Reverse Transcriptase Molecule Research Articles

Mechanism of HIV Reverse Transcriptase Inhibition by Zinc: FORMATION OF A HIGHLY STABLE ENZYME-(PRIMER-TEMPLATE) COMPLEX WITH PROFOUNDLY DIMINISHED CATALYTIC ACTIVITY

Several physiologically relevant cations including Ca(2+), Mn(2+), and Zn(2+) have been shown to inhibit HIV reverse transcriptase (RT), presumably by competitively displacing one or more Mg(2+) ions bound to RT. We analyzed the effects of Zn(2+) on reverse transcription and compared them to Ca(2+) and Mn(2+). Using nucleotide extension efficiency as a readout, Zn(2+) showed significant inhibition of reactions with 2 mM Mg(2+), even when present at only ∼5 μM. Mn(2+) and Ca(2+) were also inhibitory but at higher concentrations. Both Mn(2+) and Zn(2+) (but not Ca(2+)) supported RT incorporation in the absence of Mg(2+) with Mn(2+) being much more efficient. The maximum extension rates with Zn(2+), Mn(2+), and Mg(2+) were ∼0.1, 1, and 3.5 nucleotides per second, respectively. Zinc supported optimal RNase H activity at ∼25 μM, similar to the optimal for nucleotide addition in the presence of low dNTP concentrations. Surprisingly, processivity (average number of nucleotides incorporated in a single binding event with enzyme) during reverse transcription was comparable with Zn(2+) and Mg(2+), and single RT molecules were able to continue extension in the presence of Zn(2+) for several hours on the same template. Consistent with this result, the half-life for RT-Zn(2+)-(primer-template) complexes was 220 ± 60 min and only 1.7 ± 1 min with Mg(2+), indicating ∼130-fold more stable binding with Zn(2+). Essentially, the presence of Zn(2+) promotes the formation of a highly stable slowly progressing RT-(primer-template) complex.

Using highly active antiretroviral therapy to decrease perioperative HIV-1 transmission risk

We read with interest the novel strategy described by Drs. Papendorp and van den Berk in the March 27 issue of AIDS [1] of instituting a standard HAART regimen (tenofovir, emtricitabine, and efavirenz) plus raltegravir to reduce HIV-1 viral load rapidly in order to decrease peri-operative infectious risk. While raltegravir-containing HAART affords a more rapid viral load decline compared to other antiretroviral regimens [2], there remains a period of several weeks before virions are cleared from the plasma. This may represent an unacceptably long time to wait for urgent surgery. Building upon Drs. Papendorp and van den Berk’s innovative concept of using the pharmacodynamic properties unique to particular classes of antiretrovirals to reduce peri-operative risk, we describe an alternative strategy that theoretically offers the same benefit within a much shorter time period. Raltegravir, the first integrase inhibitor approved for clinical use, blocks incorporation of HIV-1 reverse transcripts into the host cell genome. Infected cells that have already undergone HIV-1 DNA integration at the time raltegravir is initiated will not be blocked from producing infectious virions. The rapid viral load decay seen in patients taking raltegravir may be the result of the blockade of a later stage of viral replication than other currently available antiretroviral drugs [3]. For example, it is likely that a larger population of infected cells can continue to produce virus in patients starting an efavirenz-based regimen than in patients starting a raltegravir-based regimen. With efavirenz initiation, all infected cells that have undergone reverse transcription will continue to produce virus, whereas with raltegravir only cells that have undergone viral integration will produce virus. This is likely the basis of the more rapid decay in plasma HIV-1 RNA levels in patients starting raltegravir [3]. However, with respect to occupational exposure, the critical issue is not the absolute level of plasma virus, but rather the level of infectious virus. In theory, virions produced by previously infected cells in a patient starting an integrase inhibitor will be fully infectious if transferred to another host, because integrase inhibitors act only on the integrase-viral DNA complex in newly infected cells and not on the free enzyme in the virion [4]. The situation is different in patients starting protease inhibitors. These drugs inhibit the enzyme responsible for the cleavage of immature viral proteins within the virion during and after budding from the host cell. Previously infected cells at all stages of replication will continue to produce virions when a protease inhibitor is initiated [3]. For this reason, the viral load decay on protease inhibitor regimens is not as rapid as that observed with raltegravir. However, all virions produced in the presence of therapeutic concentrations of a protease inhibitor are immature and incapable of infecting a new target. Thus, within hours of initiating a protease inhibitor-based regimen, patients will have a falling viral load composed of immature, non-infectious virions that likely represent a minimal risk for blood borne viral transmission. In the case of protease inhibitors with a slow off rate such as darunavir [5], enzyme inhibition may persist following occupational transmission of virus particles to a recipient for longer than the virion lifetime, affording complete protection. If the drug does dissociate, the virion would still need to complete the maturation process to become infectious. The non-nucleoside reverse transcriptase inhibitors potentially represent an intermediate case. In principle, they can bind to the reverse transcriptase molecules in virions, rendering them non-infectious. However, as soon as the inhibitor dissociates, reverse transcription could continue. Thus in order to minimize the probability of occupational transmission, slowly dissociating protease inhibitors offer the most rapid way to reduce infectious virus in the plasma. A potential concern of the drug regimen described by Papendorp and van den Berk is the relatively low barrier to resistance of both efavirenz and raltegravir [6], which may present a risk to the patient long after surgery. This risk is compounded by initiating HAART on an urgent basis in a setting radically different from the outpatient HIV clinic, where a support structure is ideally in place to maximize long-term positive patient outcomes. Protease inhibitor-based regimens have a significantly higher barrier to resistance than efavirenz or raltegravir, and for this reason can often be given with confidence in settings when the presence of drug-resistance mutations cannot be fully assessed. In summary, both the mechanism of action and the superior resistance profile of protease inhibitor-based HAART argue for its use in this specific setting. In regard to blood borne transmission risk, the quality as well of the quantity of virus should be considered.

HIV reverse transcriptase: structural interpretation of drug resistant genetic variants from India.

The reverse transcriptase (RT) enzyme is the prime target of nucleoside/ nucleotide (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors. Here we investigate the structural basis of effects of drug-resistance mutations in clade C RT using three-dimensional structural modeling. Apropos the expectation was for unique mechanisms in clade C based on interactions with amino acids of p66 subunit in RT molecule. 3-D structures of RT with mutations found in sequences from 2 treatment naïve, 8 failed and one reference clade C have been modeled and analyzed. Models were generated by computational mutation of available crystal structures of drug bound homologous RT. Energy minimization of the models and the structural analyses were carried out using standard methods. Mutations at positions 75,101,118,190,230,238 and 318 known to confer drug resistance were investigated. Different mutations produced different effects such as alteration of geometry of the drug-binding pocket, structural changes at the site of entry of the drug (into the active site), repositioning the template bases or by discriminating the inhibitors from their natural substrates. For the mutations analyzed, NRTI resistance was mediated mainly by the ability to discriminate between inhibitors and natural substrate, whereas, NNRTI resistance affected either the drug entry or the geometry of the active site. Our analysis suggests that different mutations result in different structural effects affecting the ability of a given drug to bind to the RT. Our studies will help in the development of newer drugs taking into account the presence of these mutations and the structural basis of drug resistance.

Slide into Action: Dynamic Shuttling of HIV Reverse Transcriptase on Nucleic Acid Substrates

The reverse transcriptase (RT) of human immunodeficiency virus (HIV) catalyzes a series of reactions to convert single-stranded viral RNA into double-stranded DNA for host cell integration. This process requires a variety of enzymatic activities, including DNA polymerization, RNA cleavage, and strand displacement synthesis. As a major target for anti-HIV therapy, RT has been the subject of extensive research. Nonetheless, how the enzyme-substrate complex acquires specific functional configurations and switches between different functional modes remains unclear. Here, we used single-molecule fluorescence resonance energy transfer to probe the interactions between RT and nucleic acid substrates in real time (1). Surprisingly, RT was observed to slide on nucleic acid duplexes, rapidly shuttling between opposite termini of the duplex. Sliding kinetics were regulated by cognate nucleotides and non-nucleoside RT inhibitors, a major class of anti-HIV drugs, which stabilized and destabilized the polymerization mode, respectively. These long-range translocation activities facilitate multiple stages of the reverse transcription pathway. First, sliding allows RT to target the polymerization site by one-dimensional search. Remarkably, upon reaching the polymerization site, an RT molecule originally bound in the opposite orientation can spontaneously flip into the polymerization orientation without dissociation, enhancing the target search efficiency. Furthermore, sliding helps RT to actively disrupt secondary structures on the substrate and kinetically access the polymerization site, thereby facilitating strand displacement synthesis. It is remarkable that an enzyme could have such large-scale translational and orientational dynamics. This type of dynamic flexibility may be a general design principle for multi-functional enzymes, helping them to rapidly access different configurations required for different functions. Reference: 1. S. Liu, E.A. Abbondanzieri, J.W. Rausch, S.F.J. Le Grice, X. Zhuang, “Slide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates”, Science (in press).

Lentivirus-Like Particles Without Reverse Transcriptase Elicit Efficient Immune Responses

Following infection by HIV or SIV, reverse transcriptase (RT) directs the conversion of the single-stranded RNA genome into a double-stranded DNA molecule that integrates into the host cell genome. RT encodes for several immunogenic epitopes that are desirable for inclusion in a human vaccine for HIV infection, however, issues of safety have dampened enthusiasm for inclusion of an enzymatically-active RT molecule into an AIDS vaccine. In this study, virally-regulated, replication-incompetent lentiviral particles were expressed from DNA plasmids. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted and mutations were engineered into capsid to decreases RNA packaging. Virus-like particles incorporated no RT (HIV-VLP DeltaRT or SHIV-VLP DeltaRT) or contained a full-length enzymatically-inactivated RT molecule (HIV-VLP or SHIV-VLP). Each secreted VLP was enveloped with a lipid bilayer derived from primate cells with embedded, native viral envelopes in similar concentrations as infectious virions. BALB/c mice were vaccinated (weeks 0, 3, and 6) with purified VLPs via intranasal inoculation in the presence of cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs). All VLPs, with or without RT, elicited both robust humoral and cellular immune responses to Gag, Pol, and Env antigens. Therefore, the lack of RT enhances the safety of these VLPs for use in future human clinical trials without a significant reduction in the overall immunogenicity of these VLP immunogens.

Stepping Statistics of Single HIV-1 Reverse Transcriptase Molecules during DNA Polymerization

DNA polymerases are protein machines that processively incorporate complimentary nucleotides into a growing double-stranded DNA (ds-DNA). Single-base nucleotide incorporation rates have been determined by stalling and restarting various polymerases, but intrinsic processive rates have been difficult to obtain, particularly for polymerases with low processivity, such as the human immunodeficiency virus type 1 reverse transcriptase (HIV RT) polymerase. Here we find, using a new fluorescence-based single-molecule polymerization assay, that the intrinsic processive DNA-dependent polymerization of HIV RT is approximately Poissionian (i.e. each nucleotide is added sequentially) with a rate of about 100 bases per second at 21 degrees C. From the same experiments, based on the stepping statistics of polymerization, we also estimate the rates for HIV RT early termination and final release of completely replicated primer-template DNA. In addition, by measuring the rate of polymerization as a function of temperature, we have estimated the activation energy for processive nucleotide incorporation.

The mature reverse transcriptase molecules in virions of mouse mammary tumor virus possess protease-derived sequences

Our efforts to express in bacteria the enzymatically active reverse transcriptase (RT) of mouse mammary tumor virus (MMTV) have shown that the RT is active only after adding 27 amino acid residues, which are derived from the end of the pro gene, to the amino-terminus of the RT (Biochem, J. (1998) 329, 579–587). In the present study we have tested whether the mature RT found in virions is also fused to protease-derived sequences. To this end, we have analyzed the RT molecules in virions of MMTV by using two antisera directed against peptides, derived from either the carboxyl-terminus of MMTV protease or the middle of MMTV RT. The data suggest that the mature RT, located in virions, contains at its amino-terminus sequences from the carboxyl-terminus of the protease protein. This finding supports previous suggestions that MMTV RT is a transframe protein (derived from both pro and pol reading frames of MMTV) and that amino acid residues located at the carboxyl-terminus of the protease have a dual usage as integral parts of both the protease and the RT enzymes.

Solution structure of the RNase H domain of the HIV-1 reverse transcriptase in the presence of magnesium.

This paper presents the first solution structure of the RNase H domain of HIV-1 reverse transcriptase (RT) determined by NMR methods. The solution conditions in this study were at physiological pH in the presence of Mg(2+). An investigation of the dependence of the (1)H-(15)N HSQC spectrum of the RNase H domain on [Mg(2+)] indicates that Mg(2+) produces significant, global effects on the amide chemical shifts, implying that divalent metal ion binding is important for stabilizing the structure of the isolated domain in solution. Analysis of amide shift data as a function of MgCl(2) concentration using either a single- or two-site binding model indicated that the latter provided a significantly improved fit, with the K(D) for site A = 2.7-3.2 mM and K(D) for site B approximately 35 mM, calculated on the assumption that site A is already occupied. Resonances of the [U-(13)C,(15)N]RNase H domain, measured at pH 6.8, in 80 mM MgCl(2), were assigned and NOESY data collected in order to determine the structure. Assignment of the NOESY spectra using the ARIA program resulted in a high-resolution structure for residues 6-114 which was similar to the crystal structure of the isolated domain,. The data were insufficient to define a compact structure for the C-terminal residues after 114. Residues I134-L138 located at the C-terminus are highly disordered and give rise to relatively sharp and intense amide resonances, while the amide resonances for the segment from E124 to A132 appear to be largely absent and are presumably subject to significant exchange broadening between different conformational states. Comparisons with crystal structure data for the full reverse transcriptase molecule indicate that the corresponding region is absent in nearly all of the crystal structures determined for the P2(1)2(1)2(1) space group, while these residues adopt an alpha-helix in structures determined for other symmetry groups. This structural heterogeneity indicates that significant conformational variability exists for this segment of the full reverse transcriptase enzyme as well, and the structure of the C-terminal peptide can be selected or deselected, depending on crystallization conditions. This analysis, along with the structural characterization contained herein, challenges the previous paradigm that the dynamic behavior of the isolated RNase H domain differs substantially from the behavior in the intact enzyme. The poor Mg(2+) binding and conformational flexibility of residues located near the active site indicate that substrate binding is a precondition for metal ion binding and for selecting the active site conformation of the RNase H domain.

Physical Mapping of HIV Reverse Transcriptase to the 5′ End of RNA Primers

Enzymatic analysis of RNA cleavage products has suggested that human immunodeficiency virus (HIV) reverse transcriptase (RT) binds to the 5' end of RNAs that are recessed on a longer DNA template (RNA primers) yet binds to the 3' end of DNA primers. One concern is that RT molecules bound at the 3' end of RNA would not be easily detected because RT may not catalyze substantial RNA extension or cleavage when bound to the 3' end. We used physical mapping to show that RT binds preferentially to the 5' end of RNA primers. An HIV-RT that lacked RNase H activity (HIV-RT(E478Q)) was incubated with the RNA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT protected a approximately 23-base region at the 5' end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal structure of HIV-RT. No protection of the RNA 3' end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wild-type HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.

Replication of Phenotypically Mixed Human Immunodeficiency Virus Type 1 Virions Containing Catalytically Active and Catalytically Inactive Reverse Transcriptase

The amount of excess polymerase and RNase H activity in human immunodeficiency virus type 1 virions was measured by using vectors that undergo a single round of replication. Vectors containing wild-type reverse transcriptase (RT), vectors encoding the D110E mutation to inactivate polymerase, and vectors encoding mutations D443A and E478Q to inactivate RNase H were constructed. 293 cells were cotransfected with different proportions of plasmids encoding these vectors to generate phenotypically mixed virions. The resulting viruses were used to infect human osteosarcoma cells, and the relative infectivity of the viruses was determined by measuring transduction of the murine cell surface marker CD24, which is encoded by the vectors. The results indicated that there is an excess of both polymerase and RNase H activities in virions. Viral replication was reduced to 42% of wild-type levels in virions with where half of the RT molecules were predicted to be catalytically active but dropped to 3% of wild-type levels when 25% of the RT molecules were active. However, reducing RNase H activity had a lesser effect on viral replication. As expected, based on previous work with murine leukemia virus, there was relatively inefficient virus replication when the RNase H and polymerase activities were encoded on separate vectors (D110E plus E478Q and D110E plus D443A). To determine how virus replication failed when polymerase and RNase H activities were reduced, reverse transcription intermediates were measured in vector-infected cells by using quantitative real-time PCR. The results indicated that using the D11OE mutation to reduce the amount of active polymerase reduced the number of reverse transcripts that were initiated and also reduced the amounts of products from the late stages of reverse transcription. If the E478Q mutation was used to reduce RNase H activity, the number of reverse transcripts that were initiated was reduced; there was also a strong effect on minus-strand transfer.

Reverse transcriptase activity in mature spermatozoa of mouse.

We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.

Sequence analysis of the polymerase domain of HIV-1 reverse transcriptase in naive and zidovudine-treated individuals reveals a higher polymorphism in alpha-helices as compared with beta-strands.

We report a statistical analysis of genetic heterogeneity of the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1. Both newly determined sequences and sequences contained in the data banks have been examined. For the calculations, the viral samples and the regions within the RT molecule were divided in two groups. The viral samples were split into those from patients not subjected to antiretroviral therapy and those from patients treated with zidovudine (AZT, 3'-azido-3'-deoxythymidine) alone or in combination with other RT inhibitors. The RT-coding region was divided into segments encoding beta-strands and segments encoding alpha-helices. A significantly lower heterogeneity was observed in beta-strands relative to the alpha-helix coding segments. Application of the D test of Tajima has provided evidence of operation of negative (or purifying) selection in sequences from viruses of patients not subjected to antiretroviral treatment as well as in treated patients. In the group of untreated individuals, regions encoding beta-strands are subjected to stronger negative selection than those encoding alpha-helices. It is likely that the observed differences reflect stronger functional constraints in beta-strands than in alpha-helices of RT.

Endogenous reverse transcriptase assays reveal synergy between combinations of the M184V and other drug resistance-conferring mutations in interactions with nucleoside analog triphosphates

Resistance of HIV-1 reverse transcriptase (RT) to nucleoside analogs (e.g. AZT, ddC and 3TC) is conferred by various amino acid substitutions or combinations thereof on the RT molecule. The M184V mutation, that confers high and low-level resistance to 3TC and ddC, respectively, can restore sensitivity to AZT when introduced into RT against a background of AZT-resistance. The K65R mutation, that confers low level resistance to both 3TC and ddC, can also restore sensitivity to AZT. This information is of potential utility in choosing combinations of anti-viral drugs for clinical use. To explore this subject further, we have used an endogenous RT reaction to study mutated viruses containing M184V alone or M184V combined with each of the K65R, E89G or both the M41L and T215Y substitutions. Endogenous assays possess the advantage of utilizing genomic RNA as template in a reaction mixture that includes each of tRNA Lys.3 and viral nucleocapsid protein, necessary for specific initiation of reverse transcription, as well as all other viral proteins that might impact on this process. We now show that viruses containing both M184V and K65R displayed synergistic resistance to 3TC triphosphate (3TCTP), while the same combination yielded the same level of resistance to ddC triphosphate (ddCTP) as that manifested by K65R alone. The combination of M184V and E89G displayed synergistic resistance against ddCTP but not 3TCTP, while viruses containing only E89G were highly resistant to 3TCTP and displayed low-level resistance to ddCTP. The results show that endogenous RT assays can reveal variable synergistic, antagonistic, or neutral effects in regard to drug sensitivity, depending on the presence of specific amino acid substitutions in RT itself.

Elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay

Three highly sensitive reverse transcriptase (RT) assays were recently published that are at least one million times more sensitive than conventional RT assays. These assays derive their high sensitivities through the ability to amplify the complementary DNA (cDNA) product of the RT reaction by the polymerase chain reaction (PCR). We describe a modified PCR-basecl RT (PBRT) assay that retains the high sensitivities of the original assays while reducing their inherent background signals. The background signal of the PBRT assay was found to be due to an intrinsic RNA-dependent DNA polymerase activity of the Taq DNA polymerase, the enzyme used for the PCR. It could be eliminated by inserting a ribonuclease digestion step prior to amplifying the cDNA product of the RT reaction by PCR and by using a thermostable DNA polymerase identified as having reduced RNA-dependent DNA polymerase activity. Comparable results were obtained using three RNA templates with two purified RT enzymes. This modified assay is capable of detecting reliably between 10 and 100 molecules of RT, which is equivalent to between 1 and 10 retrovirus particles.

Importance of the C-terminal helix to the stability and enzymatic activity of Escherichia coli ribonuclease H.

The ribonuclease H (RNase H) family of enzymes selectively degrades the RNA strand of RNA-DNA hybrids. This activity is essential for retroviruses such as HIV and resides in a domain of the larger reverse transcriptase molecule. RNase H from Escherichia coli is the best-characterized member of the family and serves as a model for structure/function studies. Despite having almost identical alpha + beta folds, the isolated domain from HIV is inactive and much less stable than the E. coli homolog. The HIV domain also shows increased disorder in its C-terminal regions (E-helix and His-containing loop). We investigated the importance of this region by studying a variant of E. coli RNase H lacking these elements (RNHdeltaE). Despite the elimination of 33 of 155 residues (including a complete helix), this C-terminal deletion mutant folds cooperatively as a subdomain. Surprisingly, this protein lacking residues near the active site retains weak Mn2+-dependent activity. A peptide corresponding to the deleted E-helix is helical in isolation and stimulates the activity of the deletion mutant in vitro. These results have implications for the catalytic mechanism of RNase H and drug design targeted to HIV reverse transcriptase.

Anti-CD3/2-chloro-5-nitrobenzoic acid costimulation differentially enhances T-cell growth without inducing HIV-1 replication: D. Kinchington*, Tony Ng, N. Mathews, M. Tisdale+ D. Devine and W. O. Ayuko+. St. Bartholomew's Medical School, London, England. +Department of Pharmaceutical and Biological Sciences, Aston University, Birmingham, England

Resistance of HIV-1 reverse transcriptase (RT) to nucleoside analogs (e.g. AZT, ddC and 3TC) is conferred by various amino acid substitutions or combinations thereof on the RT molecule. The M184V mutation, that confers high and low-level resistance to 3TC and ddC, respectively, can restore sensitivity to AZT when introduced into RT against a background of AZT-resistance. The K65R mutation, that confers low level resistance to both 3TC and ddC, can also restore sensitivity to AZT. This information is of potential utility in choosing combinations of anti-viral drugs for clinical use. To explore this subject further, we have used an endogenous RT reaction to study mutated viruses containing M184V alone or M184V combined with each of the K65R, E89G or both the M41L and T215Y substitutions. Endogenous assays possess the advantage of utilizing genomic RNA as template in a reaction mixture that includes each of tRNALys.3 and viral nucleocapsid protein, necessary for specific initiation of reverse transcription, as well as all other viral proteins that might impact on this process. We now show that viruses containing both M184V and K65R displayed synergistic resistance to 3TC triphosphate (3TCTP), while the same combination yielded the same level of resistance to ddC triphosphate (ddCTP) as that manifested by K65R alone. The combination of M184V and E89G displayed synergistic resistance against ddCTP but not 3TCTP, while viruses containing only E89G were highly resistant to 3TCTP and displayed low-level resistance to ddCTP. The results show that endogenous RT assays can reveal variable synergistic, antagonistic, or neutral effects in regard to drug sensitivity, depending on the presence of specific amino acid substitutions in RT itself.

Chapter 10 The Use of Replication--Defective Retroviruses for Cell Lineage Studies of Myogenic Cells

This chapter discusses the use of replication-defective retroviruses for cell lineage studies of myogenic cells. The chapter focuses on the application of retroviral tags for the analysis of cell lineage, migration, and gene transfer. Although retroviral tags offer many attractive features, they are hampered by potentially significant problems: they cannot be used to analyze label postmitotic cell populations; the uptake of the retrovirus and the expression of its tag may not be identical in all embryonic tissues or in every cell within a tissue; and the integration of a retrovirus may alter the properties of the cell if this integration occurs at an important locus. Even with these reservations, replication-defective retroviruses have proven to be very useful for analyzing several tissues and organ systems, such as cells of the embryonic retina, cortex, neural crest, heart, vascular system, and somite-derived skeletal muscle. Retroviruses are constructed with two principal components: (1) a central core packaging two copies of a single-stranded RNA genome with associated nucleoproteins plus reverse transcriptase molecules and (2) the membrane coat bearing specific viral envelope glycoproteins. Vectors based on the murine leukemia virus (MLV), the avian Rous sarcoma virus (RSV), and the avain spleen necrosis virus (SNV) have been widely used for cell lineage studies. Each virus type exhibits a characteristic host range spectrum affecting infectivity and expression levels of its reporter gene.

Human antibodies responsible for binding inhibition and polymerization inhibition of human immunodeficiency virus type 1 reverse transcriptase

Using a solid-phase non-radioisotopic (non-RI) reverse transcriptase (RT) assay, antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) RT activity (RTI antibody) were investigated for their ability to inhibit binding of RT to a template-primer and DNA polymerization. The RTI antibody inhibited the binding of RT to the template-primer (BI antibody), and directly reacted with the RT-template-primer complex and inhibited enzymatic activity (PI antibody). The RTI antibody interfered with formation of the RT-template-primer complex suggesting that it recognized the antigenic site involved in template-primer binding of RT molecules. Since deoxynucleotide triphosphates (dNTPs) blocked inhibition of the RT activity by the PI antibody, the antigenic site recognized by the PI antibody may be closely related to the dNTP binding site. The seropositivities of the BI and PI antibodies were 84.6% and 91.2%, respectively, in HIV-1-infected individuals; healthy individuals, HTLV-I-positive individuals, autoimmune disease patients and leukemia patients were all seronegative. No significant correlation of residual RT activities was observed when BI and PI antibodies were compared ( r = 0.688). It is possible that the epitopes recognized by the BI antibody differs from those recognized by the PI antibody. The assays described are able to detect BI and PI antibodies in the sera of HIV-1-infected individuals.

Ty3 integrase mutants defective in reverse transcription or 3'-end processing of extrachromosomal Ty3 DNA

Ty3, a retroviruslike element in Saccharomyces cerevisiae, encodes an integrase (IN) which is essential for position-specific transposition. The Ty3 integrase contains the highly conserved His-Xaa(3-7)-His-Xaa(23-32)-Cys-Xaa(2)-Cys and Asp, Asp-Xaa(35)-Glu [D,D(35)E] motifs found in retroviral integrases. Mutations were introduced into the coding region for the Ty3 integrase to determine the effects in vivo of changes in conserved residues of the putative catalytic triad D,D(35)E and the nonconserved carboxyl-terminal region. Ty3 viruslike particles were found to be associated with significant amounts of linear DNA of the approximate size expected for a full-length reverse transcription product and with plus-strand strong-stop DNA. The full-length, preintegrative DNA has at each 3' end 2 bp that are removed prior to or during integration. Such 3'-end processing has not been observed for other retroviruslike elements. A mutation at either D-225 or E-261 of the Ty3 integrase blocked transposition and prevented processing of the 3' ends of Ty3 DNA in vivo, suggesting that the D,D(35)E region is part of the catalytic domain of Ty3 IN. Carboxyl-terminal deletions of integrase caused a dramatic reduction in the amount of Ty3 DNA in vivo and a decrease in reverse transcriptase activity in vitro but did not affect the apparent size or amount of the 55-kDa reverse transcriptase in viruslike particles. The 115-kDa viruslike particle protein, previously shown to react with antibodies to Ty3 integrase, was shown to be a reverse transcriptase-IN fusion protein. These results are consistent with a role for the integrase domain either in proper folding of reverse transcriptase or as part of a heterodimeric reverse transcriptase molecule.

Escherichia coli RNase HI inhibits murine leukaemia virus reverse transcription in vitro and yeast retrotransposon Ty1 transposition in vivo.

Reverse transcription, which converts an RNA genome into double-stranded DNA, requires both the polymerase and RNase H activities of reverse transcriptase (RT). In vitro, poorly processive RT dissociates from partially copied RNA-DNA hybrids, that are usually extended by a second RT molecule. Despite similar structures, RNase HI of Escherichia coli can degrade RNA-DNA hybrids that are resistant to RNase H of RT. E. coli RNase HI is used to determine the accessibility to and requirement for RNA-DNA hybrids in reverse transcription in vivo and in vitro. In the presence of E. coli RNase HI, reverse transcription yields incomplete cDNA molecules due to degradation of RNA-DNA hybrids. Delivery of E. coli RNase HI to Ty1 particles via fusion to the capsid protein can reduce retrotransposition by more than 99%, also indicating inhibition of DNA synthesis in vivo. Inhibition of both reverse transcription in vitro and retrotransposition in vivo by E. coli RNase HI indicates that the poor processivity of RT exposes RNA-DNA hybrids critical for reverse transcription to degradation. Targeting a cellular RNase H to HIV may help define the site(s) of RNA-DNA hybrids that are susceptible to nonretroviral RNase H and may be useful for gene therapy to inhibit retroviral replication.