Evolutionary origin of human and simian immunodeficiency viruses.

From what viruses the human immunodeficiency viruses (HIVs) originated is an extremely controversial question. To address this question, we have analyzed nucleotide sequences of simian immunodeficiency viruses (SIVs) and HIVs by using the techniques for understanding molecular evolution. In particular, we compared the nucleotide sequences of whole genomes, gene region by gene region, between a given pair of viruses, including four types of SIVs--isolated from mandrills (Papio sphinx), African green monkeys (Cercopithecus aethiops), sooty mangabeys (Cercocebus atys), and rhesus macaques (Macaca mulatta)--as well as HIVs. Phylogenetic trees for all gene regions examined showed that the present HIVs may have emerged as different variants of SIVs of Old World monkeys, possibly from recombination between viruses related to SIVs.     

The emergence of simian/human immunodeficiency viruses.

Molecular evolutionary analyses strongly support the hypothesis that human immunodeficiency viruses have recently arisen from a diverse pool of nonhuman primate immunodeficiency viruses. Our understanding of the molecular phylogenetic relationships between primate and nonprimate lentiviruses is less certain, partly because key intermediate forms are still to be discovered. DNA and protein sequence comparisons reveal uncanny dissimilarities, as well as similarities, among the genetic sequences of these complex retroviruses, thereby giving rise to the notion that primate lentiviruses are participants in "fast-forward" evolution.     

Infection of a simian B cell line by human and simian immunodeficiency viruses

It is well known that HIV-1 does not establish infection in nonhuman primates, nor in cell lines derived from them, due to the existence of saturable resistance factors. In this study, we show that an in vitro established Macaca fascicularis-derived CD4(-) B cell line (F6) can be productively infected by the laboratory-adapted T-tropic HXBc2/HIV-1 strain at low multiplicity of infection, apparently because it does not express the restriction factor that has been detected in other simian cell lines. Moreover, efficient entry into F6 cells was obtained with pseudotyped recombinant HIV-1 viruses containing the laboratory-adapted T-tropic (HXBc2) or the dual-tropic (89.6) envelope glycoproteins, whereas entry of virus containing the envelope glycoproteins of the M-tropic Ba-L strain was less efficient. Virus containing primary T-tropic (Eli) envelope glycoproteins did not infect F6 cells. Furthermore, although CCR5 was not present on the cell surface and gpr15 and strl33 mRNAs were not expressed in the cells, a high level of infection of F6 cells by the M-tropic simian immunodeficiency virus SIVmac316 was observed. In contrast, F6 cells were poorly infected by T-tropic SIVmac239. Given the unique properties of the F6 cell line, i.e., that it is of simian origin yet is able to be infected by HIV-1 in a CD4-independent manner, F6 cells represent a useful model for studying cellular factors mediating resistance or permissivity to HIV-1 infection and may help to evaluate HIV-1 and SIV cell tropism.     

Population migration and the spread of types 1 and 2 human immunodeficiency viruses.

Over 14 million people are estimated to be infected with the human immunodeficiency viruses (HIV), with nearly three-fourths of the infected persons residing in developing countries. One factor responsible for dissemination of both HIV-1 and HIV-2 worldwide was the intense migration of individuals, from rural to urban centers with subsequent return migration and internationally due to civil wars, tourism, business purposes, and the drug trade. In sub-Saharan Africa, between 1960 and 1980, urban centers with more than 500,000 inhabitants increased from 3 to 28, and more than 75 military coups occurred in 30 countries. The result was a massive migration of rural inhabitants to urban centers concomitant with the spread of HIV-1 to large population centers. With the associated demographic, economic, and social changes, an epidemic of sexually transmitted diseases and HIV-1 was ignited. Migratory patterns were also responsible for the spread of endemic HIV-2 to neighboring West African countries and eventually to Europe, the Americans, and India. Although Southeast Asia was the last region in which HIV-1 was introduced, it has the greatest potential for rapid spread due to population density and inherent risk behaviors. Thus, the migration of poor, rural, and young sexually active individuals to urban centers coupled with large international movements of HIV-infected individuals played a prominent role in the dissemination of HIV globally. The economic recession has aggravated the transmission of HIV by directly increasing the population at risk through increased urban migration, disruption of rural families and cultural values, poverty, and prostitution and indirectly through a decrease in health care provision. Consequently, social and economic reform as well as sexual behavior education need to be intensified if HIV transmission is to be controlled.     

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.     

Construction of chimeric simian and human immunodeficiency viruses that produce interleukin 12

Chimeric simian and human immunodeficiency viruses (SHIVs) are useful for evaluating vaccine candidates against HIV-1 and for investigating the pathogenesis of HIV-1 in vivo. In addition, SHIVs are candidates for a vaccine against HIV-1 because attenuated SHIVs can induce long-lasting anti-HIV-1 Env humoral and cell-mediated immunity in monkeys without AIDS-like diseases. In this study, we inserted IL-12 genes in a nef-deleted SHIV to increase the ability of the SHIV to induce cell-mediated immunity against HIV-1. The SHIV vector was constructed by deleting the nef gene and replacing it with restriction enzyme sites. Since IL-12 consists of two subunit genes, p35 and p40, SHIVs with one or both of these genes were constructed. SHIVs with either one of the subunit genes could replicate without a deletion of the inserted gene, but SHIVs with two subunit genes replicated poorly and the inserted genes were rapidly deleted. Production of IL-12 was detected when both of the single-subunit SHIVs were coinfected. The production of IL-12 by the coinfection reached 800 pg/ml, and IL-12 was detected after serial passage in cell cultures, although this amount of IL-12 heterodimer was 150-1500 times less than that of the p40 subunits. These IL-12-producing SHIVs are candidates for a live-attenuated vaccine to induce effective cellular immunity against HIV-1.     

Effect of a human immunodeficiency virus protease inhibitor on human monocyte function.

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Encoded by the HIV genome are several precursor proteins that undergo proteolytic cleavage to yield functional proteins. The env precursor protein is cleaved by a cellular protease. The gag precursor protein of HIV (p55), however, is cleaved by a virally encoded aspartate protease (HIV Protease). Cleavage of p55 is required for viral maturation and infectivity. There are also several host cell aspartate proteases that serve important homeostatic functions. Cathepsins D and E are lysosomal aspartate proteases which are believed to play an important role in macrophage function, and it has been suggested that inhibition of these enzymes by an HIV protease inhibitor may exacerbate immunosuppression in AIDS patients. We have studied the effect of SK&F 107461 (a hydroxyethylene dipeptide isostere inhibitor of HIV protease), on various host defense functions of human monocytes. Pepstatin A (an inhibitor of most aspartate proteases) and leupeptin (an inhibitor of serine and cysteine proteases) were included as controls. Although less potent than the prototypic aspartate protease inhibitor pepstatin, SK&F 107461 inhibited partially purified cathepsin D in vitro. However, in cell-based assays, SK&F 107461 had no effect on the degradation of hemoglobin, antigen processing of the protein antigen streptokinase, or secretion of 17-kD IL-1 beta by monocytes at concentrations which inhibit maturation of intracellular virus in HIV infected monocytes. Furthermore, SK&F 107461 had no effect on constitutive candidacidal activity. In contrast, leupeptin and pepstatin A partially inhibited accessory cell function of monocytes in the proliferative response to the recall antigen streptokinase. In addition, leupeptin partially inhibited degradation of hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic variation of simian immunodeficiency viruses in nonhuman primates.

The generation of biologically active proviral DNA clones of simian immunodeficiency virus (SIV) that give rise to infectious virions has allowed the detailed examination of genetic variation in experimentally inoculated monkeys. Studies of nucleotide sequences derived directly from circulating leukocytes of infected monkeys show that the SIV genome undergoes rapid and dramatic variation during the course of infection. The env gene is a major site for variation, and within the Env protein, hypervariable regions analogous to those previously defined for the human immunodeficiency virus type 1 (HIV-1) env gene are apparent. A major exception is the region corresponding to the V3 domain in HIV-1, which has been highly conserved in all SIV studies to date. These data notwithstanding, the role of SIV genetic variation in the pathogenesis of AIDS in monkeys remains unclear. Genetic variation within the env gene does not appear to be sufficient for the development of AIDS since significant variation is observed in both pathogenic and nonpathogenic SIV infections. Furthermore, although it generally is believed that env gene variation might allow HIV and SIV to avoid recognition and elimination by host immune responses, this premise has not been rigorously proven. The use of molecularly cloned SIV in monkey models has provided important quantitative and qualitative information on in vivo sequence variation, and these data, in turn, have laid the groundwork for addressing the undoubtedly complex functional significance of this variation.     

Persistent infection of rhesus macaques with T-cell-line-tropic and macrophage-tropic clones of simian/human immunodeficiency viruses (SHIV).

To elucidate the functions of human immunodeficiency virus type 1 (HIV-1) genes in a nonhuman primate model, we have constructed infectious recombinant viruses (chimeras) between the pathogenic molecular clone of simian immunodeficiency virus (SIV) SIVmac239 and molecular clones of HIV-1 that differ in phenotypic properties controlled by the env gene. HIV-1SF33 is a T-cell-line-tropic virus which induces syncytia, and HIV-1SF162 is a macrophage-tropic virus that does not induce syncytia. A DNA fragment encoding tat, rev, and env (gp160) of SIVmac239 has been replaced with the counterpart genetic region of HIV-1SF33 and HIV-1SF162 to derive chimeric recombinant simian/human immunodeficiency virus (SHIV) strains SHIVSF33 and SHIVSF162, respectively. In the acute infection stage, macaques inoculated with SHIVSF33 had levels of viremia similar to macaques infected with SIVmac239, whereas virus loads were 1/10th to 1/100th those in macaques infected with SHIVSF162. Of note is the relatively small amount of virus detected in lymph nodes of SHIVSF162-infected macaques. In the chronic infection stage, macaques infected with SHIVSF33 also showed higher virus loads than macaques infected with SHIVSF162. Virus persists for over 1 year, as demonstrated by PCR for amplification of viral DNA in all animals and by virus isolation in some animals. Antiviral antibodies, including antibodies to the HIV-1 env glycoprotein (gp160), were detected; titers of antiviral antibodies were higher in macaques infected with SHIVSF33 than in macaques infected with SHIVSF162. Although virus has persisted for over 1 year after inoculation, these animals have remained healthy with no signs of immunodeficiency. These findings demonstrate the utility of the SHIV/macaque model for analyzing HIV-1 env gene functions and for evaluating vaccines based on HIV-1 env antigens.     

Vaccine protection against simian immunodeficiency virus infection.

Rhesus monkeys were immunized by multiple inoculations with purified, disrupted, noninfectious simian immunodeficiency virus (SIV) in adjuvant. Immunized monkeys developed anti-SIV antibodies detectable by whole-virus ELISA and by immunoblot reactivity; these antibodies had weak neutralizing activity. One week after the last immunization, monkeys were challenged with 200-1000 animal infectious doses of uncloned, live SIV. The same strain of SIV that was used for vaccination was also used for challenge. Anamnestic antibody responses and SIV recovery from peripheral blood were used to evaluate infection following the live virus challenge; two of six vaccinated monkeys showed no evidence of infection following the live virus challenge. Transfusion of 10 ml of whole blood from these two into uninfected, naive rhesus monkeys did not result infection of the recipients, providing further support for the lack of infection in the two previously vaccinated animals. Four of four unvaccinated control monkeys inoculated with these doses of live SIV became infected and three of these died with AIDS 118-258 days after infection. Only one of the six vaccinated monkeys has died to date. In situ hybridization with lymph node biopsy specimens suggested that the virus load was much higher in control macaques than in vaccinated macaques. These results indicate that vaccination with inactivated whole virus can protect macaques against challenge with live SIV. Furthermore, they provide hope that vaccine protection against human AIDS virus infection may be possible.     

Engineering human immunodeficiency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation.

Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection.     

Adaptation of human and simian immunodeficiency viruses for resistance to tetherin/BST-2

Tetherin (BST-2 or CD317) is an interferon-inducible cellular factor that prevents the detachment of enveloped viruses from infected cells. The primate lentiviruses have evolved different countermeasures to tetherin. The majority of SIVs use Nef to antagonize the tetherin proteins of their nonhuman primate hosts. However, due to the absence of sequences in human tetherin required for antagonism by Nef, HIV-1 Vpu and HIV-2 Env evolved to serve this function in humans. We recently identified compensatory changes in the Env cytoplasmic domain of a pathogenic nef-deleted SIV that confers resistance to rhesus macaque tetherin. These observations highlight the extraordinary plasticity of the primate lentiviruses in adapting to the tetherin proteins of their respective hosts, and reveal a prominent role for tetherin in shaping the evolution of the primate lentiviruses.     

Clinical and virological characterization of persistent human infection with simian foamy viruses

Persons occupationally exposed to nonhuman primates (NHPs) can be persistently infected with simian foamy virus (SFV). The clinical significance and person-to-person transmissibility of zoonotic SFV infection is unclear. Seven SFV-infected men responded to annual structured interviews and provided whole blood, oral, and urogenital specimens for study. Wives were tested for SFV infection. Proviral DNA was consistently detected by PCR in PBMCs of infected men and inconsistently in oral or urogenital samples. SFV was infrequently cultured from their PBMCs and throat swabs. Despite this and a long period of intimate exposure (median 20 years), wives were SFV negative. Most participants reported nonspecific symptoms and diseases common to aging. However, one of two persons with mild thrombocytopenia had clinically asymptomatic nonprogressive, monoclonal natural killer cell lymphocytosis of unclear relationship to SFV. All participants worked with NHPs before 1988 using mucocutaneous protection inconsistently; 57% described percutaneous injuries involving the infecting NHP species. SFV likely transmits to humans through both percutaneous and mucocutaneous exposures to NHP body fluids. Limited follow-up has not identified SFV-associated illness and secondary transmission among humans.     

Immunology of human immunodeficiency virus infection and the acquired immunodeficiency syndrome. An update

Recent advances in the understanding of the pathogenesis of infection with human immunodeficiency virus (HIV) stems from the demonstration that the membrane glycoprotein, CD4, is the cellular receptor for HIV. This glycoprotein is found mainly on the surface of a major subpopulation of T lymphocytes and also on macrophages, natural killer cells, some B lymphocytes, and neuronal cells. Cells infected with HIV may be destroyed or have their normal function impaired. Host immune responses to HIV are poor and are not sustained. Neutralizing antibody often is not produced, or HIV may escape from normal immunosuppressive mechanisms through the process of rapid antigenic variation. Factors and markers that may be important in the outcome or that may predict progression of HIV infection are genetic (Gc type), environmental (nutritional status or intercurrent sexually transmitted diseases sustained by the host), and immunologic (rate of decline in number and impairment of function of CD4 lymphocytes and of decline in antibody titers to HIV core protein, p24). A recombinant vaccine will probably be developed for testing in future clinical trials.     

Autoantibodies and human immunodeficiency viruses infection: a case-control study

OBJECTIVE: To determine the prevalence of organ-specific and non-specific autoantibodies in HIV-infected patients. DESIGN: A multicentric collaborative case-control study including 105 HIV patients and 100 sex- and age-matched HIV-negative healthy volunteers. METHODS: Antinuclear, anti-ds DNA, anti-histone, anti-Sm, rheumatoid factor(IgM), anti-beta 2 glycoprotein 1, antineutrophil cytoplasmic, anti-LKM1, anti-LCA1, anti-gastric parietal cell, antiplatelet, anti-intermediate filament, anti-mitotic spindle apparatus, anti-Golgi, anti-ribosome and anti-thyroid autoantibodies were screened in six European laboratories. RESULTS: Only IgG and IgM anticardiolipin, IgG antiplatelet, anti-smooth muscle and anti-thyroglobulin antibodies were statistically more frequent in HIV patients. There was no correlation with the numbers of CD4+ cells except in the case of anti-smooth muscle antibodies. We were unable to find specific autoantibodies such as anti-ds DNA, anti-Sm, AMA, anti-LKM1, anti-LCA1 or anti-beta 2 GP1 antibodies in these patients. CONCLUSIONS: Our results indicate that the autoantibody profile of HIV infections is comparable to those of other chronic viral infections. HIV does not seem to be more autoimmunogenic than other viruses.     

CD4-Pseudomonas exotoxin hybrid protein blocks the spread of human immunodeficiency virus infection in vitro and is active against cells expressing the envelope glycoproteins from diverse primate immunodeficiency retroviruses.

We previously described an unusual recombinant protein, designated CD4(178)-PE40, containing the gp120 binding region of human CD4 linked to active regions of Pseudomonas exotoxin A. The ability of this molecule to selectively inhibit protein synthesis in cells expressing the surface envelope glycoprotein of human immunodeficiency virus (HIV) suggested this molecule may be useful in treating infected individuals. To further evaluate its therapeutic potential, several in vitro properties of this hybrid toxin were examined. CD4(178)-PE40 was found to be an extremely potent cytotoxic agent, selectively killing HIV-infected cells with IC50 values around 100 pM. In a coculture system employing mixtures of HIV-infected and -uninfected cells, the hybrid toxin inhibited spread of the infection, as judged by a delay in HIV-induced cell killing and a dramatic suppression of free virus production. Experiments with control recombinant proteins indicated that this protective effect was primarily due to selective killing of the HIV-infected cells, rather than to a simple blocking effect of the CD4 moiety of the hybrid toxin. Using recombinant vaccinia viruses as expression vectors, we found the hybrid toxin to be active against cells expressing the envelope glycoproteins of divergent isolates of HIV-1, as well as HIV-2 and simian immunodeficiency virus. These results provide further support for the therapeutic potential of CD4(178)-PE40 in the treatment of HIV-infected individuals.