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M S Reitz

Publications and source records attributed to M S Reitz.

At least 73 records · Page 4Linked to original sources
PubMed1992

Viral DNA carried by human immunodeficiency virus type 1 virions.

A fundamental step in the replication of retroviruses is the reverse transcription of the viral RNA genome into a double-stranded DNA provirus. Retroviruses are believed to carry genomic information only as RNA, and synthesis of DNA is thought to start only after virus entry into the infected cell. We report here that infectious mature human immunodeficiency virus type 1 virions contain viral DNA of heterogeneous size. This heterogeneity seems to result from random stops of reverse transcription during minus- and plus-strand synthesis. The DNA carried by human immunodeficiency virus type 1 virions presumably originates from reverse transcription which takes place prior to or during formation of the mature virus particle.

Blotting, Southern↗
PubMed1992

Effect of reciprocal complementation of two defective human immunodeficiency virus type 1 (HIV-1) molecular clones on HIV-1 cell tropism and virulence.

Human immunodeficiency virus type 1 (HIV-1) displays both interstrain and intrastrain genetic variability. Virus populations with extensive microheterogeneity have been defined as swarms or quasispecies. Many of the genomes within HIV-1 swarms appear to be defective in one or more genes required for viral replication. It is unclear to what extent defective viruses play a role in the process of HIV-1 infection or in the pathogenesis of AIDS. We have isolated two biologically active HIV-1 clones: LW 12.3, which contains defects in the vif and vpr genes, and MN ST.1, which has a defect in the vpu gene. LW 12.3 is unable to replicate in peripheral blood mononuclear cells (PBMC). The growth of MN-ST.1 in SupT1 cells is marked by a 3-week lag in extracellular virus production and by the presence of unusually abundant viral buds. We demonstrate here that coinfection of PBMC with these two partially defective HIV-1 clones extends the cellular host range of LW 12.3, significantly increases the replication rate of both viral genomes, and eliminates the delay in production observed with the vpu-defective MN ST.1. When the lesions in vpr and vif of LW 12.3 are repaired, the resultant virus grows normally in PBMC. This is also the case when only vif is repaired, indicating that complementation of LW 12.3 in PBMC by MN ST.1 is mediated by vif in trans. The reciprocal complementation results in a dramatic increase of HIV-1 virulence. This two-component model represents a simplified version of the in vivo situation and illustrates one way in which interaction of defective viruses could increase the spread of infection and progression of disease.

Base Sequence↗
PubMed1992

cDNA sequencing confirms HTLV-I expression in adult T-cell leukemia/lymphoma and different sequence variations in vivo and in vitro.

Human T-lymphotropic virus type I (HTLV-I) is the etiological agent of adult T-cell leukemia/lymphoma (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). In both diseases, expression of viral message can generally only be demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. We have previously reported on the the expression of at least four types of alternatively spliced pX mRNAs in vitro and in vivo (1). The sequence variation between HTLV-I pX cDNAs cloned from two different HTLV-I-infected cell lines and from uncultured primary peripheral blood mononuclear cells (PBMC) from two ATL patients was examined. None of the cDNA clones from one of the ATL samples was completely identical to any of the previously cloned cell line messages, establishing that the demonstration of HTLV-I mRNA in ATL is not the result of PCR contamination. Sequence analysis showed that differences between samples can be clustered according to their geographic origin. Cell line cDNAs showed a more marked sequence drift than ATL cDNAs, especially in the long terminal repeat (LTR), demonstrating association of intrastrain variability with culture in vitro. Intrastrain cDNA variability in vivo also suggests ongoing viral replication in infected individuals. A premature stop codon in the pX-II open reading frame (orf) was a common finding, suggesting that the complete putative pX-II protein is not essential for T-cell immortalization or HTLV-I replication.

Base Sequence↗
PubMed1991

Relative activities of HIV-1-IIIB and HIV-1BaL LTR and tat in primary monocytes and lymphocytes.

The mechanisms determining the ability of some but not other strains of human immunodeficiency virus type 1 (HIV-1) to grow in peripheral blood monocyte-macrophages are presently unclear. The tat gene of HIV-1-IIIB which replicates poorly in human macrophages, and the tat gene of HIV-1-BaL, which replicates to high titers in the same cells in transient expression systems with their respective long terminal repeats (LTR) driving a reporter chloramphenicol acetyl transferase (CAT) gene were compared. The authors hypothesized that the tat gene and LTR of BaL might help account for its efficient growth in primary monocyte-macrophages by virtue of a high activity in these cells relative to that of the IIIB tat and LTR. Primary peripheral blood lymphocytes and monocytes were cotransfected with either the HIV-1BaL or HIV-1-IIIB LTR fused to the CAT gene and their respective tat genes. The IIIB tat and LTR were at least as active in primary lymphocytes as the BaL combination, and both tat-LTR pairs were more active in primary lymphocytes than monocytes. The same relative activities were also observed in primary monocytes after in vitro maturation to macrophages prior to transfection. These data strongly suggest that neither the tat gene nor the LTR of HIV-1-IIIB and HIV-1BaL can account for the great ability of the latter or the inability of the former to grow in monocyte-macrophages.

Amino Acid Sequence↗
PubMed1991

Lessons from caprine and ovine retrovirus infections.

Two lentiviruses that infect sheep and goats have been shown to be closely related to the human immunodeficiency virus (HIV). These ungulate lentiviruses cause a spectrum of diseases, including arthritis in their natural hosts. The molecular and cellular biology of these viruses as well as possible pathogenic mechanisms is compared to HIV-1 in order to identify common features and significant differences between the human and animals pathogens.

Animals↗
PubMed1990

Characterization of an HIV-1 point mutant blocked in envelope glycoprotein cleavage.

The envelope proteins of retroviruses are derived from a polypeptide precursor protein by cleavage adjacent to a cluster of basic amino acids. Site-specific mutagenesis was used to construct a mutant of the human immunodeficiency virus type 1 (HIV-1) in which the arginine residue at the carboxy-terminus of the gp120 was changed to a threonine residue. This single substitution was sufficient to abolish all detectable cleavage of the gp160 envelope precursor polypeptide as well as virus infectivity. The gp160 was produced in normal quantities from a biologically active clone of the mutant virus after transfection into cos-1 cells. The mutant gp160 contained N-linked oligosaccharide chains with mannose-rich cores similar to those of the gp160 produced by the wild-type clone. Immunofluorescence assays showed that gp160 was transported to the surface of transfected CD4+ HeLa cells. No envelope proteins of known size could be detected in the media of cells transfected with the mutant virus, suggesting that functional virions were not formed. Binding of the mutant gp160 to the CD4 receptor molecule was unimpaired. Despite this and the presence of gp160 on the cell surface, neither growth of mutant-transfected CD4+ HeLa cells nor cocultivation of transfected cos-1 cells with H9 cells resulted in significant syncytium formation. The data indicate that the carboxy-terminal arginine residue of HIV-1 gp120 is necessary for envelope protein cleavage and suggest cleavage is important in the virus life cycle in both functional virus release and membrane fusion.

Arginine↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Functional contribution of cysteine residues to the human immunodeficiency virus type 1 envelope.

Although the envelope gene of human immunodeficiency virus type 1 shows considerable strain variability, cysteine residues of the envelope protein are strongly conserved, suggesting that they are important to the envelope structure. We constructed and analyzed mutants of a biologically active molecular clone of human immunodeficiency virus type 1 in which different cysteines were replaced by other amino acids in order to determine their functional importance. Substitution of cysteines 296 and 331, on either side of a region recognized by type-specific neutralizing antibodies, or on either side (residues 418 and 445) of a region important for CD4 binding, resulted in noninfectious mutants. These mutants were blocked early in the viral life cycle. Their gp160 envelope precursor polypeptides were poorly cleaved, and CD4 binding was also strongly impaired. Similar substitutions in the first variable region (residue 131) or between the first and second variable regions (residue 196) also gave noninfectious mutant virus, but here the block was late in the virus life cycle; these mutants were defective for syncytium formation. Substitution of cys386, between the neutralization and CD4 binding regions, resulted in a virus which retained infectivity but which spread much more slowly than the wild type. As with the cys131 and cys196 mutants, the cys386 mutant appeared to be defective in syncytium formation. These results show that all seven of the tested cysteines are vital for envelope function and suggest that this is likely true for all envelope cysteines. The results further show that regions important for CD4 binding, proteolytic cleavage recognition, and syncytium formation are all multiple and distributed over a relatively large part of the gp120 and therefore are likely dependent on protein tertiary structure.

Amino Acid Sequence↗
PubMed1990

The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 does not constitute the neutralization epitope.

We previously reported the in vitro generation of a neutralization-resistant variant of the molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1), HXB2D. The molecular basis for the resistance was shown to be a point mutation in the env gene, causing the substitution of threonine for alanine at position 582 of gp41. Here, we show the variant to be resistant to syncytium inhibition as well as to neutralization by the immune-selecting serum. Moreover, 30% of HIV-positive human sera able to neutralize the parental virus have significantly decreased ability to neutralize the variant. As the A-to-T substitution thus has general relevance to the interaction of HIV-1 with the host immune system, we investigated further the biologic and immunologic bases for the altered properties. Synthetic peptides corresponding to the 582 region failed to compete in infectivity, neutralization, or syncytium inhibition assays and did not elicit neutralizing antibodies. Furthermore, human antibodies, affinity purified on synthetic peptide resins, bound to gp41 and peptides from the 582 region but did not possess neutralizing antibody activity. Some viral constructs in which the AVERY sequence in the 582 region was altered by site-directed mutagenesis were not infectious, indicating that the primary structure in this region is crucial for viral infectivity. Constructs predicted to possess a local secondary structure similar to that of the variant nevertheless behaved like the parental virus and remained neutralization sensitive. These results suggest that the requirements for neutralization resistance in this region are very precise. Our results with synthetic peptides show that the 582 region does not by itself constitute a neutralization epitope. Moreover, the degree of flexibility in amino acid substitution which allows maintenance of neutralization sensitivity suggests that position 582 does not form part of a noncontiguous neutralization epitope. The basis for neutralization resistance of the immune-selected variant is more likely a conformational change altering a neutralization epitope at a distant site.

Amino Acid Sequence↗
PubMed1990

Structure and expression of tat-, rev-, and nef-specific transcripts of human immunodeficiency virus type 1 in infected lymphocytes and macrophages.

Primary RNA transcripts from the human immunodeficiency virus type 1 (HIV-1) are processed into mature mRNA by a complex series of splicing events. Viral structural proteins and reverse transcriptase are translated from unspliced or singly spliced transcripts. Proteins which control virus replication, including tat, rev, and nef, are translated from transcripts which are the product of multiple splicing. We have analyzed the composition and relative abundance of the latter transcripts in long-term infected cell lines and in acutely infected peripheral blood cells by amplification with the polymerase chain reaction (PCR) followed by Southern blot, molecular cloning, and DNA sequence analyses. In H9 cells chronically infected with the HIV-1 strain HTLV-IIIB, the predominant of the three kinds of transcripts is those coding for nef. Transcripts with coding potential for rev constituted an intermediate fraction of those analyzed, while those for tat accounted for only a small minority. A similar pattern was observed with Southern blots of PCR-amplified transcripts from peripheral blood lymphocytes acutely infected with HTLV-IIIB. The same general pattern was also observed with PCR-amplified transcripts from peripheral blood monocyte-macrophages infected with an HIV-1 strain (BA-L) able to grow to high titers in macrophages. In these cells, however, the apparent major form of nef transcript contained only the first and third exons of the multiply spliced transcripts and appeared to be generated by either a single or a triple splicing mechanism. As with lymphocytes, tat-specific mRNAs were by far the least abundant. It thus appears that different cell types infected with different strains of HIV-1 maintain a similar balance of expression in which transcripts for nef vastly predominate over those for tat and that those for rev are intermediate in abundance.

Amino Acid Sequence↗
PubMed1989

Structure of simian immunodeficiency virus regulatory genes.

Three full-length cDNA clones were obtained from cells infected with the simian immunodeficiency virus (SIV) isolated from captive macaques (SIVMAC). Nucleotide sequence analyses suggested that these represented mRNA for the SIV MAC genes tat, rev (formerly, art/trs), and nef (formerly, 3'orf). The putative tat-specific clone was active in trans-activation of the SIV MAC long terminal repeat in COS-1 and Jurkat cells. In contrast, the human immunodeficiency virus 1 long terminal repeat was significantly trans-activated only in the COS-1 cells. This suggests that trans-activation by the SIV tat gene is modulated by cell-specific factors. The structure of all of the clones suggested an mRNA splicing pattern more complex than that described for human immunodeficiency virus 1.

Amino Acid Sequence↗
PubMed1989

Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus.

The gibbon ape leukemia virus, SEATO strain, and human T-cell leukemia virus type I envelope glycoproteins can be functionally assembled with a Moloney murine leukemia virus core into infectious particles. The envelope-host cell receptor interaction is the major determinant of the host cell specificity for these hybrid virions.

Animals↗
PubMed1989

Human T-lymphotropic virus I-infected T cells constitutively express lymphotoxin in vitro.

We have studied the pattern of expression of the lymphokines tumor necrosis factor (TNF alpha) and lymphotoxin (TNF beta) in T-cell lines established by transformation with human T-lymphotropic virus, type I (HTLV-I), the etiologic agent of adult T-cell leukemia (ATL). We report here that nine of nine HTLV-I-infected T-cell lines, established by in vitro infection with HTLV-I, including those with CD4+ or CD8+ as well as CD4-/CD8- phenotypes, constitutively produce high levels of TNF alpha and -beta mRNA and secrete biologically active TNF beta into the culture medium. Similar patterns of expression are seen in six of six HTLV-I-infected T-cell lines directly established from ATL patients. In contrast, several T-cell lines, either uninfected or infected with human immunodeficiency virus I, did not produce comparable levels of the TNF beta. Comparisons of a normal functional T-cell clone before and after infection with HTLV-I show that expression of TNF beta mRNA is induced in the infected cells. The high level expression in HTLV-I-infected cell lines dose not seem to involve perturbation of the TNF alpha/beta genetic loci by proviral integration. A cell line (81-66/45) nonproductively transformed with HTLV-I that produces tat-1 in the absence of viral structural proteins, produces both TNF alpha and -beta mRNA. This suggests that expression of these cytokines could be mediated in trans by the tat-1 gene product.

Blotting, Southern↗