Biomedical subjects

C D Pauza

Publications and source records attributed to C D Pauza.

72 records · Page 4Linked to original sources

PubMed1990

Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination.

Proviral integration is a required step in the retrovirus life cycle. The mechanism of integration involves specific modification of the ends of linear viral DNA and subsequent recombination with host sequences. Integration results in the limited loss of sequence information at the termini of the viral genome. The composition of the intact linear DNA termini were inferred by sequencing the 2-long terminal repeat (2-LTR) circle junction that is formed when the linear molecule undergoes intramolecular, blunt-end ligation. The junction sequence contained the nucleotides GTAC that were not present at the ends of the integrated provirus. Comparison with the sequence of the LAV-1 strain of HIV-1 demonstrated that the GT dinucleotide derived from the right-hand terminus (U5) of the linear viral DNA and the AC dinucleotide came from the left-hand terminus (U3). Therefore, the corrected size of the LAV-1 LTR is 637 bp. This conclusion was confirmed independently by assessing the structure of linear viral DNA in acutely infected T cells. A portion of the population of linear HIV-1 DNA molecules were specifically deleted at their 3' ends; the extent of this deletion was 2 bases. This result is consistent with the activity of viral integrase protein on linear viral DNA and it accounts for the structure of integrated HIV-1 proviruses.

Base Sequence↗

PubMed1990

Extrachromosomal HIV-1 DNA in persistently infected U937 cells.

Persistent human immunodeficiency virus type 1 (HIV-1) infection of U937 monocytic cells resulted in the accumulation of novel forms of extrachromosomal viral DNA. These DNA species are larger than the genome size of HIV-1 and persist indefinitely. The extrachromosomal viral DNA species (E-DNA) were shown to be structurally stable by subcloning of infected cell lines and restriction fragment analysis. Similar E-DNA structures were observed in independent infections. Persistently infected monocytic cells had low levels of viral antigens, reflecting the low levels of viral RNA that were detected. These results support a role for E-DNA in persistent HIV-1 infection of monocytic cells.

Cell Line↗

PubMed1989

Persistent human immunodeficiency virus type 1 infection of monoblastoid cells leads to accumulation of self-integrated viral DNA and to production of defective virions.

Cell-free virus preparations from persistently infected monoblastoid cells (HU937) become progressively less infectious during long-term passage. This effect is specific for cell lines derived from U937 and is not observed in persistently infected T-cell lines. Reduced infectivity is correlated with accumulation of unusual, high-molecular-weight, extrachromosomal forms of the human immunodeficiency virus type 1 (HIV-1) DNA. These DNA molecules contain multiple copies of the viral genome, and their structures are highly variable. Of 17 subclones of the HU937 cell line, 15 unique restriction fragment patterns were observed for the HIV-1 viral DNA. Structural analysis of these viral DNA species indicated that they were formed by sequential rounds of long terminal repeat-mediated integration of one circular DNA form into preexisting monomeric or multimeric structures. These viral DNA structures are termed nested self-integrates. Once formed, self-integrates prove to be stable and can be maintained for several months in culture. The unusual structures of HIV-1 DNA in persistently infected monoblastoid cells attest to an alternative to the accepted retrovirus life cycle. The self-integrated viral DNA species reported here may explain some aspects of the mechanism controlling establishment and maintenance of persistent HIV-1 infection in cells of the monocyte/macrophage lineage.

Cells, Cultured↗

PubMed1988

HIV persistence in monocytes leads to pathogenesis and AIDS.

An hypothesis of the pathogenic mechanism leading to acquired immunodeficiency syndrome (AIDS) that places special emphasis on the potential for infected monocytes to act as the reservoir of a persistent human immunodeficiency virus (HIV) infection has been developed. Monocytes may mediate directly the infection and ultimate destruction of helper T cells; this establishes a direct relationship between antigen presentation and HIV dissemination, thus accounting for the cytopathogenic effects and immune system debilitation associated commonly with AIDS. The possibility that this mode of virus dissemination can account for the depletion of helper-T-cell subsets based on their antigen specificity is considered and may explain why the cellular immune response to the virus is ineffective. This concept and may also elucidate the role of intercurrent infections in the development of disease and it suggests mechanistic explanations for the conversion from prodromal to fulminant AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Human immunodeficiency virus infection of T cells and monocytes proceeds via receptor-mediated endocytosis.

The rates of internalization and uncoating of 32P-labelled human immunodeficiency virus (HIV) in the human T lymphoid cell line CEM are consonant with a receptor-mediated endocytosis mechanism of entry. This interpretation was affirmed by electron microscopic observation of virions within endosomes. Virus binding and infectivity were inhibited to the same extent by pretreatment with OKT4A antibody, therefore, the CD4 receptor-dependent pathway of internalization appears to be the infectious route of entry. The pattern of internalization by the human monoblastoid cell line U937 proved to be more complex, involving rapid and efficient CD4-independent internalization. Electron microscopy revealed the presence of large intracellular vesicles, each containing several virions. Antibody against the CD4 receptor for virus efficiently blocked infection, but did not reduce significantly HIV binding or internalization in the U937 cell line. Consequently, U937 cells have a CD4-independent pathway of virus internalization that does not coincide with the route of entry for infectious HIV.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

Human immunodeficiency virus infection of monoblastoid cells: cellular differentiation determines the pattern of virus replication.

Stringent control of human immunodeficiency virus (HIV) replication was observed in the human monoblastoid cell line U937. A low-multiplicity infection of these cells by the LAV1 strain of HIV was productive for 2.5 days; then virus replication became restricted and no further evidence of virion production was observed. The dramatic decrease in HIV production was due in part of reduced accumulation of cytoplasmic viral RNA and occurred in the absence of evident cytopathic effects. In contrast, infected cells induced to differentiate by phorbol ester, vitamin D3, or lymphokine supernatant did not release markers of HIV despite the accumulation of significant levels of cytoplasmic viral RNA. HIV infection altered the pattern of c-myc RNA accumulation in U937 cells. Expression of this gene changes normally in response to the state of cellular differentiation; in infected cells the level of c-myc expression was correlated to the levels of viral RNA accumulation and not to cellular differentiation. These results suggest that restricted replication of HIV in monocytes might be an important mechanism of virus persistence and demonstrate a relationship between HIV replication and monocyte differentiation.

Cell Differentiation↗

PubMed1987

Regulation of human T-lymphocyte gene expression by interleukin 2: immediate-response genes include the proto-oncogene c-myb.

Antigen-stimulated human T lymphocytes must bind the immunoregulatory hormone interleukin 2 (IL-2) if they are to transit from the G1 to the S phase of the cell cycle. Indirect methods, such as the measurement of thymidine uptake rates, were previously the only means available for exploring the mechanism of action of IL-2. Several cDNA clones have been isolated which are expressed subsequent to IL-2 binding, and the expression of two of these genes. Tact52 and Tact75, is regulated directly at the level of transcription; expression of the proto-oncogene c-myb is also regulated directly by IL-2 binding. These genes thus constitute a set which is coordinately regulated in the course of the transition from G1 to S phase of human T lymphocytes, and their expression depends on IL-2 binding.

Cell Cycle↗

PubMed1986

Characterization of the human c-myc protein using antibodies prepared against synthetic peptides.

Synthetic peptides with amino acid sequences based on inferred sequences encoded by the human c-myc proto-oncogene have been used as immunogens to produce polyclonal and monoclonal antibodies. These peptide-specific antibodies have then been used to identify the intact human c-myc protein. By immunoprecipitation and immunoblotting analysis we have defined the human c-myc protein as a phosphoprotein with an apparent molecular mass of 62 kDa on a sodium dodecyl sulphate-polyacrylamide gel. The protein is present in both normal and transformed cells but the steady-state levels of p62c-myc are elevated in the transformed cells by comparison with normal ones. We have used our anti-c-myc peptide antibodies to study the subcellular localization of p62c-myc. We find the protein to be loosely associated with cell nuclei by an interaction which is highly sensitive to variations in ionic strength within the physiological range. In response to in vitro or in vivo heat-shock the protein becomes sequestered in an insoluble complex associated with the nucleus. This complex contains a discrete subset of nuclear proteins, of which p62c-myc is a member. Immunofluorescence microscopy of p62c-myc shows it to be localized in a defined and previously unobserved subnuclear structure. A role for p62c-myc as an intracellular messenger is suggested.

Amino Acid Sequence↗

PubMed1984

The control of transferrin receptor synthesis in mitogen-stimulated human lymphocytes.

Human lymphocytes cultured in the presence of the plant mitogenic lectin phytohemagglutin in (PHA) become activated and leave the G0 phase of the cell cycle. In the presence of PHA and lymphokines produced in situ the cells will enter S phase and undergo cell division. We have determined the time course of appearance for the receptor for transferrin as an initial attempt to understand the molecular mechanisms regulating the onset of lymphocyte differentiation and proliferation in the 48 h following PHA addition. Using three different assay methods we have shown that the increase in the number of surface receptor molecules is due to the accumulation of newly synthesized receptor and not to the redistribution of a previously existing pool of receptor molecules. The total amount of transferrin receptor increased at least four-fold. In vitro translation of RNA from activated lymphocytes indicates that the new receptor synthesis is due, at least in part, to increased availability of mRNA encoding the transferrin receptor.

Cells, Cultured↗

PubMed1984

Location of amino acid alterations in mutants of aspartate transcarbamoylase: Structural aspects of interallelic complementation.

Recent genetic studies of the pyrB locus of Escherichia coli resulted in the characterization of 29 mutant strains harboring defects in the structural gene that encodes the catalytic chains of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2). Three alleles, pyrB554, pyrB730, and pyrB748, have been cloned, and their nucleotide sequences have been determined along with that of the wild-type pyrBI operon in order to locate the sites of the alterations in the catalytic chains. Missense mutation pyrB554 leads to replacement of serine-52 by phenylalanine, and the inactive mutant enzyme has properties similar to those of wild-type aspartate transcarbamoylase. The amber mutation pyrB730 results in unstable truncated polypeptide chains 27 amino acids shorter than wild-type chains. Deletion mutation pyrB748 causes the removal of 181 amino acids. Combining these results with knowledge of the crystallographic structure of the wild-type enzyme provides a basis for tentative structural mechanisms for the observed complementation behavior of the mutant proteins.

Alleles↗

PubMed1982

Genes encoding Escherichia coli aspartate transcarbamoylase: the pyrB-pyrI operon.

In both Escherichia coli and Salmonella typhimurium there is approximately balanced synthesis of the six catalytic and six regulatory polypeptide chains of the regulatory enzyme aspartate transcarbamoylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2). This control is achieved by the contiguous pyrB and pyrI genes, which encode the catalytic and regulatory chains, respectively. Evidence for this single transcriptional unit was obtained by a study of various deletion mutations and from the DNA sequence of the intercistronic region between pyrB and pyrI. One pyrB deletion, pyrB748, produced a normal level of regulatory chains even though it removed a substantial portion of the pyrB gene. Another deletion, pyrB740, shares a similar terminus at one end within pyrB, but the promoter region is removed. In this deletion mutation, there is no production of the regulatory polypeptide, indicating that a single region adjacent to pyrB controls transcription of pyrI as well. Molecular cloning and subsequent DNA analysis demonstrated that the pyrB and pyrI genes are contiguous with pyrI as the distal gene in the operon. The cistrons are separated by a 15-nucleotide untranslated region containing a sequence capable of interacting with the 16S ribosomal RNA and allowing translation of the pyrI cistron.

Amino Acid Sequence↗

PubMedDate not supplied

Cellular immune responses in rhesus macaques infected rectally with low dose simian immunodeficiency virus.

Monkeys infected rectally with low dose simian immunodeficiency virus (SIV) were resistant to high dose challenge with SIV. Peripheral blood mononuclear cells (PBMC) from two of four challenged monkeys were unable to support SIV replication in vitro unless cultures were depleted of CD8+ lymphocytes. Monkeys that had survived high dose rectal infection with SIV also suppressed virus replication in cultured PBMC. PBMC from uninfected monkeys supported virus replication in both unfractionated and CD8-depleted cultures. Virus-suppressive activity of PBMC may be an important correlate of protective immunity in AIDS.

Animals↗

PubMedDate not supplied

Antiviral activity of primate gamma delta T lymphocytes isolated by magnetic cell sorting.

The exposure of human or rhesus monkey peripheral blood mononuclear cells (PBMCs) to interleukin 2 (IL-2) in vitro resulted in a selective outgrowth of gamma delta lymphocytes. Using positive selection by monoclonal antibodies and magnetic beads, gamma delta T lymphocytes were isolated from these cultures. Without priming by viral antigens, the purified gamma delta T lymphocytes lyse immunodeficiency virus-infected cells substantially better than the uninfected counterparts.

Animals↗

PubMedDate not supplied

An Hsp60 related protein is associated with purified HIV and SIV.

The possible physical association of heat shock proteins (Hsp's) with immunodeficiency viruses (HIV and SIV) has been examined. The virions were purified by a) polyethylene glycol (PEG) precipitation and Sepharose 4B filtration, b) PEG precipitation and centrifugation over a Renografin gradient, or c) PEG precipitation and Matrex Cellufine Sulfate affinity chromatography. Western blotting revealed an Hsp60 related protein associated with HIV and SIV. Other Hsp's (such as Hsp70) were not detected, suggesting a specific interaction between Hsp60 and viral factors.

Antibodies, Monoclonal↗

PubMedDate not supplied

Immunization of Macaca mulatta with aroA attenuated Salmonella typhimurium expressing the SIVp27 antigen.

Attenuated bacteria expressing foreign antigens stimulate both systemic and mucosal immune responses to the recombinant protein. We studied the infection of rhesus macaques with an attenuated Salmonella typhimurium expressing the simian immunodeficiency virus p27 capsid protein. Juvenile rhesus macaques were inoculated by intragastric intubation with doses ranging from 3 to 9 x 10(9) viable aroA attenuated S. typhimurium. The bacterial infection was self-limiting with no overt clinical signs. Salmonella were shed in the feces of macaques for approximately five days. Salmonella were isolated from fecal material to examine the in vivo stability of both the attenuating mutation and the integrated SIVp27 expression cassette. All Salmonella isolates retained both the attenuating mutation and the recombinant expression construct. In vitro analysis showed that a minimum of 7.2 microg of p27 was delivered by a single oral dose with attenuated, recombinant S. typhimurium.

Amino Acid Sequence↗

PubMedDate not supplied

Macaque models for early immunosuppression and T cell loss during acute immunodeficiency virus infections.

The interval of acute infection with immunodeficiency viruses is critically important for determining the long-term rate of disease progression. The steps of initial infection, systemic dissemination, and explosive replication of pathogenic SIV or SHIV in macaques are being mapped to show the mechanisms responsible for remodeling host immunity, for establishing the persistent infection, and for promoting disease progression. Here, we describe recent studies on two ways in which CD4+ T cell populations are depleted during acute infection. Initially, we discuss recent work on the mechanisms for CD4+ T cell-mediated, MHC-unrestricted cytolysis. This mechanism shows how even soluble viral antigens such as the envelope glycoprotein, can prime CD4+ lymphocytes to be both effector and target cells in an unrestricted cytolysis mechanism. The consequence of unrestricted cytolysis is a more rapid destruction of the CD4+ T cell population. Secondly, we discuss the broader issue of T cell hyperactivation during acute infection. Inappropriate activation of this lymphocyte population renders cells susceptible to activation induced cell death and also increases the rate of virus replication. Macaque immunization studies have shown a clear role for extracellular Tat in hyperactivation. These two mechanisms, unrestricted cytolysis and T cell hyperactivation, are components of the acute infection that remodel host immunity and dictate the rate of progression to AIDS.

Animals↗

PubMedDate not supplied

Are gamma delta T cells important for the elimination of virus-infected cells?

Rhesus monkeys (Macaca mulatta) gamma delta T cells were identified using a monoclonal antibody. The relative representation of gamma delta T lymphocytes in the peripheral blood, lymph nodes, and spleen resembles that of Homo sapiens. The analysis of function and specificity revealed further significant similarities between the simian and human gamma delta T-cell systems. Since both human and monkey gamma delta T lymphocytes can effectively lyse cells infected with immunodeficiency viruses, it is possible that the primate gamma delta T-cell systems contribute to antiviral immunosurveillance.

Animals↗

PubMedDate not supplied

Pathogenesis of SIVmac251 after atraumatic inoculation of the rectal mucosa in rhesus monkeys.

Intrarectal inoculation of rhesus monkeys with low doses of SIVmac led to a prolonged clinical and virological latency that was not observed for high intrarectal doses or for intravenous inoculation. Animals infected intrarectally with low virus doses remained negative for serum antibody responses to SIV for at least one year even though they readily transferred SIV to naive recipients via transfusion of whole blood.

Animals↗