-alpha (TNF-α) plays a key role in inducing necroptosis and HIV-1 Envelope and Tat proteins function as its co-factors. Taken together,necroptosis can function as an alternative cell death pathway in lieu of apoptosis during HIV-1 infection, thereby also contributing to HIV-1-induced cytopathic effects. Our results reveal that in addition to apoptosis, necroptosis also plays an important role in HIV-1-induced pathogenesis.     

A Human CD4+ T Cell Epitope in the Influenza Hemagglutinin Is Cross-Reactive to Influenza A Virus Subtypes and to Influenza B Virus

The hemagglutinin protein (HA) of the influenza virus family is a major antigen for protective immunity. Thus, it is a relevant target for developing vaccines. Here, we describe a human CD4(+) T cell epitope in the influenza virus HA that lies in the fusion peptide of the HA. This epitope is well conserved in all 16 subtypes of the HA protein of influenza A virus and the HA protein of influenza B virus. By stimulating peripheral blood mononuclear cells (PBMCs) from a healthy adult donor with peptides covering the entire HA protein based on the sequence of A/Japan/305/1957 (H2N2), we generated a T cell line specific to this epitope. This CD4(+) T cell line recognizes target cells infected with influenza A virus seasonal H1N1 and H3N2 strains, a reassortant H2N1 strain, the 2009 pandemic H1N1 strain, and influenza B virus in cytotoxicity assays and intracellular-cytokine-staining assays. It also lysed target cells infected with avian H5N1 virus. We screened healthy adult PBMCs for T cell responses specific to this epitope and found individuals who had ex vivo gamma interferon (IFN-γ) responses to the peptide epitope in enzyme-linked immunospot (ELISPOT) assays. Almost all donors who responded to the epitope had the HLA-DRB1*09 allele, a relatively common HLA allele. Although natural infection or standard vaccination may not induce strong T and B cell responses to this highly conserved epitope in the fusion peptide, it may be possible to develop a vaccination strategy to induce these CD4(+) T cells, which are cross-reactive to both influenza A and B viruses.     

Human Immunodeficiency Virus Type 1-Specific CD8+ T-Cell Responses during Primary Infection Are Major Determinants of the Viral Set Point and Loss of CD4+ T Cells

Primary HIV-1 infection (PHI) is marked by a flu-like syndrome and high levels of viremia that decrease to a viral set point with the first emergence of virus-specific CD8⁺ T-cell responses. Here, we investigated in a large cohort of 527 subjects the immunodominance pattern of the first virus-specific cytotoxic T-lymphocyte (CTL) responses developed during PHI in comparison to CTL responses in chronic infection and demonstrated a distinct relationship between the early virus-specific CTL responses and the viral set point, as well as the slope of CD4⁺ T-cell decline. CTL responses during PHI followed clear hierarchical immunodominance patterns that were lost during the transition to chronic infection. Importantly, the immunodominance patterns of human immunodeficiency virus type 1 (HIV-1)-specific CTL responses detected in primary, but not in chronic, HIV-1 infection were significantly associated with the subsequent set point of viral replication. Moreover, the preservation of the initial CD8⁺ T-cell immunodominance patterns from the acute into the chronic phase of infection was significantly associated with slower CD4⁺ T-cell decline. Taken together, these data show that the specificity of the initial CTL response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.In the first weeks after human immunodeficiency virus type 1 (HIV-1) acquisition, viral loads peak at high levels, accompanied by a flu-like syndrome (15). A rapid depletion of the CD4⁺ T-cell population occurs during this acute infection, in particular, within the gastrointestinal tract-associated lymphoid tissue (6, 19, 20), marking a nonrecoverable scar on the immune system. With the resolution of the clinical syndromes, viral loads decrease to a set point, which persists at this level for months to years until progressive CD4⁺ T-cell decline results in the onset of AIDS. It has been shown that the initial viral set point following primary infection is a very strong predictor of the disease-free period until the onset of AIDS (18, 21, 22).The initial decrease in the viral load during primary HIV-1 infection (PHI) is temporally associated with the first emergence of virus-specific CD8⁺ T-cell responses, and several studies have provided strong evidence that HIV-1-specific CD8⁺ T-cell responses are capable of controlling viral replication (5, 16, 24, 25, 27, 31, 33). However, significant numbers of virus-specific CD8⁺ T cells are detectable both in chronically infected individuals who progress rapidly to AIDS and in those who do not experience HIV-1 disease progression for decades (1, 11), and the characteristics that define a protective HIV-1-specific CD8⁺ T-cell response are not known. In particular, the level of control over viral replication is not predicted by the overall breadth, magnitude, or function of virus-specific CD8⁺ T-cell responses in chronic HIV-1 infection (1, 4, 11, 26, 28).Here, we demonstrate in a large cohort of individuals identified during PHI that immunodominance patterns of virus-specific CD8⁺ T-cell responses detected in PHI, but not in chronic HIV-1 infection, are strongly associated with the subsequent set point of viral replication. These data show that the specificity of the initial CD8⁺ T-cell response to HIV is critical for the subsequent control of viremia and have important implications for the rational selection of antigens for future HIV-1 vaccines.     

Nef-Induced CD4 Endocytosis in Human Immunodeficiency Virus Type 1 Host Cells: Role of p56lck Kinase

Human immunodeficiency virus type 1 (HIV-1) Nef interferes with the endocytic machinery to modulate the cell surface expression of CD4. However, the basal trafficking of CD4 is governed by different rules in the target cells of HIV-1: whereas CD4 is rapidly internalized from the cell surface in myeloid cells, CD4 is stabilized at the plasma membrane through its interaction with the p56^lck kinase in lymphoid cells. In this study, we showed that Nef was able to downregulate CD4 in both lymphoid and myeloid cell lines but that an increase in the internalization rate of CD4 could be observed only in lymphoid cells. Expression of p56^lck in nonlymphoid CD4-expressing cells restores the ability of Nef in order to increase the internalization rate of CD4. Concurrent with this observation, the expression of a p56^lck-binding-deficient mutant of CD4 in lymphoid cells abrogates the Nef-induced acceleration of CD4 internalization. We also show that the expression of Nef causes a decrease in the association of p56^lck with cell surface-expressed CD4. Regardless of the presence of p56^lck, the downregulation of CD4 by Nef was followed by CD4 degradation. Our results imply that Nef uses distinct mechanisms to downregulate the cell surface expression levels of CD4 in either lymphoid or myeloid target cells of HIV-1.Besides proteins that are essential for proper virus processing and assembly, the genomes of primate lentiviruses such as human immunodeficiency virus type 1 (HIV-1) encode auxiliary proteins that modulate viral infectivity. The 27-kDa auxiliary protein Nef is a key element in the progression of primary HIV-1 infection toward AIDS. Cases of patients infected with HIV-1 strains harboring a deletion in the nef gene or a defective nef allele have been reported. Some of these patients exhibit asymptomatic or slow progression toward the disease (6, 17, 37). In vitro, Nef facilitates viral replication and enhances the infectivity of viral particles (13, 47, 69). The mechanisms involved in the Nef-induced increase of viral infectivity remain elusive; however, it is a multifactorial process related to the ability of Nef to alter the trafficking of host cell proteins.Indeed, the most documented effect of Nef during the course of viral infection is its ability to disturb the clathrin-dependent trafficking machinery involved in the transport of transmembrane proteins through endosomal compartments. This leads to the modulation of the level of cell surface expression for some receptors, including CD4, which is the primary receptor of HIV-1 (35) and major histocompatibility complex class I (reviewed in references 22 and 27). The downregulation of CD4, which results in the impairment of the immunological synapse (72) and the downregulation of major histocompatibility complex class I molecules (reviewed in reference 16), is believed to contribute to the escape of HIV-1-infected cells from immunosurveillance. Moreover, the downregulation of CD4 helps avoid superinfection of cells, which would be deleterious to the virus (reviewed in reference 21), and has a direct impact on viral fitness by allowing better incorporation of the functional envelope in viral particles produced from CD4-expressing cells (3, 36, 53).Nef-induced cell surface downregulation of CD4 is efficient in all CD4-expressing cells and depends on the integrity of a di-Leu motif at position 164/165 of the C-terminal flexible loop of HIV-1 Nef (2, 9, 25). This di-Leu motif allows for the interaction with clathrin-associated adaptor protein (AP) complexes that participate in the clathrin-dependent vesicular transport within the endocytic pathway. The AP type 2 (AP-2) complex is localized at the plasma membrane and is essential to the assembly and function of clathrin-coated pits involved in the internalization of receptors from the cell surface (59). The interaction of Nef with AP-2 is well delineated and has been proposed to enhance the targeting of CD4 to clathrin-coated pits and its internalization (10, 12, 26, 32, 39).Helper T lymphocytes are the predominant cell type that expresses CD4; however, CD4 is also present at the surfaces of monocytes and macrophages (70), where its function is yet to be elucidated. Whereas cell surface CD4 is rapidly internalized in myeloid cells, CD4 is stabilized at the plasma membrane in lymphoid cells through its interaction with the Src family protein tyrosine kinase p56^lck. Cys residues located at positions 420/422 in the CD4 cytoplasmic tail are essential to the constitutive association with p56^lck (73). Besides its role in signal transduction, this interaction also correlates with an accumulation of CD4 in lipid rafts and enhanced exclusion of CD4 from clathrin-coated pits (50).In T cells, treatment with phorbol esters such as phorbol 12-myristate 13-acetate (PMA) provokes the phosphorylation of Ser residues found in the cytoplasmic tail of CD4. This correlates with a decreased association of p56^lck with CD4 and the internalization of the receptor (24, 32-34, 41, 45, 48, 52, 56, 61, 66-68). Nef-induced CD4 downregulation is known to be independent of Ser phosphorylation (20) and is therefore governed by mechanisms different from those involved in PMA-induced CD4 downregulation. However, the Leu-based sorting motif in the CD4 cytoplasmic tail is critical for both PMA and Nef-induced CD4 downregulation (2, 5, 24, 31, 56, 60, 68), thus indicating that despite being different, the mechanisms involved in Nef- and PMA-induced CD4 downregulation partially overlap.In the present study, we investigated whether the mechanisms used by Nef to downregulate CD4 are cell type-dependent processes. We looked at the trafficking and steady-state expression of CD4 in the main target cells of HIV-1, CD4-positive T lymphocytes, and cells of the monocyte/macrophage lineage. Our results demonstrate that the presence of p56^lck has a direct impact on the mechanisms used by Nef to downregulate CD4 from the cell surface of T lymphocytes. They also reveal that Nef uses distinct pathways to decrease levels of cell surface expression of CD4 in lymphoid or myeloid target cells of HIV-1.     

Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.