Mechanism of enzyme inhibition mediated by anti-reverse transcriptase antibodies from HIV type 1-infected individuals.

We have examined the mechanisms of reverse transcriptase (RT) inhibition mediated by anti-RT antibodies, isolated by affinity chromatography, from four HIV-1-positive individuals. In kinetics assays, anti-RT immunoglobulin (Ig) obtained from three of the sera mediated a noncompetitive type of inhibition against template primer; two of these three also mediated noncompetitive inhibition with respect to deoxyribonucleoside triphosphate. Such inhibition did not require that the Ig be preincubated with RT prior to the addition of reaction components. In contrast, a more complicated pattern of inhibition was exhibited by anti-RT Ig from the fourth serum. Preincubation of this Ig with enzyme markedly enhanced the inhibition. The results demonstrate that the specificities of RT-inhibiting antibodies vary among HIV-1-infected individuals, but that one prevalent mechanism of inhibition involves interactions with epitopes outside of the enzyme active site.

Strain-specific neutralizing determinant in the transmembrane protein of simian immunodeficiency virus.

Monoclonal antibody SF8/5E11, which recognizes the transmembrane protein (TMP) of simian immunodeficiency virus of macaque monkeys (SIVmac), displayed strict strain specificity. It reacted with cloned and uncloned SIVmac251 but not with cloned SIVmac142 and SIVmac239 on immunoblots. This monoclonal antibody neutralized infection by cloned, cell-free SIVmac251 and inhibited formation of syncytia by cloned SIVmac251-infected cells; these activities were specific to cloned SIVmac251 and did not occur with the other viruses. Site-specific mutagenesis was used to show that TMP amino acids 106 to 110 (Asp-Trp-Asn-Asn-Asp) determined the strain specificity of the monoclonal antibody. This strain-specific neutralizing determinant is located within a variable region of SIVmac and human immunodeficiency virus type 2 (HIV-2) which includes conserved, clustered sites for N-linked glycosylation. The determinant corresponds exactly to a variable, weak neutralizing epitope in HIV-1 TMP which also includes conserved, clustered sites for N-linked glycosylation. Thus, the location of at least one neutralizing epitope appears to be common to both SIVmac and HIV-1. Our results suggest a role for this determinant in the viral entry process. Genetic variation was observed in this neutralizing determinant following infection of a rhesus monkey with molecularly cloned SIVmac239; variant forms of the strain-specific, neutralizing determinant accumulated during persistent infection in vivo. Selective pressure from the host immune response in vivo may result in sequence variation in this neutralizing determinant.

Myristoylation of gag proteins of HIV-1 plays an important role in virus assembly.

The gag proteins of HIV-1 are modified by the addition of myristic acid to the amino terminal glycine residue. Site-directed mutagenesis was used to construct a mutant of HIV-1 in which this glycine residue was changed to an alanine. Upon transfection into cos-1 cells, the mutant genome directed the synthesis of the full complement of HIV-1 proteins, but p17 and p17-containing polyproteins were not myristoylated. The cells transfected with the mutant DNA did not release any virus particles and no viral cores were visible by electron microscopy. Furthermore, supernatant from these transfected cells failed to infect CEM cells. The expression and function of gp120 on the surface of cells transfected with the mutant DNA was unaffected as these cells formed syncytia comparable in both size and number to the ones obtained with wild-type DNA.

Monoclonal antibodies to HTLV-III451 gp41: delineation of an immunoreactive conserved epitope in the transmembrane region of divergent isolates of HIV-1.

We report on the development of monoclonal antibodies directed against the transmembrane portion of the envelope of HTLV-III451 gp41. One of these monoclonal antibodies, designated M71/2B4, was found to cross-react with transmembrane proteins from other independent isolates of HIV-1, namely IIIB, MN, and RF. Thus, this monoclonal antibody identifies an epitope located in a region of gp41 that is conserved among all these isolates. To identify this conserved region a series of E. coli recombinant proteins were screened in immunoblot with M71/2B4. From these results the epitope recognized by this antibody appears to map at the amino terminus of gp41, in the region indicated between the cleavage site with gp120 (aa 508) and the HindIII site (aa647).

Identification of simian immunodeficiency virus SIVMAC env gene products.

A monoclonal antibody recognizing an antigenic determinant on the env transmembrane protein, gp32 of simian immunodeficiency virus SIVMAC has been developed and designated SF8/5E11. The reactivity of this antibody was found to be type specific, since it did not cross-react with either SIVSMM or SIVMNe transmembrane proteins. The availability of both this antibody and the complete nucleotide sequence of SIVMAC allowed us to define the organization of the env gene products of this virus. Radiolabel sequencing of the amino termini of both gp160 and gp32 confirmed the positions of both cleavage sites predicted by alignment of the inferred amino acid sequences of the SIVMAC and human immunodeficiency virus type 1 env genes. The cleavage site between the signal peptide and the external env glycoprotein resides between the cysteine residue at position 21 and the threonine residue at position 22, starting from the first residue after the env gene initiator methionine. The env precursor polyprotein gp160 is cleaved between arginine 526 and glycine 527 to give rise to the external glycoprotein and the transmembrane of SIVMAC.

Purification and partial characterization of human immunodeficiency virus type 2 reverse transcriptase.

We have raised a rabbit monospecific antibody against a synthetic peptide derived from a sequence within the COOH-terminal portion of the reverse transcriptase (RT) of HIV-1. This sequence was also found to be conserved in the predicted amino acid sequence of HIV-2. The antibody, designated C2003, cross-reacted with HIV-2 RT on immunoblots of HIV-2 virus extract and directly inhibited HIV-2 RT activity. Fractionation of HIV-2 RT by immunoaffinity chromatography with C2003 antibody yielded a pair of viral proteins of 68 and 55 kD associated with both RT and RNAse H activities. Both proteins were found to be highly immunogenic, recognized by 11 of 11 human sera that previously tested positive for antibodies to HIV-2 antigens in immunoblot assays.

Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24.

Human immunodeficiency virus (HIV) p24 was purified to homogeneity and subjected to NH2-terminal sequencing. The sequence determined perfectly corresponded to the amino acid sequence predicted from the nucleotide sequence of a middle portion of the HIV first open frame: the gag gene. Edman degradation of purified HIV p17 revealed instead a blocked NH2 terminus. Hybridomas secreting monoclonal antibodies to p24 and p17 were developed and used to immunologically characterize these two HIV gag gene products. They identified two gag precursor polyproteins in the cytoplasm of HIV-infected cells: Pr53gag, which corresponds to the primary translational product, and Pr39gag, which corresponds to an intermediate product of cleavage of Pr53gag. These monoclonal antibodies allowed us also to study posttranslational modification of HIV p24 and p17. p24 was found to be phosphorylated, which is a very unusual feature for a major retroviral core protein. p17 was found to be myristylated, as are all NH2-terminal gag proteins of the known human retroviruses.

High prevalence of serum antibodies to reverse transcriptase in HIV-1-infected individuals.

The HIV immunoblot profiles of 700 HIV-antibody-positive sera were examined to determine the frequency of antibody reactivity with p66/p51, the reverse transcriptase of HIV. We report a remarkably high seroprevalence of antibodies to p66/p51, detected in 79% of the sera. Only gp41 is recognized more frequently in these assays. The level of anti-p66/p51 seroreactivity varies only slightly among the clinical stages of HIV infection.

Immunological and chemical analysis of P6, the carboxyl-terminal fragment of HIV P15.

The first open reading frame of the HIV genome has been identified as the gag gene. The proteins encoded by this gene are p17 as the amino-terminal protein, p24 as the middle peptide, and p15 as the carboxyl-terminal end. A monoclonal antibody recognizing an antigenic determinant on a fragment of p15 has been developed and designated M35/2F8. This monoclonal has been instrumental in radiosequencing the carboxyl-terminal product of p15, p6, and in determining the cleavage site between this protein and the amino-terminal product, p7. By immunoaffinity chromatography it was also possible to purify p6 from HIV lysates and all p6 containing polyproteins from HIV-infected cells. These results gave more insight into the composition and processing of the HIV gag gene.

Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene.

Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41.