Macrophage tropism of human immunodeficiency virus type 1 facilitates in vivo escape from cytotoxic T-lymphocyte pressure

Early after seroconversion, macrophage-tropic human immunodeficiency virus type 1 (HIV-1) variants are predominantly found, even when a mixture of macrophage-tropic and non-macrophage-tropic variants was transmitted. For virus contracted by sexual transmission, this is presently explained by selection at the port of entry, where macrophages are infected and T cells are relatively rare. Here we explore an additional mechanism to explain the selection of macrophage-tropic variants in cases where the mucosa is bypassed during transmission, such as blood transfusion, needle-stick accidents, or intravenous drug abuse. With molecularly cloned primary isolates of HIV-1 in irradiated mice that had been reconstituted with a high dose of human peripheral blood mononuclear cells, we found that a macrophage-tropic HIV-1 clone escaped more efficiently from specific cytotoxic T-lymphocyte (CTL) pressure than its non-macrophage-tropic counterpart. We propose that CTLs favor the selective outgrowth of macrophage-tropic HIV-1 variants because infected macrophages are less susceptible to CTL activity than infected T cells.     

Feline immunodeficiency virus tropism

Feline immunodeficiency virus (FIV) induces a disease similar to acquired immunodeficiency syndrome (AIDS) in cats, yet in contrast to human immunodeficiency virus (HIV), CD4 is not the viral receptor. We identified a primary receptor for FIV as CD134 (OX40), a T cell activation antigen and costimulatory molecule. CD134 expression promotes viral binding and renders cells permissive for viral entry, productive infection, and syncytium formation. Infection is CXCR4-dependent, analogous to infection with X4 strains of HIV. Thus, despite the evolutionary divergence of the feline and human lentiviruses, both viruses use receptors that target the virus to a subset of cells that are pivotal to the acquired immune response. Further, we applied the new method for FIV receptor to Ebola virus entry factors with some modifications, and identified receptor-type tyrosine kinases, Axl and Dtk (members of Tyro3 family). Distribution of the molecules matches well with the Ebola virus tropism.     

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.     

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid

Retroviral tropism is determined in part by cellular restriction factors that block infection by targeting the incoming viral capsid. Indeed, human immunodeficiency virus type 1 (HIV-1) infection of many nonhuman primate cells is inhibited by one such factor, termed Lv1. In contrast, a restriction factor in humans, termed Ref1, does not inhibit HIV-1 infection unless nonnatural mutations are introduced into the HIV-1 capsid protein (CA). Here, we examined the infectivity of a panel of mutant HIV-1 strains carrying substitutions in the N-terminal CA domain in cells that exhibit restriction attributable to Lv1 or Ref1. Manipulation of HIV-1 CA could alter HIV-1 tropism, and several mutations were identified that increased or decreased HIV-1 infectivity in a target-cell-specific manner. Many residues that affected HIV-1 tropism were located in the three variable loops that lie on the outer surface of the modeled HIV-1 conical capsid. Some tropism determinants, including the CypA binding site, coincided with residues whose mutation conferred on HIV-1 CA the ability to saturate Ref1 in human cells. Notably, a mutation that reverses the infectivity defect in human cells induced by CypA binding site mutation inhibits recognition by Ref1. Overall, these findings demonstrate that exposed variable loops in CA and a partial CypA "coat" can modulate restriction and HIV-1 tropism and suggest a model in which the exposed surface of the incoming retroviral capsid is the target for inhibition by host cell-specific restriction factors.     

Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.     

Influence of gag on human immunodeficiency virus type 1 species-specific tropism

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is due in part to dominant acting restriction factors in humans (Ref1) and monkeys (Lv1). Here we show that gag encodes determinants of species-specific lentiviral infection, related in part to such restriction factors. Interaction between capsid and host cyclophilin A (CypA) protects HIV-1 from restriction in human cells but is essential for maximal restriction in simian cells. We show that sequence variation between HIV-1 isolates leads to variation in sensitivity to restriction factors in human and simian cells. We present further evidence for the importance of target cell CypA over CypA packaged in virions, specifically in the context of gp160 pseudotyped HIV-1 vectors. We also show that sensitivity to restriction is controlled by an H87Q mutation in the capsid, implicated in the immune control of HIV-1, possibly linking immune and innate control of HIV-1 infection.     

Continuous viral escape and selection by autologous neutralizing antibodies in drug-naive human immunodeficiency virus controllers

We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication.Antibodies capable of neutralizing a subject''s own virus, called autologous neutralizing antibodies (ANAbs), have been the subject of recent studies redefining the timing and character of this response. ANAbs develop early in essentially all seropositive subjects and increase in titer during the first few months and years of infection (15, 30). Previously published data were obtained using an assay that measures ANAbs against the complete quasispecies without an analysis of the individual envelope protein (Env) sequences to which these ANAb responses were directed (10). The contemporaneous virus pool was poorly neutralized, leading to an assumption that contemporaneous ANAbs are ineffective in controlling viremia. In chronic infection, ANAbs generally have been difficult to detect (3, 29, 31, 40), but there is ample evidence for selection by NAb and resulting virus env evolution in the host (12, 30, 38). The titers of ANAbs measured against clinical or autologous isolates cultured in peripheral blood mononuclear cells typically have been low in chronic infection (31, 40), while other studies indicated the presence of strong ANAbs (2). Although ANAbs may be ineffective in subjects with high virus loads due to the continuous generation of escape variants, their role in maintaining low viral loads in human immunodeficiency virus (HIV) controllers is not known.NAbs that recognize heterologous isolates to which the subject has never been exposed, called heterologous NAbs (HNAbs), are found later in infection, and not all subjects develop this broadening of the response (5). In studies that utilized easy-to-neutralize laboratory or primary viruses, titers of HNAbs can be high (5, 6, 26, 29). Early work had shown that polyclonal HNAbs in HIV-infected subjects are directed to conserved conformational determinants on gp120 (32), including the CD4-binding site (CD4bs) (22). Several human neutralizing monoclonal antibodies with broad activity also are directed to conserved conformational determinants on Env proteins, such as the CD4bs (4) and V3 (17). However, the mechanisms that lead to the development of broad HNAbs are unknown. Their development likely is dependent upon the specific autologous Env proteins to which the subject is exposed, and these proteins are variants of the original infection in these subjects, except for cases of superinfection. Thus, we reasoned that a detailed analysis of the neutralization of individual autologous variants in subjects with broad responses and viral control could be informative.The purpose of this study was to examine the autologous neutralizing responses against autologous viral variants in the plasma of HIV-positive subjects that were controlling infection for many years. These subjects have moderate HNAbs against the quasispecies of other subjects (27). We compared longitudinal samples from five chronically infected, antiretroviral treatment-naive adults late in infection. Despite the short time frame between the sample time points, the amount of env variation was surprisingly high, indicating continuous viral evolution in controllers; contemporaneous ANAbs were present and maintained in all except one elite controller. We cloned individual env gp160 plasma variants and analyzed sequence changes related to the autologous neutralization sensitivity or resistance. We systematically examined the ANAb response directed to individual variants using contemporaneous and noncontemporaneous plasma samples and observed patterns that have not been previously reported. Mutations that were significantly associated with sensitivity or resistance to ANAbs were found on parts of the envelope that are exposed and thus may be accessible to antibodies, consistently with a role in escape and containment by NAbs.     

Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways.     

Human immunodeficiency virus pseudotypes with expanded cellular and species tropism.

One mechanism for expanding the cellular tropism of a virus is through the formation of phenotypically mixed particles or pseudotypes, a process commonly occurring during viral assembly in cells infected with two or more viruses. We report here that dual infection of cells with human immunodeficiency virus (HIV) and a murine amphotropic retrovirus leads to the production of HIV pseudotypes that have acquired the host range of the amphotropic retrovirus and are capable of infecting not only CD4- human cells but also mouse cells. The replication of the HIV pseudotypes in the various CD4- cells was determined by measuring the appearance of HIV antigens in the supernatants, by cocultivation of CD4+ CEM cells with the infected CD4- cells, and in some cases by assaying the culture supernatants directly for infectious virus. Of the cells tested, human foreskin fibroblasts were the best host cells, and by in situ cytohybridization, we were able to document that all cells in the culture were infected. In addition, the temporal appearance of HIV-specific proteins in the HIV pseudotype-infected fibroblasts was similar to that seen in CD4+ CEM cells. If the human fibroblasts were first infected with the amphotropic retrovirus, they demonstrated the property of superinfection exclusion and were resistant to subsequent infection by the HIV pseudotype. In other cell lines, including the human glioblastoma-derived cell line U373MG, HeLa cells, BALB/c mouse embryo cells, and SC-1 wild mouse cells, although the HIV pseudotype infection appeared to be less efficient, substantial amounts of HIV were nevertheless produced. These results indicate that the HIV (amphotropic retrovirus) pseudotypes may be useful for studying the molecular biology of HIV infections in a wide range of cells.     

V3-independent determinants of macrophage tropism in a primary human immunodeficiency virus type 1 isolate.

Human immunodeficiency virus type 1 isolates differ in their ability to productively infect macrophages, and several groups have mapped the genetic basis for macrophage tropism to regions of env that include the third hypervariable region (V3 loop). We recently described a primary isolate (89.6) which is highly macrophage tropic and yet differs from other macrophage-tropic strains studied in that it is cytopathic in T cells. Genetic mapping of macrophage tropism determinants in this virus was done by using chimeras generated with the prototypic non-macrophage-tropic strain HXB2. Replacement of a 2.7-kb env-containing region of HXB with corresponding sequences from 89.6 conferred the macrophage-tropic phenotype, but insertion of the 89.6 V3 loop along with V4/V5 sequences did not. Conversely, placement of HXB sequences that included V3 into 89.6 did not impair this strain's ability to replicate in macrophages. Sequence analysis of V3 shows that 89.6 differs markedly from previously described macrophage-tropic consensus sequences and that it is more similar to highly charged non-macrophage-tropic strains. This suggests either that macrophage tropism is defined by structural determinants resulting from complex interactions among multiple env regions rather than V3 sequence-specific requirements or that there are multiple mechanisms by which different strains may establish productive macrophage infection. In addition, because the HXB V3 loop supports productive macrophage infection in the background of 89.6, phenotypic characterization of V3 sequences should be considered specific to the viral context in which they are placed.     

Generation of hybrid human immunodeficiency virus utilizing the cotransfection method and analysis of cellular tropism.

Human immunodeficiency viruses (HIV) isolated from infected individuals show tremendous genetic and biologic diversity. To delineate the genetic determinants underlying specific biologic characteristics, such as rate of replication, cytopathic effects, and ability to infect macrophages and T4 lymphoid cells, generation of hybrid HIV using viruses which exhibit distinct biologic features is essential. To develop methods for generating hybrid HIV, we constructed truncated HIV proviral DNA plasmids. Upon digestion with restriction enzymes, these plasmid DNAs were cotransfected into human rhabdomyosarcoma cells to generate hybrid HIV. The hybrid HIVs derived by this method were infectious upon transmission to both phytohemagglutinin-stimulated peripheral blood lymphocytes and established human leukemic T-cell lines. The virus derived from molecular clone pHXB2 (HIVHTLV-III) productively infected CEMx174 cells. On the other hand, molecular clone pARV (HIVSF2)-derived virus did not show productive infection of CEMx174 cells when used as a cell-free virus. The hybrid HIV containing the 3' end of the genome from pARV and the 5' end of the genome from pHXB2 was effective in infecting CEMx174 cells, but the converse hybrid containing 5' pARV and 3' pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef play a role in the ability of the virus to infect a certain cell type. The intracellular ligation method should be useful in the analysis of related and unrelated HIV-1 isolates with common restriction enzyme cleavage sites.     

Macrophage tropism of human immunodeficiency virus type 1 and utilization of the CC-CKR5 coreceptor.

The recent identification of the CC-CKR5 beta chemokine receptor as a major cofactor for entry of macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1) raises the question of whether macrophage tropism is determined by utilization of this chemokine receptor. We observe that in addition to macrophage-tropic isolates of clades A, B, and E, macrophage-tropic isolates of clade F also utilize the CC-CKR5 molecule for entry. However, using single-round replication-competent reporter viruses carrying the envelope genes of T-cell line-tropic or macrophage-tropic phenotypic recombinant and mutant HIV-1 strains in infection of stable cell lines that coexpress the CD4 and chemokine receptors, we were unable to establish a strict correlation between macrophage tropism and utilization of the CC-CKR5 chemokine receptor. This latter finding suggests that a cofactor other than CC-CKR5 serves to determine entry into primary macrophages.     

Complex determinants of macrophage tropism in env of simian immunodeficiency virus.

Macrophage-tropic virus variants evolved during the course of infection of individual rhesus monkeys with cloned, non-macrophagetropic simian immunodeficiency virus. Specific changes in the envelope gene (env) were found to be primarily responsible for the dramatic increase in the ability of the virus to replicate in macrophages. Cloned viruses differing at nine amino acid positions in env exhibited a more than 100-fold difference in replicative capacity for primary cultures of rhesus monkey alveolar macrophages. At least five of the nine amino acid changes contributed to macrophage tropism. These determinants were distributed across the full length of env, including both the gp120 and gp41 products of the env gene. Furthermore, the emergence of macrophagetropic variants in vivo was associated with specific pathologic manifestations in which the macrophage is the major infected cell type. Thus, major determinants of macrophage tropism reside in env, they can be complex in nature, and the presence of macrophage-tropic virus variants in vivo can influence the disease course and disease manifestations.     

HLA class I-driven evolution of human immunodeficiency virus type 1 subtype c proteome: immune escape and viral load

Human immunodeficiency virus type 1 (HIV-1) mutations that confer escape from cytotoxic T-lymphocyte (CTL) recognition can sometimes result in lower viral fitness. These mutations can then revert upon transmission to a new host in the absence of CTL-mediated immune selection pressure restricted by the HLA alleles of the prior host. To identify these potentially critical recognition points on the virus, we assessed HLA-driven viral evolution using three phylogenetic correction methods across full HIV-1 subtype C proteomes from a cohort of 261 South Africans and identified amino acids conferring either susceptibility or resistance to CTLs. A total of 558 CTL-susceptible and -resistant HLA-amino acid associations were identified and organized into 310 immunological sets (groups of individual associations related to a single HLA/epitope combination). Mutations away from seven susceptible residues, including four in Gag, were associated with lower plasma viral-RNA loads (q < 0.2 [where q is the expected false-discovery rate]) in individuals with the corresponding HLA alleles. The ratio of susceptible to resistant residues among those without the corresponding HLA alleles varied in the order Vpr > Gag > Rev > Pol > Nef > Vif > Tat > Env > Vpu (Fisher's exact test; P < or = 0.0009 for each comparison), suggesting the same ranking of fitness costs by genes associated with CTL escape. Significantly more HLA-B (chi(2); P = 3.59 x 10(-5)) and HLA-C (chi(2); P = 4.71 x 10(-6)) alleles were associated with amino acid changes than HLA-A, highlighting their importance in driving viral evolution. In conclusion, specific HIV-1 residues (enriched in Vpr, Gag, and Rev) and HLA alleles (particularly B and C) confer susceptibility to the CTL response and are likely to be important in the development of vaccines targeted to decrease the viral load.     

Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, the emergence of mutations in the gene being targeted could lead to the rapid escape from the siRNA. Here, we simulate viral escape by systematically introducing single-nucleotide substitutions in all 19 HIV-1 residues targeted by an effective siRNA. We found that all mutant viruses that were tested replicated better in the presence of the siRNA than in the presence of the wild-type virus. The antiviral activity of the siRNA was completely abolished by single substitutions in 10 (positions 4 to 11, 14, and 15) out of 16 positions tested (substitution at 3 of the 19 positions explored rendered nonviable viruses). With the exception of the substitution observed at position 12, substitutions at either the 5' end or the 3' end (positions 1 to 3, 16, and 18) were better tolerated by the RNA interference machinery and only in part affected siRNA inhibition. Our results show that optimal HIV-1 gene silencing by siRNA requires a complete homology within most of the target sequence and that substitutions at only a few positions at the 5' and 3' ends are partially tolerated.