Molecular characterization of a highly divergent HIV type 1 isolate obtained early in the AIDS epidemic from the Democratic Republic of Congo.

Numerous complete human immunodeficiency virus type 1 (HIV-1) genomes have been characterized for contemporary viruses, but few isolates obtained early in the HIV-1 epidemic have been studied. In this article, we describe the molecular characterization of an HIV-1 isolate (83CD003) that was obtained from an AIDS patient in Kinshasa, Democratic Republic of Congo (DRC) in 1983. The complete 83CD003 genome was sequenced in its entirety and found to encode uninterrupted open reading frames for all viral genes. Phylogenetic analysis revealed 83CD003 was a member of the major (M) group of HIV -1, but did not group with any of the known subtypes. Rather, it formed an independent lineage in all regions of its genome that was roughly equidistant from representatives of all other subtypes. Similarly, 83CD003 also did not cluster with any of several unclassified group M sequences that have been reported more recently to circulate in the DRC, suggesting that it may represent an early group M lineage thai is either rare or has gone extinct. The molecular clone of 83CD003 yielded an infectious virus after transfection into mammalian cells and its biological properties can be further studied.

Mutations in both gp120 and gp41 are responsible for the broad neutralization resistance of variant human immunodeficiency virus type 1 MN to antibodies directed at V3 and non-V3 epitopes.

The escape of human immunodeficiency virus type 1 from effects of neutralizing antibodies was studied by using neutralization-resistant (NR) variants generated by growing the neutralization-sensitive (NS) wild-type MN virus in the presence of human serum with neutralizing antibodies, more than 99% of which were directed at the V3 region of gp120. The variants obtained had broad neutralization resistance to human sera, without limitation with respect to the V3 specificity of the sera. The molecular basis for the resistance was evaluated with molecularly cloned viruses, as well as with pseudoviruses expressing envelope glycoproteins of the NS and NR phenotypes. Nucleotide sequence analyses comparing NS and NR clones revealed a number of polymorphisms, including six in the V1/V2 region, two in C4/V5 of gp120, three in the leucine zipper (LZ) domain of gp41, and two in the second external putative alpha-helix region of gp41. A series of chimeras from NS and NR env genes was constructed, and each was presented on pseudoviruses to locate the domain(s) which conferred the phenotypic changes. The neutralization phenotypes of the chimeric clones were found to be dependent on mutations in both the C4/V5 region of gp120 and the LZ region of gp41. Additionally, interaction between mutations in gp120 and gp41 was demonstrated in that a chimeric env gene consisting of a gp120 coding sequence from an NS clone and a gp41 sequence from an NR clone yielded a pseudovirus with minimal infectivity. The possible significance of predicted amino acid changes in these domains is discussed. The results indicate that polyvalent antibodies predominantly directed against V3 can induce NR through selection for mutations that alter interactions of other domains in the envelope complex.

Isolation and characterization of a syncytium-inducing, macrophage/T-cell line-tropic human immunodeficiency virus type 1 isolate that readily infects chimpanzee cells in vitro and in vivo.

Fresh human immunodeficiency virus type 1 (HIV-1) isolates from patients with AIDS were screened for infectivity in chimpanzee peripheral blood mononuclear cells (PBMC) to identify strains potentially able to generate high virus loads in an inoculated animal. Only 3 of 23 isolates obtained were infectious in chimpanzee cells. Of these three, only one (HIV-1DH12) was able to initiate a productive infection in PBMC samples from all 25 chimpanzees tested. HIV-1DH12 tissue culture infections were characterized by extremely rapid replication kinetics, profound cytopathicity, and tropism for chimp and human PBMC, primary human macrophage, and several human T-cell lines. An infection was established within 1 week of inoculating a chimpanzee with 50 50% tissue culture infective doses of HIV-1DH12; cell-free virus was recovered from the plasma at weeks 1, 2, and 4 and was associated with the development of lymphadenopathy. Virus loads during the primary infection and at 6 months postinoculation were comparable to those reported in HIV-1-seropositive individuals.

Integration is required for productive infection of monocyte-derived macrophages by human immunodeficiency virus type 1.

Certain human immunodeficiency virus type 1 (HIV-1) isolates are able to productively infect nondividing cells of the monocyte/macrophage lineage. We have used a molecular genetic approach to construct two different HIV-1 integrase mutants that were studied in the context of an infectious, macrophage-tropic HIV-1 molecular clone. One mutant, HIV-1 delta D(35)E, containing a 37-residue deletion within the central, catalytic domain of integrase, was noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages. The HIV-1 delta D(35)E mutant, however, exhibited defects in the assembly and/or release of progeny virions in transient transfection assays, as well as defects in entry and/or viral DNA synthesis during the early stages of monocyte-derived macrophage infection. The second mutant, HIV-1D116N/8, containing a single Asp-to-Asn substitution at the invariant Asp-116 residue of integrase, was also noninfectious in both peripheral blood mononuclear cells and monocyte-derived macrophages but, in contrast to HIV-1 delta D(35)E, was indistinguishable from wild-type virus in reverse transcriptase production. PCR analysis indicated that HIV-1D116N/8 entered monocyte-derived macrophages efficiently and reverse transcribed its RNA but was unable to complete its replication cycle because of a presumed block to integration. These data are consistent with the hypothesis that integration is an obligate step in productive HIV-1 infection of activated peripheral blood mononuclear cells and primary human macrophage cultures.

Characterization of epitopes defining two major subclasses of polytropic murine leukemia viruses (MuLVs) which are differentially expressed in mice infected with different ecotropic MuLVs.

Polytropic murine leukemia viruses (MuLVs) arise in mice by recombination of ecotropic MuLVs with endogenous retroviral envelope genes and have been implicated in the induction of hematopoietic proliferative diseases. Inbred mouse strains contain many endogenous sequences which are homologous to the polytropic env genes; however, the extent to which particular sequences participate in the generation of the recombinants is unknown. Previous studies have established antigenic heterogeneity among the env genes of polytropic MuLVs, which may reflect recombination with distinct endogenous genes. In the present study, we have examined many polytropic MuLVs and found that nearly all isolates fall into two mutually exclusive antigenic subclasses on the basis of the ability of their SU proteins to react with one of two monoclonal antibodies, termed Hy 7 and MAb 516. Epitope-mapping studies revealed that reactivity to the two antibodies is dependent on the identity of a single amino acid residue encoded in a variable region of the receptor-binding domain of the env gene. This indicated that the two antigenic subclasses of MuLVs arose by recombination with distinct sets of endogenous genes. Evaluation of polytropic MuLVs in mice revealed distinctly different ratios of the two subclasses after inoculation of different ecotropic MuLVs, suggesting that individual ecotropic MuLVs preferentially recombine with distinct sets of endogenous polytropic env genes.

Amino acid substitutions in the human immunodeficiency virus type 1 gp120 V3 loop that change viral tropism also alter physical and functional properties of the virion envelope.

The third variable (V3) region within the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) has been reported to be an important determinant of viral tropism. In this study a series of isogenic recombinant HIV-1 viruses, containing V3 regions from fresh isolates, were examined to ascertain if a relationship exists between viral tropism and specific properties of the virion-associated envelope. All of the viruses were able to infect CD4+ primary lymphocytes, although with different infection kinetics. Several recombinants, however, were unable to infect a continuous CD4+ T-cell line permissive for the parental virus and exhibited a marked decrease in the kinetics of virion-associated gp120 binding to a soluble form of CD4. A known macrophage-tropic HIV-1 isolate, also unable to infect the T-cell line, bound CD4 with similarly slow reaction kinetics. Although the inability to infect T-cell lines is a commonly observed property of macrophage-tropic isolates of HIV-1, the loss of T-cell line tropism by the V3 recombinants was not accompanied by a substantial infectivity for monocyte-derived macrophages, as monitored by reverse transcriptase production. Additional analyses of the recombinant virion gp120s indicated that most of the V3 substitutions increased the inherent stability of the virion gp120-gp41 envelope complex. These results indicate that V3-induced alterations in viral tropism are associated with changes in physical and functional properties of the virion envelope.

A novel HIV-1 isolate containing alterations affecting the NF-kappa B element.

Three molecular clones of HIV-1, derived from a single isolate (AL1), exhibited distinct replicative and cytopathic properties during propagation in a human T cell line. The phenotypic differences observed were attributable, in large part, to changes affecting the viral LTR. Nucleotide sequence and PCR analyses demonstrated the presence of novel duplications or deletions involving the NF-kappa B motif. These changes in the enhancer element were identified in the original AL1 virus stock. Subcloning of the variant NF-kappa B segments into LTR-driven CAT expression vectors confirmed a correlation between promoter activity and replicative/cytopathic capacity.

Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T lymphocytes.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) contains three binding sites for the transcriptional factor Sp1. In order to investigate the role that the Sp1-binding sites play in regulation of HIV replication, we have introduced a deletion of all three Sp1-binding sites into the LTR of an infectious molecular clone of HIV. Viral stocks have been prepared from this mutant virus, designated dl-Sp, and these stocks have been used to study its replicative ability in human T cells. The dl-Sp virus replicated efficiently in MT4 cells and in phytohemagglutinin-stimulated human peripheral blood lymphocytes, but it replicated poorly and with delayed kinetics in A3.01 (CEM) T cells unless those cells had been treated with the cytokine tumor necrosis factor alpha. Gel retardation assays to study the levels of DNA-binding proteins present in these cells showed that NF-kappa B activity could be detected in the nuclei of MT4 cells but not in A3.01 cells unless they had been treated with tumor necrosis factor alpha. Thus, the presence of NF-kappa B activity appeared to be required for efficient replication of an HIV whose LTR Sp1-binding sites had been deleted. This suggests that NF-kappa B can functionally compensate for Sp1 in activating HIV replication. The HIV LTR is therefore similar to the promoter-enhancer units of other viruses in that it is composed of multiple functional elements that may contribute differently to viral replication depending on the levels of DNA-binding proteins present in the target cells.

Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gp120.

A previously reported amino acid substitution within the second conserved domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope results in the production of noninfectious particles. Molecular characterization of spontaneous revertant viruses, which arose during long-term cocultures of this env mutant, revealed that an amino acid change within another region of gp120 could functionally compensate for the mutation and restore infectivity. In the current study, we have introduced a conservative amino acid substitution at this second-site revertant codon and observed a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared which contained an amino acid change within a variable region of gp120 which restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the HIV-1 envelope, suggest that at least three discrete regions of gp120 may interact during the establishment of a productive viral infection. This critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to or concomitant with the fusion of viral and cellular membranes.

In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity.

Site-specific mutagenesis was used to introduce amino acid substitutions at the asparagine codons of four conserved potential N-linked glycosylation sites within the gp120 envelope protein of human immunodeficiency virus (HIV). One of these alterations resulted in the production of noninfectious virus particles. The amino acid substitution did not interfere with the synthesis, processing, and stability of the env gene polypeptides gp120 and gp41 or the binding of gp120 to its cellular receptor, the CD4 (T4) molecule. Vaccinia virus recombinants containing wild-type or mutant HIV env genes readily induced syncytia in CD4+ HeLa cells. These results suggest that alterations involving the second conserved domain of the HIV gp120 may interfere with an essential early step in the virus replication cycle other than binding to the CD4 receptor. In long-term cocultures of a T4+ lymphocyte cell line and colon carcinoma cells producing the mutant virus, revertant infectious virions were detected. Molecular characterization of two revertant proviral clones revealed the presence of the original mutation as well as a compensatory amino acid change in another region of HIV gp120.

Molecular characterization of a polymerase mutant human immunodeficiency virus.

A cell line (8E5) containing a single defective copy of human immunodeficiency virus proviral DNA and producing noninfectious viral particles lacking reverse transcriptase (RT) and endonuclease proteins has recently been described (Folks, et al., (1986b) J. Exp. Med. 164, 280-290). In this report, the mutation in a full-length molecular clone of the provirus (p8E5) was mapped to a 1931-bp region of the pol gene encoding RT. The nucleotide sequence of this segment revealed a 1-base deletion 301 codons from the common amino terminus of the 64- and 51-kDa RTs. Expression of the p8E5 RT segment in Escherichia coli generated an enzymatically inactive and truncated 33-kDa protein.

Biologic and molecular characterization of two newly isolated ras-containing murine leukemia viruses.

A murine sarcoma virus (MSV) was recovered from an (NFS X NS.C58v-1) F1 mouse which developed splenic sarcoma and erythroleukemia 6 months after inoculation with a mink cell focus-inducing murine leukemia virus (MuLV) isolated from an NFS mouse infected with a wild mouse ecotropic MuLV. The MSV, designated NS.C58 MSV-1, induced foci of transformation in mouse and rat fibroblasts, and inoculation of mice of various strains 2 weeks of age or younger resulted in erythroleukemia and sarcomatous lesions in spleen, lymph node, and brain. The MSV provirus was molecularly cloned from a genomic library prepared from transformed non-producer rat cells. The 8.8-kilobase proviral DNA contained a 1.0-kilobase p21 ras coding segment which replaced most of the gp70-encoding portion of an MuLV, most likely the endogenous C58v-1 ecotropic virus. The ras oncogene is closely related to v-Ha-ras by hybridization, expression of p21 protein, and nucleotide sequence. It is nearly identical in sequence to v-bas, the only previously described transduced, activated mouse c-ras. At position 12 in the p21 coding region, arginine is substituted for the naturally occurring glycine present in c-ras. A second MSV isolate is described which is similar to NS.C58 MSV-1 except for a 100- to 200-base-pair deletion in the noncoding region of the ras-containing insert.

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus.

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.

Relationship between a retroviral germ line reintegration and a new mutation at the ashen locus in B10.F mice. Retroviral integration and an ashen mutation.

In a litter of B10.F mice a single mouse was found with a "dilute-like" coat-color mutation. F2 matings were then carried out to establish sufficient numbers of mice carrying this mutation in a homozygous form. The coat-color mutation was found to be allelic to the ashen locus which is closely linked to the dilute locus on chromosome 9. Further analysis of the ashen mice showed they contained an additional ecotropic provirus integrated into their chromosomal DNA. This result initially suggested that retroviral integration was responsible for the mutation. However, backcross studies showed the mutation and provirus were genetically unlinked. The reintegrated provirus was molecularly cloned and transfection studies showed the virus was B tropic in host range. The restriction map of the cloned provirus was found to be similar to previously described B-tropic viruses. The results are discussed in view of the low probability of two rare and unlinked events occurring in a single mouse.

Streptolysin S activation by lipoteichoic acid.

Lipoteichoic acid from streptococci and Staphylococcus aureus both activated membrane-bound precursor streptolysin S and induced the formation of extracellular streptolysin S. Lipoteichoic acid could replace other substances, such as yeast ribonucleic acid core, that act as carriers for hemolysin but which are not components of the streptococcus or the host. Lipoteichoic acid may play a role as the physiological carrier of streptolysin S to host tissues.

Polypeptides of simian rotavirus (SA-11) determined by a continuous polyacrylamide gel electrophoresis method.

Simian rotavirus (SA-11) isolated from infected African green monkey kidney cells was separated into two virus fractions in a CsCl density gradient and their proteins analysed on a continuous phosphte buffered polyacrylamide gel electrophoresis system. One peak (buoyant density 1.37 g/ml) contained double capsid virus particles which were radioimmunoassay (RIA)- and haemagglutinin (HA)- positive and yielded eight polypeptides whose mol. wt. ranged from 48,000 to 128,000. The second peak (buoyant density 1.39 g/ml) which contained 70% single capsid particles and was RIA-positive but HA-negative, yielded only five polypeptides. The three polypeptides missing in the second peak are associated presumably with the outer capsid of SA-11 virus particles and one or more of these is assumed to be the HA of SA-11 rotavirus.