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Biomedical subjects

M S Reitz

Publications and source records attributed to M S Reitz.

At least 55 records · Page 3Linked to original sources
PubMed1995

Spontaneous reversion of human immunodeficiency virus type 1 neutralization-resistant variant HXB2thr582: in vitro selection against cytopathicity highlights gp120-gp41 interactive regions.

Spontaneous revertants of the immune-selected variant HXB2thr582, which resists neutralization by certain conformationally dependent antibodies specific for the CD4-binding site on gp120 (such as F105), appeared after long-term culture in the absence of immune-selecting serum. Molecular analysis showed some of the viruses in the revertant stock contained a simple back mutation, whereas others retained the Thr-582 codon but contained a substitution of serine for phenylalanine in gp41 at position 673. Neutralization sensitivity to the selecting serum and to F105 of infectious clones containing either the back mutation or the compensatory mutation, HXB2thr582ser673, was confirmed. HXB2thr582-infected cells have a greater propensity for syncytium formation and single cell killing than do either the parental HXB2 or the revertant HXB2thr582ser673. This suggests that the revertant arose by selection in vitro for a less cytopathic virus. Our results link three envelope regions shown to influence virus-cell fusion as well as neutralization by antibody: the CD4-binding region, the leucine zipper domain, and a region hidden to antipeptide antibodies upon envelope oligomerization. Taken together they illustrate the functional importance of the gp120-gp41 interaction and emphasize the impact of the interplay between envelope regions on overall conformation and function and on recognition by neutralizing antibodies.

Amino Acid Sequence↗
PubMed1995

Growth of macrophage-tropic and primary human immunodeficiency virus type 1 (HIV-1) isolates in a unique CD4+ T-cell clone (PM1): failure to downregulate CD4 and to interfere with cell-line-tropic HIV-1.

Human immunodeficiency virus type 1 (HIV-1) isolates derived directly from clinical samples are usually unable to grow in cytokine-independent continuous cell lines, thus hindering the study of their biological features and their sensitivity to humoral and cellular protective immunity. To overcome these limitations, we have derived from the Hut78 T-cell line a CD4+ clone (PM1) characterized by a unique susceptibility to a wide range of HIV-1 isolates, including primary and biologically pure macrophage (M phi)-tropic isolates (e.g., HIV-1BaL), which are unable to infect other human T- or promonocytic cell lines. Both primary and M phi-tropic HIV-1 establish persistent infection in PM1, with sustained levels of virus replication for prolonged periods. Experiments with chimeric viruses containing envelope fragments of HIV-1BAL inserted into the genetic framework of HXB2, a molecular clone derived from the cell-line-tropic isolate HIV-1IIIB, showed the third hypervariable domain (V3) of gp120 to be a critical determinant of the cell line tropism of HIV-1. Nevertheless, the V3 loop of HIV-1BaL was not sufficient to confer on the chimeras a bona fide M phi tropism. The biological characteristics of HIV-1BaL and of a primary isolate (HIV-1(573)) were investigated by using the PM1 clone. Infection of PM1 by HIV-1BaL was critically dependent on the CD4 receptor, as shown by competition experiments with an anti-CD4 monoclonal antibody (OKT4a) or with soluble CD4. However, the amount of soluble CD4 required for inhibition of HIV-1BaL was approximately 100-fold higher than for HIV-1IIIB, suggesting that the affinity of HIV-1BaL for CD4 is significantly lower. Infection of PM1 with either HIV-1BaL or HIV-1(573) failed to induce downregulation of surface CD4 expression and syncytium formation. Analogous results were obtained with a chimeric virus (HXB2[BaL PvuII-BamHI]) encompassing a large portion of gp120 and gp41 of HIV-1BaL, indicating that the env genes contain critical determinants for CD4 downregulation and syncytium formation. Consistent with the lack of CD4 downregulation, persistent infection of PM1 by HIV-1BaL or HIV-1(573) failed to interfere with HIV-1IIIB superinfection, as revealed by the expression of a type-specific V3 loop epitope (M77) and by the induction of extensive syncytium formation. This lack of interference suggests that a direct viral interaction may occur in vivo between biologically diverse HIV-1 strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

A Rev-inducible mutant gag gene stably transferred into T lymphocytes: an approach to gene therapy against human immunodeficiency virus type 1 infection.

One strategy for somatic gene therapy for human immunodeficiency virus type 1 (HIV-1) infection is based on the regulated expression of dominant negative mutants of the HIV-1 gag gene. To limit expression of the mutant Gag polypeptide to HIV-1-infected cells, we have constructed a replication-defective retroviral vector that contains a Rev-responsive element. By using this construct we have obviated problems that can be associated with constitutive expression of an exogenous gene, an important step toward developing a human therapy. In uncloned T lymphocytes infected (transduced) with this retroviral construct, HIV-1 replication was inhibited by 94% with a concomitant decrease in the cytopathic effects of the virus. In addition, simian immunodeficiency virus (SIV) replication was also shown to be significantly inhibited, suggesting that this mutant Gag protein may have antiviral efficacy against a broad range of primate lentiviruses and that an SIV/macaque model can be used for further in vivo studies. These results have important implications in assessing the potential of somatic gene therapy in the treatment of HIV-1 infection.

Amino Acid Sequence↗
PubMed1994

Phylogenesis and genetic complexity of the nonhuman primate retroviridae.

The three known groups of nonhuman primate retroviruses (simian immunodeficiency virus, simian T cell lymphotropic/leukemic virus type I, and simian foamy virus) are thought to have equivalent human counterparts. This is clearly the case with human immunodeficiency virus types 1 and 2, the causative agents of acquired immunodeficiency syndrome, and with human T cell lymphotropic/leukemia virus type I (HTLV-I), which causes T cell leukemia and a progressive form of myelopathy (tropical spastic paraparesis/HTLV-I-associated myelopathy), and HTLV-II. However, the presence of spumaviruses (foamy viruses) in humans remains uncertain. Data accumulated in the last 5 years suggest the possibility that the human retroviruses are indeed the result of transmission of simian retroviruses to humans. In this article we attempt to parallel the genetic features of the simian retroviridae with their human counterparts and argue for the possibility of horizontal transmission of these viruses from monkeys to humans.

Animals↗
PubMed1994

Viral variability and serum antibody response in a laboratory worker infected with HIV type 1 (HTLV type IIIB).

Molecular clones of HIV-1 were obtained from isolates cultured from peripheral blood mononuclear cells (PBMCs) and directly from uncultured PBMCs from a laboratory worker accidentally infected with the HIV-1 laboratory strain, HIV-1(HTLV-IIIB). Envelope sequences corresponding to the first 752 amino acids of HIV-1(HTLV-IIIB) clone BH10 were obtained from clones of cultured virus and sequenced. Three env clones obtained shortly after infection differed among themselves at only seven nucleotide positions, resulting in one amino acid substitution and one frameshift mutation. These envelope sequences were as similar to the envelope sequences of various IIIB clones as the latter were to each other. env divergence increased over the course of infection. However, the overall diversity in env clones obtained two or more years after infection was still comparable to that among IIIB env clones from the original IIIB culture. Multiple clones of partial env gene sequences containing the V3 loop were also obtained directly from uncultured PBMCs by polymerase chain reaction amplification. The env sequences of these clones were generally similar to those of the cultured viruses. Within the V3 region, the earliest isolates retained the sequence of the HXB2 clone from IIIB. Clones obtained later showed a progressive divergence in V3. An A-to-T substitution within the GPGRAF sequence at the tip of the V3 loop was observed within 1 year after infection, and this mutation predominated in all subsequent isolates. Antibodies against the V3 loops of IIIB and divergent 1987 and 1990 LW isolates appeared simultaneously in laboratory worker serum and persisted with no significant differences in titer. Furthermore, neutralization studies with autologous sequential sera suggested selection for the A-to-T change in V3 was not due to V3-directed antibodies. These results demonstrate a surprising homogeneity among env sequences of HIV-1 from an infected laboratory worker, perhaps because the initial infection originated from a relatively homogeneous population of tissue culture-adapted virus.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Alteration of V3 loop context within the envelope of human immunodeficiency virus type 1 enhances neutralization.

Neutralization of a chimeric human immunodeficiency virus (HIV) type 1, containing the V3 loop of the MN isolate substituted within the HXB2 envelope, was enhanced up to 20-fold compared with the HXB2 or MN parental isolates by human HIV-positive sera. MN V3 loop-specific monoclonal antibodies were better able to recognize the chimeric virus compared with MN, staining a greater percentage of infected cells and exhibiting slight increases in relative affinity with a concomitant increase in neutralization titer. Competition analysis revealed that enhanced neutralization by human HIV-positive sera of the chimera was attributable in some cases to better reactivity with the linear V3 loop epitope but in others to conformational loop epitopes or previously cryptic or poorly recognized epitopes outside the loop region. Mice primed with a vaccinia virus-chimeric envelope recombinant and boosted with gp160 developed a spectrum of antibodies different from that of mice similarly immunized with HXB2 or MN recombinants or that of naturally infected humans. The chimeric envelope elicited antibodies with enhanced binding to the native MN V3 loop; however, the sites seen by the BALB/c mice were not neutralizing epitopes. Nevertheless, similar to the observations made with use of human sera, the chimeric virus was more readily neutralized by all of the immune mouse sera, an effect apparently mediated by non-V3 loop epitopes. These studies illustrate that not only the V3 loop sequence and conformation but also its context within the viral envelope influence neutralization.

AIDS Vaccines↗
PubMed1993

Loss of a neutralizing epitope by a spontaneous point mutation in the V3 loop of HIV-1 isolated from an infected laboratory worker.

The third hypervariable region, or V3 loop, represents the principal neutralizing domain of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1). Sequential viral isolates from a laboratory worker (LW) accidentally infected with HIV-1IIIB in 1985 were analyzed using type-specific neutralizing monoclonal antibodies directed to the V3 loop. A single amino acid substitution, Ala-->Thr at position 21 in the V3 loop of HIV-1LW isolated in 1987, was shown to determine the loss of the neutralizing epitope recognized by one of the monoclonal antibodies (M77). However, this antibody efficiently recognized linear V3 loop peptides containing either the Ala or Thr residue at position 21, indicating that a local change in conformation was responsible for the epitope loss in the native gp120. Molecular modeling studies, experimentally supported by different amino acid replacements at position 21, indicated that the Ala-->Thr substitution leads to a drastic change in the domain of the V3 loop, which contains the complementary surface for antibody binding. These results provide evidence for the first time that a conformation-dependent epitope within the V3 loop of HIV-1 is involved in the generation of neutralization escape mutants in vivo.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Human T-lymphotropic virus type I tax regulates the expression of the human lymphotoxin gene.

Human T-lymphotropic virus type-I (HTLV-I)-infected T-cell lines constitutively produce high levels of lymphotoxin (LT). To analyze the mechanisms that lead to the expression of LT in HTLV-I-infected cell lines, we studied regulatory regions of the human LT promoter involved in the activation of the human LT gene. As determined by deletional analysis, sequences between +137 and -116 (relative to the transcription initiation site) are sufficient to direct expression of a reporter gene in the HTLV-I-infected cell line MT-2. Site-directed mutation of a of the single kappa B-like motif present in the LT promoter region (positions -99 to -89) completely abrogated LT promoter activity in MT-2 cells, suggesting that this site plays a critical role in the activation of the human LT gene. Transfection of LT constructs into HTLV-I-uninfected and -unstimulated Jurkat and U937 cell lines showed little to no activity of the LT promoter. Cotransfection of the same constructs with a tax expression plasmid into Jurkat cells led to detectable promoter activity, which could be significantly increased by stimulation of the cells with phorbol myristate acetate (PMA). Similarly, cotransfection of the LT promoter constructs and the tax expression plasmid into U937 cells led to significant promoter activity upon stimulation with PMA. These data suggest that HTLV-I tax is involved in the upregulation of LT gene expression in HTLV-I-infected cells.

Animals↗
PubMed1993

An immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 (HXB2-Env:Ala 582(-->Thr)) decreases viral neutralization by monoclonal antibodies to the CD4-binding site.

An immune-selected point-mutation (HXB2-Env:Ala582(-->Thr)) in the transmembrane protein, gp41, of the human immunodeficiency virus type 1 confers relative insensitivity to neutralization by a number of sera from HIV-1-positive persons. Affinity-purified human antibodies to continuous epitopes spanning Ala582 do not neutralize the virus (C. Wilson, M. S. Reitz, Jr., K. Aldrich, P. J. Klasse, J. Blomberg, R. C. Gallo, and M. J. Robert-Guroff, J. Virol. 64, 3240-3248, 1990). The specificity of the antibodies that the mutation renders less active has not previously been determined. We now report that this substitution in gp41 reduces the neutralizing activity of monoclonal antibodies to discontinuous gp120 epitopes, which overlap with the CD4-binding site. There was no such difference in sensitivity to neutralization by soluble CD4, CD4-immunoglobulin, or by two monoclonal antibodies to the V3 region of gp120. Furthermore, the ability of 10 human HIV-1-positive sera to block the binding of soluble CD4 to mammalian-recombinant gp120 correlated weakly with their differentiation of neutralization between the wild-type and the Env:Ala582(-->Thr)-mutant virus. We thus suggest that the substitution in gp41 modulates the conformation of gp120 so as to decrease viral sensitivity to one category of antibodies which is partly responsible for the group-specific neutralization of HIV-1 by human sera.

Amino Acid Sequence↗
PubMed1993

Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways.

Amino Acid Sequence↗
PubMed1993

A rapid method for site-specific mutagenesis using larger plasmids as templates.

To facilitate the introduction of specific point mutations in plasmids that are too large to be amplified efficiently by a single PCR, we have developed a method for site-directed mutagenesis by generating partial plasmid fragments, which introduces changes as simply as conventional techniques. Plasmids containing a fragment of the human immunodeficiency virus type-1 (HIV-1) envelope gene were subjected to PCR with four pairs of PCR primers for each desired point mutation. One primer in each of two of these four pairs contained the desired mutation. The four pairs of primers were designed so that four overlapping fragments were amplified from the plasmid template, two of which contained the mutation. These fragments were then reannealed and electroporated directly into Escherichia coli. The desired mutation was typically found in 66% to 83% of the resulting colonies. The technique is almost as simple as previous techniques, shows similar efficiency and is applicable to plasmids that would normally be too large for efficient site-specific mutagenesis. The entire procedure, from PCR amplification to transfection into E. coli, can be completed in one day.

Base Sequence↗
PubMed1992

Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus.

An independent strain (JI) of human herpesvirus 7 (HHV-7) was isolated from a patient with chronic fatigue syndrome (CFS). No significant association could be established by seroepidemiology between HHV-7 and CFS. HHV-7 is a T-lymphotropic virus, infecting CD4+ and CD8+ primary lymphocytes. HHV-7 can also infect SUP-T1, an immature T-cell line, with variable success. Southern blot analysis with DNA probes scanning 58.8% of the human herpesvirus 6 (HHV-6) genome and hybridizing to all HHV-6 strains tested so far revealed homology to HHV-7 with only 37.4% of the total probe length. HHV-7 contains the GGGTTA repetitive sequence, as do HHV-6 and Marek's disease chicken herpesvirus. DNA sequencing of a 186-base-pair fragment of HHV-7(JI) revealed an identity with HHV-6 and human cytomegalovirus of 57.5% and 36%, respectively. Oligonucleotide primers derived from this sequence (HV7/HV8, HV10/HV11) amplified HHV-7 DNA only and did not amplify DNA from other human herpesviruses, including 12 different HHV-6 strains. Southern blot analysis with the p43L3 probe containing the 186-base-pair HHV-7 DNA fragment hybridized to HHV-7 DNA only. The molecular divergence between human cytomegalovirus, on the one hand, and HHV-6 and HHV-7, on the other, is greater than between HHV-6 and HHV-7, which, in turn, is greater than the difference between HHV-6 strains. This study supports the classification of HHV-7 as an additional member of the human beta-herpesviruses.

Amino Acid Sequence↗
PubMed1992

Expression of alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers.

Although human T-cell lymphotropic virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), the role of viral gene expression in the progression to and maintenance of the leukemic state in vivo is unclear because of the inability of most previous studies to readily detect HTLV-I RNA in infected individuals. By using the reverse transcriptase-polymerase chain reaction, we detected spliced messages for the HTLV-I pX regulatory genes in primary uncultured cells from ATL patients and healthy asymptomatic carriers. In addition to the expected doubly spliced pX message, three alternatively spliced mRNAs were demonstrated (pX delta 17, pX-p21rex, and pX-orfII mRNAs, where orf = open reading frame). The same splice sites were shown in the messages from uncultured ATL cells and from the HTLV-I-producing C10/MJ cell line. Alternatively spliced pX mRNAs have the potential to code for known and putative pX gene products. Among the transcripts is a monocistronic mRNA likely to code for p21rex (pX-p21rex mRNA). Since alternative splicing of HTLV-I pX mRNA can be found in primary uncultured cells, it is likely to have a functional significance in vivo. This suggests possible roles for HTLV-I gene expression in the progression to and maintenance of ATL, as well as in the phase preceding it.

Base Sequence↗
PubMed1992

No detection of HTLV-I DNA in punch skin biopsies from patients with cutaneous T-cell lymphoma by the polymerase chain reaction.

In this study we have tried to take advantage of the high genetic homology between conserved regions of human T-cell lymphotropic virus (HTLV) types I and II, hoping that a possible retrovirus in patients with cutaneous T-cell lymphoma (CTCL) would have regions with homology to types I/II. DNA was extracted from punch skin biopsies from 21 patients and subjected to the polymerase chain reaction (PCR), using primer sets designed to match conserved regions in the HTLV-I/II genome. The PCR products were subjected to agarose gel electrophoresis with subsequent Southern blotting and hybridization to an HTLV-I probe. No bands of exogenous origin were seen on the agarose gel or by hybridization. If a retrovirus is present in the skin in CTCL patients, it is either not related to HTLV-I/II, present at a copy number below the PCR detection limit, or has been cleared from the skin before the clinical symptoms appear.

Base Sequence↗