Reverse Transcriptase Release Research Articles

Release of human immunodeficiency virus by THP-1 cells and human macrophages is regulated by cellular adherence and activation

Macrophage adherence, an important regulatory signal, has the potential to affect human immunodeficiency virus (HIV) production either directly or by priming monocytes to respond to other activating signals. We have investigated the role of adherence as an activator of HIV-1 transcription and release. The effects of adherence on HIV-1 transcription were examined by using THP-1 cells, a human monocytic cell line, transfected with HIV long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs. The effects of adherence on release of HIV-1 were investigated in both HIV-1-infected THP-1 cells and human peripheral blood monocyte-derived macrophages (MDM). Adherence of lipopolysaccharide (LPS)-stimulated THP-1 cells to either tissue culture plastic or endothelial cells was crucial for enhanced HIV-1 transcription as measured by LTR-CAT expression. Such increased LTR-CAT expression did not occur with an HIV LTR construct containing mutated NF-kappa B binding sites. In contrast, release of whole HIV, measured by reverse transcriptase (RT) activity in tissue culture medium, was reduced upon adherence of stimulated HIV-1-infected THP-1 cells without suppression of HIV LTR-CAT transcription or p24 release. This finding suggested that activation of adherent monocytic cells interfered with HIV assembly and release. Although the reduction of RT activity following activation of HIV-1-infected MDM was independent of adhesion, adherence alone of nonstimulated HIV-infected MDM to endothelial cells was sufficient to induce a reduction in RT release. This study demonstrates that LPS stimulation of monocytic cells enhances HIV LTR transcription under adherent conditions. In contrast, activation of adherent monocytic cells infected with HIV reduced viral release.

Mutational analysis of the envelope gene of Moloney murine leukemia virus.

The env gene products of Moloney murine leukemia virus are required for binding and entry of the virus into the target cell. Thirty-three linker insertion mutations were constructed throughout the env gene of Moloney murine leukemia virus. Twenty of the mutations were located in the surface protein (SU), and the remaining thirteen were located in the transmembrane protein (TM). The viability of the viruses containing these env gene mutations was determined by performing transient transfections and screening for the release of reverse transcriptase. Eleven viable mutants were isolated, nine in SU and two in TM. Three of the viable mutants were temperature sensitive. Four of the viable mutants were clustered in the carboxy terminus of SU. The env gene products of transfected cell lines which produced viable virus were analyzed. Our results indicated two regions of SU important for the stability of the SU/TM heteropolymer and one region important for the interaction of the env gene products with the viral core.

Enhanced neutralization of feline immunodeficiency virus by complement viral lysis

The ability of complement to inactivate feline immunodeficiency virus (FIV) was examined. Treatment of virus with complement plus sub-neutralizing titers of antiserum resulted in a significant reduction in the virus titer compared with treatment of the virus with complement or antibody alone. One of the mechanisms by which cat complement inactivates FIV was shown to be by viral lysis as determined by a reverse transcriptase release assay. Kinetic studies revealed that viral lysis is initiated soon after the addition of complement to a mixture of virus and antiserum. Treatment of FIV with normal non-complement-inactivated human serum resulted in virus inactivation and release of viral RT in the absence of specific antiserum. It appears that FIV activates complement directly through the classical pathway and that integrity of the membrane attack components is a requirement for FIV lysis by human serum. The vulnerability of two distinct isolates of FIV to complement lysis was compared using complement from different species. Oradell isolate was more sensitive to complement lysis than the Petaluma isolate as assessed by reverse transcriptase release. It appears that factors intrinsic to the virus isolate may influence the amplitude of complement-dependent viral lysis.

Mutational analysis of the N-linked glycosylation sites of the SU envelope protein of Moloney murine leukemia virus.

The role of the N-linked glycosylation sites in the major envelope glycoprotein, SU (gp70), of Moloney murine leukemia virus has been examined. By using site-specific oligonucleotide-directed mutagenesis, each of the seven glycan addition sites has been individually eliminated. Mutations resulting in the loss of a single glycosylation site produced, intracellularly, stable precursor SU-TM proteins which were 4 to 5 kDa smaller than the wild-type virus SU-TM protein. Mutant delta 1,4,7, a trimutant lacking three N-linked glycan addition sites, resulted in a viable, infectious virus with a stable SU-TM protein approximately 12 to 15 kDa smaller than the wild-type SU-TM protein. Five of the seven single-site mutations resulted in viable virus as judged by the release of reverse transcriptase in transient-expression assays and XC syncytium assays. Mutations at two of the sites resulted in a detectable phenotype. Virus mutated at position 2 was temperature sensitive in Rat2 cells; viable virus was produced at 32 degrees C but not at 37 degrees C. Virus mutated at position 3 was noninfectious and yielded virions lacking detectable mature SU protein. The mutation results in the block of transport of the protein to the cell surface and assembly into virion particles.

Neutralization of human immunodeficiency virus type 1 by complement occurs by viral lysis

The ability of complement to inactivate human immunodeficiency virus (HIV) in the presence of specific antibody was evaluated. HIV was treated with complement and/or antibody, and then its titer was determined on the CD4+ H9 cell line. While complement alone had no effect on the HIV titer, complement plus subneutralizing levels of antibody resulted in titer reductions. Complement sources deficient in membrane attack component C5 or C8 did not inactivate antibody-treated HIV, suggesting that neutralization occurred via lysis. This possibility was investigated by assessing release of reverse transcriptase (RT) from the virion. Antibody plus complement, but neither reagent alone, released RT from HIV in a dose-dependent manner. Release of RT did not occur with C5- or C8-deficient sera, also indicating a requirement for membrane attack components. These studies show that complement can neutralize HIV via the classical complement pathway and that this neutralization occurs via C5b-9-mediated viral lysis. Thus, complement may play a major role in resistance to disease by lysing HIV and preventing infection of Fc- and complement receptor-positive cells, as well as CD4+ cells.

Antibodies to the primary immunodominant domain of human immunodeficiency virus type 1 (HIV-1) glycoprotein gp41 enhance HIV-1 infection in vitro

Previous experiments had shown that two human monoclonal antibodies (huMAbs) directed against human immunodeficiency virus type 1 (HIV-1) enhanced HIV-1 infection in vitro (Robinson et al., Proc. Natl. Acad. Sci. USA, 87:3185-3189, 1990). This complement-mediated, antibody-dependent enhancement (C'-ADE) of HIV-1 infection caused 12-fold increases in reverse transcriptase released from MT-2 cells. In the study reported here, it was demonstrated that both of these huMAbs, 86 and V10-9, bound to an immunodominant peptide in gp41 (amino acids 586 to 620). This peptide blocked C'-ADE of HIV-1 infection in vitro regardless of whether huMAb 86 or human polyclonal anti-HIV was used as the source of anti-HIV antibody. Blockade of enhanced infections was characterized by decreases in antigen synthesis, cytopathic effect, and reverse transcriptase release. The ability of the huMAbs to enhance infection was determined to be dependent upon specific peptide reactivity and not dependent upon immunoglobulin subclass, complement fixation, or gross antigen reactivity. Since the peptide to which enhancing antibodies bind is immunodominant and does not bind neutralizing antibodies, it may be worthwhile to investigate deletion of this 35-amino-acid peptide from candidate anti-HIV vaccines.

Complement-mediated antibody-dependent enhancement of HIV-1 infection requires CD4 and complement receptors

In this study it is demonstrated that complement-mediated antibody-dependent enhancement (C'-ADE) of HIV-1 infection in vitro is blocked by murine monoclonal antibodies to CD4 and complement receptor type 2 (CR2) while HIV-1 infection in the absence of C'-ADE is blocked by anti-CD4 but not anti-CR2 monoclonal antibodies. The anti-CR2 murine monoclonal antibody, OKB7, blocked C'-ADE of HIV-1 infection at concentrations greater than 1 μg/ml. The anti-CD4 monoclonal antibody, OKT4a, but not OKT4f blocked C'-ADE at concentrations greater than 0.06 μg/ml. HIV-1 infections were quantitated by cytopathic effect, indirect immunofluorescence, and reverse transcriptase release. It appears from these in vitro studies that C'-ADE of HIV-1 infection requires both CD4 and complement receptors while HIV-1 infection in the absence of antibody and complement requires only CD4.

Antibody-dependent enhancement of simian immunodeficiency virus (SIV) infection in vitro by plasma from SIV-infected rhesus macaques

Plasma from two rhesus macaques (Macaca mulatta) experimentally infected with the simian immunodeficiency virus (SIV; isolate SIVmac251) enhanced SIVmac infection of a human CD4+ lymphoblastoid cell line, MT-2. Prechallenge plasma samples from these animals and serum from SIV-negative macaques did not enhance infection. Compared with controls, infection enhancement was characterized by the rapid appearance of syncytium formation (3 to 4 days sooner), reverse transcriptase release (10-fold increase), and cytopathic effect (60% cell killing). Enhancement of activity was dependent on the presence of diluted, fresh SIV-negative macaque serum as a source of complement. A requirement for complement was shown by the absence of enhancement in heat-inactivated serum and by dose-dependent inhibition of enhancement in the presence of polyclonal antibody to monkey complement component C3. Monoclonal antibody to CD4 (OKT4a) blocked enhancement completely, while monoclonal antibody to the human complement component C3d receptor CR2 (OKB7) reduced enhancement by greater than 50%, indicating a requirement for CD4 and CR2 in mediating this phenomenon. SIV infection-enhancing activity appeared in macaques soon after experimental inoculation (28 days). The titer increased over time and peaked just prior to the death of both macaques from opportunistic infections and lymphoma. In vitro SIV infection enhancement is nearly identical to the in vitro complement-mediated, antibody-dependent enhancing (C'-ADE) activity observed in human immunodeficiency virus-positive human sera (Robinson et al., Lancet i:790-794, 1988; Robinson et al., J. Acq. Immun. Def. Synd. 2:33-42, 1989). These observations validate the macaque-SIV model for studies of C'-ADE.

Comparison of the Human Immunodeficiency Virus Type 1 and 2 Proteases by Hybrid Gene Construction and trans-Complementation

To determine the cleavage specificity of the proteases of the type 1 and 2 human immunodeficiency viruses (HIV-1, HIV-2), we interchanged this domain of the polymerase (pol) genes and analyzed the maturation programs of the chimeric polyproteins in an Escherichia coli expression system. In both cases, release of reverse transcriptase and integrase was observed, together with the respective 10-kDa protease form resulting from autocatalysis, although the maturation proceeded less efficiently compared to the homologous system. In further experiments, the ability of both HIV-1 and HIV-2 proteases to release in vivo gag p24 from an in-frame fusion of the full length gag and protease precursors was analyzed. In either case, p24 was released, albeit with greater efficiency in the heterologous gene construction. In vitro mixed lysate experiments with the HIV-1 gag precursor furthermore demonstrate that both enzymes respond to the aspartyl proteinase inhibitor pepstatin A. Taken together, these results illustrate that although different cleavage recognition sequences exist for HIV-1 and -2, they are amenable to the proteases of both viruses, but additionally that subtle differences in the mode of action of both enzymes are observable.

Interferon-α But Not AZT Suppresses HIV Expression in Chronically Infected Cell Lines

Promonocytic (U1) and T lymphocytic (ACH-2) cell lines chronically infected with human immunodeficiency virus type 1 (HIV-1) constitutively express low levels of virus, but expression can be induced by phorbol esters and cytokines. Whereas ACH-2 cells produce infectious virions, U1 cells produce defective, noninfectious particles. Although 3'-azido-3'-deoxythimidine (AZT) prevented acute HIV infection of susceptible cells, it did not prevent the induction of HIV expression in the infected cell lines. In contrast, interferon alpha (IFN-alpha) inhibited the release of reverse transcriptase and viral antigens into the culture supernatant after phorbol ester stimulation of both cell lines. Further, IFN-alpha suppressed the production or release (or both) of whole HIV virions, but had no effect on the amount of cell-associated viral proteins. Also, after phorbol ester stimulation of ACH-2 cells, IFN-alpha reduced the number of infectious viral particles secreted into the culture supernatant, but had no effect on the infectivity of cell-associated virus. These findings lend support to the combined use of antiviral agents that have action at both the early (AZT) and the late (IFN-alpha) stages of HIV replication.

Antibody-independent, complement-mediated enhancement of HIV-1 infection by mannosidase I and II inhibitors

Human immunodeficiency virus type 1 (HIV-1) infectivity and cytopathic effect require proper maturation of the viral envelope glycoprotein carbohydrate moieties. We have found that fresh human serum enhances the infectivity of HIV-1 in MT-2 cell infection assays when virus is synthesized in the presence of the mannosidase I inhibitor, 1-deoxymannojirimycin, or the mannosidase II inhibitor, swainsonine, but has no enhancing effect on virus synthesized in the presence of the glucosidase I inhibitors, castanospermine and 1-deoxynojirimycin, or the glucosidase II inhibitor, bromoconduritol. Enhanced infections were characterized by cytopathic effect, antigen synthesis and reverse transcriptase release, all which occurred sooner than in control-infected cultures. This enhancement of infection was also observed in C1q-deficient serum but was not observed in serum that was heatinactivated or depleted of complement components C3 or factor B, thus suggesting a requirement for the alternate pathway of complement.

Complement‐mediated lysis of type‐C virus: Effect of primate and human sera on various retroviruses

AbstractAntibody‐independent complement‐mediated lysis of type‐C RNA tumor viruses (retroviruses) was measured in the sera of humans and other primates using the reverse transcriptase release assay. In this assay, preincubated mixtures of test serum and virus are assayed for the presence of viral reverse transcriptase released from disrupted virions. Serum decomplemented by heating possesses no such activity. Sera containing non‐specific heat‐labile factors inhibitory to the reverse transcriptase release assay are excluded from consideration. According to this assay, both normal and leukemic human sera, as well as sera from several other primate species including apes, Old World monkeys and New World monkeys, are all capable of lysing both endogenous and infectious primate and murine type‐C viruses. At least two non‐primate species (cat and rabbit) also possess this activity, although several other non‐primates do not. There is no appreciable viral specificity; each test serum possessing complement‐mediated virolytic activity can lyse all classes of retroviruses against which it is tested. Those primate species which spontaneously release retroviruses (baboon, squirrel monkey), as well as those primate species which are infected with exogenous retroviruses (rhesus, gibbon ape), are all capable of lysing their respective viruses. In addition, the relative efficiency of complement‐mediated viral lysis correlates better with primate phylogeny than with the relative ease of type‐C virus release or transmission. This would seem to suggest that complement‐mediated viral lysis does not, in fact, play a primary role in blocking the release or transmission of type‐C viruses among primates.