Frequent intrapatient recombination between human immunodeficiency virus type 1 R5 and X4 envelopes: implications for coreceptor switch

Emergence of human immunodeficiency virus type 1 (HIV-1) populations that switch or broaden coreceptor usage from CCR5 to CXCR4 is intimately coupled to CD4+ cell depletion and disease progression toward AIDS. To better understand the molecular mechanisms involved in the coreceptor switch, we determined the nucleotide sequences of 253 V1 to V3 env clones from 27 sequential HIV-1 subtype B isolates from four patients with virus populations that switch coreceptor usage. Coreceptor usage of clones from dualtropic R5X4 isolates was characterized experimentally. Sequence analysis revealed that 9% of the clones from CXCR4-using isolates had originated by recombination events between R5 and X4 viruses. The majority (73%) of the recombinants used CXCR4. Furthermore, coreceptor usage of the recombinants was determined by a small region of the envelope, including V3. This is the first report demonstrating that intrapatient recombination between viruses with distinct coreceptor usage occurs frequently. It has been proposed that X4 viruses are more easily suppressed by the immune system than R5 viruses. We hypothesize that recombination between circulating R5 viruses and X4 viruses can result in chimeric viruses with the potential to both evade the immune system and infect CXCR4-expressing cells. The broadening in cell tropism of the viral population to include CXCR4-expressing cells would gradually impair the immune system and eventually allow the X4 population to expand. In conclusion, intrapatient recombination between viruses with distinct coreceptor usage may contribute to the emergence of X4 viruses in later stages of infection.     

Antigenic variation within the CD4 binding site of human immunodeficiency virus type 1 gp120: effects on chemokine receptor utilization

To assess the antigenicity of envelope glycoproteins derived from primary human immunodeficiency virus type 1 populations, their interactions with the receptor CD4, and their coreceptor usage, we have cloned and expressed multiple gp120 proteins from a number of primary virus isolates. Characterization of these proteins showed a high degree of antigenic polymorphism both within the CD4 binding site and in defined neutralization epitopes, which may partially account for the general resistance of primary isolates to neutralizing agents. Furthermore, chimeric viruses expressing gp120 proteins with reduced CD4 binding abilities are viable, suggesting that primary viruses may require a less avid interaction with the receptor CD4 to initiate infection than do their laboratory-adapted counterparts. The coreceptor usage of chimeric viruses was related to the ability of the virus to bind CD4, with reduced CD4 binding correlating with preferential usage of CXCR4. Changes in coreceptor usage mapped to sequence changes in the C2 and V4 regions, with no changes seen in the V3 region.     

Characterization of a macaque recombinant monoclonal antibody that binds to a CD4-induced epitope and neutralizes simian immunodeficiency virus

A potent neutralizing Fab fragment from a long-term survivor of simian immunodeficiency virus (SIVsm) infection was used to construct a recombinant macaque immunoglobulin G1kappa (IgG1kappa) molecule, designated IgG1-201. A Chinese hamster ovary cell line expressing IgG1-201 was derived by stable transfection and optimized for antibody secretion by methotrexate selection and dihydrofolate reductase gene amplification. IgG1-201 effectively neutralized the homologous, molecularly cloned SIVsmH4 virus but had no activity against the heterologous SIVmac251/BK28 virus. The previously characterized, neutralization-resistant SIVsmE543-3 virus was also not neutralized by IgG1-201. Binding to SIVsmH4 gp120 was enhanced in the presence of recombinant soluble CD4, suggesting that IgG1-201 bound a CD4-induced epitope. IgG1-201 immunoprecipitated the SIVsmH4 but not the SIVsmE543-3 envelope despite a close relationship between these two clones. Immunoprecipitation of a panel of SIVsmH4/SIVsmE543-3 chimeric viruses tentatively assigned the neutralization epitope to the third constant domain, immediately C terminal to the V3 loop. These findings suggest the presence of at least one CD4-induced neutralization epitope on SIV, as is the case with human immunodeficiency virus type 1.     

Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.

Infection of macaque monkeys with simian immunodeficiency virus (SIV) is probably the best animal model currently available for studying acquired immunodeficiency syndrome. In this report, we describe three infectious molecular clones of SIVmac and one of human immunodeficiency virus type 2 (HIV-2) and their use in the study of cell and species specificity, animal infection, and the relationship of gene sequence to function. Replication of the cloned viruses in different cell lines varied dramatically. Some human CD4+ cell lines (HUT 78 and MT-4) supported the replication of SIVmac and HIV-2, while others (CEM and Jurkat-T) supported the replication of HIV-2 but not SIVmac. Growth of cloned virus in macaque lymphocytes in vitro was predictive of macaque infection in vivo. Macaque lymphocytes supported the replication of SIVmac239 and SIVmac251 but not SIVmac142 or HIV-2ROD. Using virus recovery and antibody response as criteria for infection, macaques that received cloned SIVmac251 and SIVmac239 became infected, while macaques receiving cloned SIVmac142 and HIV-2ROD did not become infected. Nucleotide sequences from the envelope region of all four cloned viruses demonstrated that there is considerable flexibility in the location of the translational termination (stop) signal. These infectious molecular clones will be very useful for future studies directed at the molecular basis for persistence, pathogenicity, tropism, and cell and species specificity.     

Suppression of simian immunodeficiency virus replication in vitro by CD8+ lymphocytes

The AIDS-like disease in rhesus monkeys induced by the simian immunodeficiency virus (SIV) has been used as a model to explore the nature of the T lymphocyte response after infection with viruses of the human immunodeficiency virus family. Activated CD8+ lymphocytes are present in increased numbers in the paracortex of lymph nodes of SIV-infected rhesus monkeys with a lymphadenopathy syndrome. We demonstrate that SIV is more readily isolated from CD8+ lymphocyte-depleted PBL of SIV-infected animals than from their unfractionated PBL. Rather than reflecting the fact that the CD8+ lymphocyte-depleted cell populations are simply enriched for CD4+ lymphocytes, this indicates that CD8+ cells themselves are critical in this regulatory interaction. In fact, CD8+ lymphocytes from SIV-infected but not uninfected rhesus monkeys can block SIV replication in vitro in PBL populations. A T lymphocyte population that blocks replication of viruses of the HIV family may contribute to containing the progression of AIDS.     

Control of viremia and prevention of simian-human immunodeficiency virus-induced disease in rhesus macaques immunized with recombinant vaccinia viruses plus inactivated simian immunodeficiency virus and human immunodeficiency virus type 1 particles

An effective vaccine against the human immunodeficiency virus type 1 (HIV-1) will very likely have to elicit both cellular and humoral immune responses to control HIV-1 strains of diverse geographic and genetic origins. We have utilized a pathogenic chimeric simian-human immunodeficiency virus (SHIV) rhesus macaque animal model system to evaluate the protective efficacy of a vaccine regimen that uses recombinant vaccinia viruses expressing simian immunodeficiency virus (SIV) and HIV-1 structural proteins in combination with intact inactivated SIV and HIV-1 particles. Following virus challenge, control animals experienced a rapid and complete loss of CD4(+) T cells, sustained high viral loads, and developed clinical disease by 17 to 21 weeks. Although all of the vaccinated monkeys became infected, they displayed reduced postpeak viremia, had no significant loss of CD4(+) T cells, and have remained healthy for more than 15 months postinfection. CD8(+) T-cell and neutralizing antibody responses in vaccinated animals following challenge were demonstrable. Despite the control of disease, virus was readily isolated from the circulating peripheral blood mononuclear cells of all vaccinees at 22 weeks postchallenge, indicating that immunologic control was incomplete. Virus recovered from the animal with the lowest postchallenge viremia generated high virus loads and an irreversible loss of CD4(+) T-cell loss following its inoculation into a na?ve animal. These results indicate that despite the protection from SHIV-induced disease, the vaccinated animals still harbored replication-competent and pathogenic virus.     

CD8+ and CD20+ lymphocytes cooperate to control acute simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys: modulation by major histocompatibility complex genotype

We have previously described two isogenic molecularly cloned simian immunodeficiency virus/human immunodeficiency virus chimeric viruses (SHIVs) that differ from one another by 9 amino acids and direct distinct clinical outcomes in inoculated rhesus monkeys. SHIV(DH12R-Clone 7), like other highly pathogenic CXCR4-tropic SHIVs, induces rapid and complete depletions of CD4+ T lymphocytes and immunodeficiency in infected animals. In contrast, macaques inoculated with SHIV(DH12R-Clone 8) experience only partial and transient losses of CD4+ T cells, show prompt control of their viremia, and remain healthy for periods of time extending for up to 4 years. The contributions of CD8+ and CD20+ lymphocytes in suppressing the replication of the attenuated SHIV(DH12R-Clone 8) and maintaining a prolonged asymptomatic clinical course was assessed by treating animals with monoclonal antibodies that deplete each lymphocyte subset at the time of virus inoculation. The absence of either CD8+ or CD20+ cells during the SHIV(DH12R-Clone 8) acute infection resulted in the rapid, complete, and irreversible loss of CD4+ T cells; sustained high levels of postpeak plasma viremia; and symptomatic disease in Mamu-A*01-negative Indian rhesus monkeys. In Mamu-A*01-positive animals, however, the aggressive, highly pathogenic phenotype was observed only in macaques depleted of CD8+ cells; SHIV(DH12R-Clone 8) was effectively controlled in Mamu-A*01-positive monkeys in the absence of B lymphocytes. Taken together, these results indicate that both CD8+ and CD20+ B cells contribute to the control of primate lentiviral infection in Mamu-A*01-negative macaques. Furthermore, the major histocompatibility complex genotype of an infected animal, as exemplified by the Mamu-A*01 allele in this study, has the additional capacity to shift the balance of the composite immune response.     

Generation of a chimeric human and simian immunodeficiency virus infectious to monkey peripheral blood mononuclear cells.

We constructed five chimeric clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVMAC) and four SIVMAC mutants by recombinant DNA techniques. Three chimeric clones and all mutants with an alteration in either the vif, vpx, vpr, or nef gene were infectious to human CD4-positive cell lines. The susceptibility of macaque monkey peripheral blood mononuclear cells (PBMC) to infection by these mutants and chimeras was examined in vitro. Macaque PBMC supported the replication of wild-type and vpx, vpr, and nef mutant SIVMAC strains. A chimera carrying the long terminal repeats (LTRs), gag, pol, vif, and vpx of SIVMAC and tat, rev, vpu, and env of HIV-1 was also replication competent in PBMC. In contrast, HIV-1, the vif mutant of SIVMAC, a chimera containing rev and env of SIVMAC, and a chimera containing vpx, vpr, tat, rev, and env of SIVMAC did not grow in PBMC. Western immunoblotting analysis of the replicating chimera in PBMC confirmed the hybrid nature of the virus. These data strongly suggested that the sequence important for macaque cell tropism lies within the LTR, gag, pol, and/or vif sequences of the SIVMAC genome.     

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.     

High level of surface CD4 prevents stable human immunodeficiency virus infection of T-cell transfectants.

CD4 is the principal receptor for the human immunodeficiency virus (HIV). We have isolated and studied CD4-expressing tumor cell clones made by expressing CD4 in the T-cell tumor line HSB. Two clones, one designated HSBCD4, a clone expressing low levels of CD4, and the other, HSB10xCD4, a high-expresser CD4+ clone, were studied for their ability to bind and replicate HIV. In contrast to many other CD4+ cells that down-modulate CD4 following HIV infection, the HSB10xCD4 clones continued to express high levels of surface CD4 following infection with HIV. Unlike infection of HSBCD4 or many other human CD4+ cells, HIV infection of HSB10xCD4 clone was short lived: p24 antigen, provirus, or coculturable virus was present for less than 14 days following infection with several strains of HIV-1 or with HIV-2. When infection was initiated by transfection of proviral DNA, high and low CD4 expressers initially produced p24 antigen at approximately the same level. However, high CD4 expressers produced coculturable virus only during the first few days following transfection, whereas low CD4 expressers transfected with HIV continued to produce virus beyond 6 weeks. Monoclonal antibody-mediated down-modulation of CD4 surface expression on HSB10xCD4 clones permitted these formerly HIV-resistant cells to become persistently infected with HIV. Thus, high concentrations of CD4 on the surface of an HIV-infected cell prevent persistent HIV infection of CD4+ cells.     

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) genome in relation to HIV-1 and simian immunodeficiency virus SIV (AGM).

We constructed an infectious molecular clone of the human immunodeficiency virus type 2 (HIV-2) and generated nine frameshift mutants corresponding to nine open reading frames identified so far. Three structural (gag, pol, env) and two regulative (tat, rev) gene mutants were not infectious, whereas vif, vpx, vpr, and nef genes were dispensable for infectivity. All of the mutants except env and rev were cytopathic in CD4+ human leukemia cells. In transfection assays, the expression of HIV-2 long terminal repeat was activated by infectious clones of HIV-1, HIV-2, and simian immunodeficiency virus from African green monkey but not by the tat mutants. However, an HIV-2 tat mutant could produce small amounts of virus proteins and particles in contrast to a rev mutant, which directed no detectable synthesis of virus proteins and virions.     

The envelope gp120 gene of human immunodeficiency virus type 1 determines the rate of CD4-positive T-cell depletion in SCID mice engrafted with human peripheral blood leukocytes.

We have used envelope recombinant viruses generated between two molecular clones of human immunodeficiency virus type 1 (HIV-1), T-cell-tropic HIV-1SF2 and macrophage-tropic HIV-1SF162, to assess pathogenic potential in the human peripheral blood leukocyte-reconstituted severe combined immune deficiency mouse model. Recombinant HIV-1SF2 viruses expressing the envelope gp120 gene of HIV-ISF162 caused as rapid a CD4+ T-cell depletion as did HIV-1SF162. The reciprocal HIV-1SF162 recombinant virus with the HIV-1SF2 envelope caused slower CD4+ T-cell loss. Although changing the V3 loop sequence of HIV-1SF162 to that of HIV-1SF2 did not change the rate of CD4+ T-cell depletion, replacing the V3 of HIV-1SF2 with the sequence of HIV-1SF162 resulted in virus that was poorly infectious in vivo but not in vitro. These studies suggest that the envelope gene determines properties important for pathogenesis in vivo as well as for cell tropism in vitro. HIV-1 infection in vivo may have more stringent requirements for envelope conformation.     

Mutational analysis of the human immunodeficiency virus vpr open reading frame.

Mutations were introduced by recombinant DNA techniques into the vpr open reading frame of an infectious molecular clone of human immunodeficiency virus type 1. The effect of these changes on the replicative and cytopathologic properties of the virus recovered from transfected cells was studied in several human CD4+ lymphocyte cell lines. In all cases, mutant viruses were infectious and cytopathic. However, when a low-input dose was used, mutants grew significantly more slowly than the wild-type virus. The growth kinetics of vpr mutants were distinct from those of vif and vpu mutants.     

Replacement of murine leukemia virus readthrough mechanism by human immunodeficiency virus frameshift allows synthesis of viral proteins and virus replication

Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model.     

The cytoplasmic and transmembrane domains of the vaccinia virus B5R protein target a chimeric human immunodeficiency virus type 1 glycoprotein to the outer envelope of nascent vaccinia virions.

The outer envelope of the extracellular form of vaccinia virus (EEV) is derived from the Golgi membrane and contains at least six viral proteins. Transfection studies indicated that the EEV protein encoded by the B5R gene associates with Golgi membranes when synthesized in the absence of other viral products. A domain swapping strategy was then used to investigate the possibility that the B5R protein contains an EEV targeting signal. We constructed chimeric genes encoding the human immunodeficiency virus (HIV) type 1 glycoprotein with the cytoplasmic and transmembrane domains replaced by the corresponding 42-amino-acid C-terminal segment of the B5R protein. Recombinant vaccinia viruses that stably express a chimeric B5R-HIV protein or a control HIV envelope protein with the original cytoplasmic and transmembrane domains were isolated. Cells infected with recombinant vaccinia viruses that expressed either the unmodified or the chimeric HIV envelope protein formed syncytia with cells expressing the CD4 receptor for HIV. However, biochemical and microscopic studies demonstrated that the HIV envelope proteins with the B5R cytoplasmic and transmembrane domains were preferentially targeted to the EEV. These data are consistent with the presence of EEV localization signals in the cytoplasmic and transmembrane domains of the B5R protein.     

Pathogenesis of primary R5 human immunodeficiency virus type 1 clones in SCID-hu mice

We studied the replication and cytopathicity in SCID-hu mice of R5 human immunodeficiency virus type 1 (HIV-1) biological clones from early and late stages of infection of three patients who never developed MT-2 cell syncytium-inducing (SI; R5X4 or X4) viruses. Several of the late-stage non-MT-2 cell syncytium-inducing (NSI; R5) viruses from these patients depleted human CD4(+) thymocytes from SCID-hu mice. Earlier clones from the same patients did not deplete CD4(+) thymocytes from SCID-hu mice as well as later clones. We studied three R5 HIV-1 clones from patient ACH142 in greater detail. Two of these clones were obtained prior to the onset of AIDS; the third was obtained following the AIDS diagnosis. In GHOST cell infection assays, all three ACH142 R5 HIV-1 clones could infect GHOST cells expressing CCR5 but not GHOST cells expressing any of nine other HIV coreceptors tested. Furthermore, these patient clones efficiently infected stimulated peripheral blood mononuclear cells from a normal donor but not those from a homozygous CCR5Delta32 individual. Statistical analyses of data obtained from infection of SCID-hu mice with patient ACH142 R5 clones revealed that only the AIDS-associated clone significantly depleted CD4(+) thymocytes from SCID-hu mice. This clone also replicated to higher levels in SCID-hu mice than the two earlier clones, and a significant correlation between viral replication and CD4(+) thymocyte depletion was observed. Our results indicate that an intrinsic property of AIDS-associated R5 patient clones causes their increased replication and cytopathic effects in SCID-hu mice and likely contributes to the development of AIDS in patients who harbor only R5 quasispecies of HIV-1.     

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.     

Functional domains within the human immunodeficiency virus type 2 envelope protein required to enhance virus production

Primate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.     

Soluble CD4 enhances simian immunodeficiency virus SIVagm infection.

The CD4 molecule is expressed on T-helper cells and serves as the cellular receptor for the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for the simian immunodeficiency viruses SIVmac and SIVagm. HIV-1, HIV-2, and SIVmac infectivity can be blocked by monoclonal antibodies (MAbs) directed against the CD4 molecule and by soluble CD4 proteins (sCD4). In the present study, we demonstrated not only lack of inhibition, but 10- to 100-fold sCD4-dependent enhancement of SIVagm infectivity of human T-cell lymphoma lines, although SIVagm infection was blocked by MAbs OKT4a and Leu3a. SIVagm enhancement with sCD4 was suppressed by MAbs OKT4a and Leu3a to levels observed without addition of sCD4. The infectivity of all four tested SIVagm variants was enhanced by sCD4 on all tested lymphoma cell lines. These results suggest a second step (second or secondary receptor) required for enhancing virus entry into the cell and may have serious implications for approaches to the treatment of acquired immunodeficiency syndrome on the basis of modified sCD4 molecules.