Reverse Transcriptase Processivity Research Articles

Anti-HIV-1 activity of the G-quadruplex ligand BRACO-19.

A dynamic G-quadruplex region has been previously shown to form in the long terminal repeat (LTR) promoter of the HIV-1 integrated DNA genome. Inhibition of promoter activity and antiviral effects have been observed when this region was stabilized by BRACO-19, a trisubstituted acridine derivative that binds G-quadruplexes. Here, we aimed at characterizing the antiviral mechanism of action of BRACO-19 by analysing its activity towards a broad range of HIV-1 strains, host cells and infection modes. The antiviral activity of BRACO-19 in cell lines and primary cells infected or persistently infected by HIV-1 strains was evaluated at different times post-infection. Virucidal, viral binding, time-dependent and drug-dependent assays were performed to identify the viral target step. Circular dichroism, UV spectroscopy and a reverse transcriptase (RT) stop assay were used to assess RNA G-quadruplex folding and inhibition of RT processing. Thorough virological assays demonstrated that BRACO-19 acts both at the reverse transcription and the post-integration level during the virus life cycle. This behaviour was rationalized by the observation that a G-quadruplex-forming sequence identical to that of the LTR DNA is present at the 3'-end of the virus RNA genome. Biophysics and biomolecular testing showed that this region has the ability to fold into very stable G-quadruplex structures that are even more stabilized by BRACO-19, therefore inhibiting the reverse transcription process at the template level. Our findings strongly support the activity of BRACO-19 at the viral G-quadruplex level and therefore strengthen the use of viral G-quadruplexes as new anti-HIV-1 targets.

Biochemical interactions between HIV-1 integrase and reverse transcriptase

Human immunodeficiency virus (HIV) infection yields a high level of non-integrated viral DNA in the infected cells and up to 99% of total viral DNA can be capable of transcription. This capability of non-integrated viral DNA is reducing the efficacy of anti-HIV drug development approaches that solely focus on the integration reactions of viral replication. Using kinetic modeling, we show the transient coordinated regulation of viral DNA production by retrovirus-encoded interacting enzymes reverse transcriptase (RT) and integrase (IN), and thus we identify a possible mechanism for reducing overall efficiency of viral replication. The results indicate that both IN·RT and IN·DNA complexes and their formation rates affect RT processivity and thereby the viral DNA expression level within the pre-integration complex.

HIV-1 Reverse Transcriptase (RT) Polymorphism 172K Suppresses the Effect of Clinically Relevant Drug Resistance Mutations to Both Nucleoside and Non-nucleoside RT Inhibitors

Polymorphisms have poorly understood effects on drug susceptibility and may affect the outcome of HIV treatment. We have discovered that an HIV-1 reverse transcriptase (RT) polymorphism (RT(172K)) is present in clinical samples and in widely used laboratory strains (BH10), and it profoundly affects HIV-1 susceptibility to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) when combined with certain mutations. Polymorphism 172K significantly suppressed zidovudine resistance caused by excision (e.g. thymidine-associated mutations) and not by discrimination mechanism mutations (e.g. Q151M complex). Moreover, it attenuated resistance to nevirapine or efavirenz imparted by NNRTI mutations. Although 172K favored RT-DNA binding at an excisable pre-translocation conformation, it decreased excision by thymidine-associated mutation-containing RT. 172K affected DNA handling and decreased RT processivity without significantly affecting the k(cat)/K(m) values for dNTP. Surface plasmon resonance experiments revealed that RT(172K) decreased DNA binding by increasing the dissociation rate. Hence, the increased zidovudine susceptibility of RT(172K) results from its increased dissociation from the chain-terminated DNA and reduced primer unblocking. We solved a high resolution (2.15 Å) crystal structure of RT mutated at 172 and compared crystal structures of RT(172R) and RT(172K) bound to NNRTIs or DNA/dNTP. Our structural analyses highlight differences in the interactions between α-helix E (where 172 resides) and the active site β9-strand that involve the YMDD loop and the NNRTI binding pocket. Such changes may increase dissociation of DNA, thus suppressing excision-based NRTI resistance and also offset the effect of NNRTI resistance mutations thereby restoring NNRTI binding.

Compensation by the E138K Mutation in HIV-1 Reverse Transcriptase for Deficits in Viral Replication Capacity and Enzyme Processivity Associated with the M184I/V Mutations

Recently, several phase 3 clinical trials (ECHO and THRIVE) showed that E138K and M184I were the most frequent mutations to emerge in patients who failed therapy with rilpivirine (RPV) together with two nucleos(t)ide reverse transcriptase inhibitors, emtricitabine (FTC) and tenofovir (TDF). To investigate the basis for the copresence of E138K and M184I, we generated recombinant mutated and wild-type (WT) reverse transcriptase (RT) enzymes and HIV-1(NL4-3) infectious clones. Drug susceptibilities were determined in cord blood mononuclear cells (CBMCs). Structural modeling was performed to analyze any impact on deoxynucleoside triphosphate (dNTP) binding. The results of phenotyping showed that viruses containing both the E138K and M184V mutations were more resistant to each of FTC, 3TC, and ETR than viruses containing E138K and M184I. Viruses with E138K displayed only modest resistance to ETR, little resistance to efavirenz (EFV), and no resistance to either FTC or 3TC. E138K restored viral replication capacity (RC) in the presence of M184I/V, and this was confirmed in cell-free RT processivity assays. RT enzymes containing E138K, E138K/184I, or E138K/184V exhibited higher processivity than WT RT at low dNTP concentrations. Steady-state kinetic analysis demonstrated that the E138K mutation resulted in decreased K(m)s for dNTPs. In contrast, M184I/V resulted in an increased K(m) for dNTPs compared to those for WT RT. These results indicate that the E138K mutation compensates for both the deficit in dNTP usage and impairment in replication capacity by M184I/V. Structural modeling shows that the addition of E138K to M184I/V promotes tighter dNTP binding.

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus

BackgroundThe major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in β3-β4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses.MethodsProviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74.ResultsReplication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT.ConclusionsThe improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

The mutation T477A in HIV-1 reverse transcriptase (RT) restores normal proteolytic processing of RT in virus with Gag-Pol mutated in the p51-RNH cleavage site

BackgroundThe p51 subunit of the HIV-1 reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. Our previous work showed that mutations in the RT p51↓RNH cleavage site resulted in virus with defects in proteolytic processing of RT and significantly attenuated infectivity. In some cases, virus fitness was restored after repeated passage of mutant viruses, due to reversion of the mutated sequences to wild-type. However, in one case, the recovered virus retained the mutated p51↓RNH cleavage site but also developed an additional mutation, T477A, distal to the cleavage site. In this study we have characterized in detail the impact of the T477A mutation on intravirion processing of RT.ResultsWhile the T477A mutation arose during serial passage only with the F440V mutant background, introduction of this substitution into a variety of RT p51↓RNH cleavage site lethal mutant backgrounds was able to restore substantial infectivity and normal RT processing to these mutants. T477A had no phenotypic effect on wild-type HIV-1. We also evaluated the impact of T477A on the kinetics of intravirion Gag-Pol polyprotein processing of p51↓RNH cleavage site mutants using the protease inhibitor ritonavir. Early processing intermediates accumulated in p51↓RNH cleavage site mutant viruses, whereas introduction of T477A promoted the completion of processing and formation of the fully processed RT p66/p51 heterodimer.ConclusionsThis work highlights the extraordinary plasticity of HIV-1 in adapting to seemingly lethal mutations that prevent RT heterodimer formation during virion polyprotein maturation. The ability of T477A to restore RT heterodimer formation and thus intravirion stability of the enzyme may arise from increased conformation flexibility in the RT p51↓RNH cleavage site region, due to loss of a hydrogen bond associated with the normal threonine residue, thereby enabling proteolytic cleavage near the normal RT p51↓RNH cleavage site.

Comparative analysis of in vitro processivity of HIV-1 reverse transcriptases containing mutations 65R, 74V, 184V and 65R + 74V

While HIV-1 reverse transcriptase (RT) mutations of M to V at position 184 are commonly observed in the clinic, the double mutation of 65R + 74V is rarely seen. It has been demonstrated that rapid R → K reversion occurs at RT codon 65 during replication of HIV-1 in human peripheral blood mononuclear cells containing 65R + 74V mutations and that processivity of the RT is reduced relative to wild type. However, clinical studies show that M184V can be detected after several months of therapy interruption, suggesting more effective processivity. Herein, the in vitro RT processivity of genetically engineered M184V and double mutant 65R + 74V was compared. Virion-associated RTs of WT pNL4-3, K65R, L74V, M184V and 65R + 74V were used to perform RT processivity assays in the presence of trap, poly(rC)-oligo(dG). Both RTs with 184V and 65R + 74V mutations exhibited similar processivity when compared with each other and a significantly decreased processivity as compared to WT RT. Both mutant RTs synthesized shorter cDNA molecules (37–42 nt) relative to WT RT, which made longer (65–70 nt) cDNA molecules. Since these surprising biochemical results cannot explain the clinical phenotype, a hypothesis is presented to explain the discrepancy and suggest new approaches for future testing.

In vitro Synthesis of Long DNA Products in Reactions with HIV-RT and Nucleocapsid Protein

In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.

Antihuman Immunodeficiency Virus Type 1 (HIV-1) Activity of Rare Earth Metal Complexes of 4-Hydroxycoumarins in Cell Culture.

The cerium Ce(III), lanthanum La(III), and neodymium Nd(III) complexes with 4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one (warfarin) (W) and 3,3′-benzylidenebis[4-hydroxycoumarin] (1) were synthesized and studied for the first time for cytotoxicity (on MT-2 cells) and as anti-HIV agents under acute and chronic infection. The complexes were characterized by different physicochemical methods: mass spectrometry, 1H NMR, 13C NMR, and IR spectroscopy. The spectra of the complexes were interpreted on the basis of comparison with the spectrum of the free ligands. Anti-HIV effect of the complexes/ligands was measured in MT-2 cells by microtiter infection assay. Detection of endogenous reverse transcriptase (RT) activity and RT processivity by PCR indicative for proviral DNA synthesis demonstrated that anti-HIV activity has not been linked to early stages of viral replication. No effect on late steps of viral replication has been found using cells chronically producing HIV-1LAI virus. La(W) demonstrated anti-HIV activity (IC50=21.4 μM) close to maximal nontoxic concentration. Nd(W), Ce(1), and Nd(1) demonstrated limited anti-HIV potency, so none of the complexes seems appropriate to be used in clinic. Further targeting of HIV-1 inhibition by La(W) is under progress.

Virion instability of human immunodeficiency virus type 1 reverse transcriptase (RT) mutated in the protease cleavage site between RT p51 and the RT RNase H domain.

Each of the human immunodeficiency virus type 1 (HIV-1) pol-encoded enzymes, protease (PR), reverse transcriptase (RT), and integrase (IN), is active only as a dimer (or higher-order oligomer in the case of IN), but only RT comprises subunits of different masses. RT is a heterodimer of 66-kDa and 51-kDa subunits. The latter is formed by HIV PR-catalyzed cleavage of p66 during virion maturation, resulting in the removal of the RNase H (RNH) domain of a p66 subunit. In order to study the apparent need for RT heterodimers in the context of the virion, we introduced a variety of mutations in the RT p51-RNH protease cleavage site of an infectious HIV-1 molecular clone. Surprisingly, rather than leading to virions with increased RT p66 content, most of the mutations resulted in significantly attenuated virus that contained greatly decreased levels of RT that in many cases was primarily p51 RT. IN levels were also reduced in several mutants. However, most mutants showed normal levels of the Pr160(gag-pol) precursor polyprotein, suggesting that reduced virion RT arose from proteolytic instability rather than decreased incorporation. Mutant virion p24 Gag levels were equivalent to wild type, indicating that Gag incorporation and processing were not affected. Repeated passage of MT-2 cells exposed to mutant viruses led to the appearance of virus with improved replication capacity; these virions contained normally processed RT at near-wild-type levels. These results imply that additional proteolytic processing of RT to the p66/p51 heterodimer is essential to provide proteolytic stability of RT during HIV-1 maturation.

Characterization of the protease domain of Rice tungro bacilliform virus responsible for the processing of the capsid protein from the polyprotein

BackgroundRice tungro bacilliform virus (RTBV) is a pararetrovirus, and a member of the family Caulimoviridae in the genus Badnavirus. RTBV has a long open reading frame that encodes a large polyprotein (P3). Pararetroviruses show similarities with retroviruses in molecular organization and replication. P3 contains a putative movement protein (MP), the capsid protein (CP), the aspartate protease (PR) and the reverse transcriptase (RT) with a ribonuclease H activity. PR is a member of the cluster of retroviral proteases and serves to proteolytically process P3. Previous work established the N- and C-terminal amino acid sequences of CP and RT, processing of RT by PR, and estimated the molecular mass of PR by western blot assays.ResultsA molecular mass of a protein that was associated with virions was determined by in-line HPLC electrospray ionization mass spectral analysis. Comparison with retroviral proteases amino acid sequences allowed the characterization of a putative protease domain in this protein. Structural modelling revealed strong resemblance with retroviral proteases, with overall folds surrounding the active site being well conserved. Expression in E. coli of putative domain was affected by the presence or absence of the active site in the construct. Analysis of processing of CP by PR, using pulse chase labelling experiments, demonstrated that the 37 kDa capsid protein was dependent on the presence of the protease in the constructs.ConclusionThe findings suggest the characterization of the RTBV protease domain. Sequence analysis, structural modelling, in vitro expression studies are evidence to consider the putative domain as being the protease domain. Analysis of expression of different peptides corresponding to various domains of P3 suggests a processing of CP by PR. This work clarifies the organization of the RTBV polyprotein, and its processing by the RTBV protease.

A surface-mediated origin of the RNA world: biogenic activities of clay-adsorbed RNA molecules

The involvement of clay surfaces in the origin of the first genetic molecules on Earth has long been suggested. However, the formation of these polymers was not sufficient by itself to initiate the evolutionary process leading to the appearance of life. These macromolecules had to persist in primeval habitats so that their biological potentiality could be expressed. In this study, we assess the possibility of development of the RNA world on a clay substrate by investigating the capacity of different RNA molecules adsorbed/bound on the clay minerals montmorillonite (M) and kaolinite (K) to persist in the presence of a degrading agent (RNase-A), to interact specifically with complementary RNA strands, and to transmit the information contained in their nucleotide sequences. The RNase-A degradation of clay-adsorbed 23S rRNA from Escherichia coli was significantly slower (75–80%) than that observed for free rRNA, and the complete digestion of nucleic acid in the presence of clay was obtained in 2 vs. 1 h. Clay-adsorbed Poly[A] homopolymer was able to recognize the complementary Poly[U] homopolymer present in the surrounding water solution and to establish a specific interaction (association) with it, possibly leading to the formation of double strands. Reverse transcription and amplification (RT–PCR) amplification of free and clay-adsorbed 16S indicated that the presence of clay particles partially reduced the efficiency and processivity of reverse transcriptase but did not inhibit its activity, demonstrating that clay-adsorbed RNA is still available for enzymatic replication. These findings indicate that primordial genetic molecules adsorbed on clay minerals would have been protected against degrading agents present in the environment and would have been in the right conditions to undergo evolutionary processes.

Effects of Unpaired Nucleotides within HIV-1 Genomic Secondary Structures on Pausing and Strand Transfer

Reverse transcriptase-mediated RNA displacement synthesis is required for DNA polymerization through the base-paired stem portions of secondary structures present in retroviral genomes. These regions of RNA duplex often possess single unpaired nucleotides, or "bulges," that disrupt contiguous base pairing. By using well defined secondary structures from the human immunodeficiency virus, type 1 (HIV-1), genome, we demonstrate that removal of these bulges either by deletion or by introducing a complementary base on the opposing strand results in increased pausing at specific positions within the RNA duplex. We also show that the HIV-1 nucleocapsid protein can increase synthesis through the pause sites but not as efficiently as when a bulge residue is present. Finally, we demonstrate that removing a bulge increases the proportion of strand transfer events to an acceptor template that occur prior to complete replication of a donor template secondary structure. Together our data suggest a role for bulge nucleotides in enhancing synthesis through stable secondary structures and reducing strand transfer.

Single Unpaired Nucleotides Facilitate HIV-1 Reverse Transcriptase Displacement Synthesis through Duplex RNA

During reverse transcription of viral RNA, HIV-1 reverse transcriptase (RT) encounters RNA stem-loop structures that require displacement synthesis activity in which RT disrupts the RNA helix to access the template strand. A primer extension assay was developed to assess HIV-1 RT RNA displacement synthesis activity in vitro. Initial results revealed that HIV-1 RT performs only limited amounts of RNA displacement through long stretches of RNA duplex, with the majority of synthesis stalling at sequence-dependent pause positions. DNA displacement synthesis through the same sequence, however, proceeded rapidly to the end of the template. The RNA folding algorithm mfold indicated that the presence of an unpaired nucleotide, or "bulge," along the RNA duplex would promote helix melting ahead of the DNA primer terminus to create a small gap of nondisplacement synthesis. Primer extension assays using substrates possessing single-nucleotide bulges in the nontemplate strand near pause sites resulted in diminished pausing at positions within the predicted melted region. Surprisingly, the bulges also reduced pausing distal to the bulge at positions that are expected to remain base-paired. Further analysis revealed that stalling during RNA displacement synthesis results from the displaced RNA re-annealing to the template strand thus forcing the primer terminus to become unpaired and, therefore, not extendable. Introduction of a bulge facilitates displacement synthesis through distal regions by increasing RT processivity in the vicinity of a bulge and reducing the impact of branch migration on primer extension.

Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag–pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.

Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag–pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.

The M184V substitution in human immunodeficiency virus type 1 reverse transcriptase delays the development of resistance to amprenavir and efavirenz in subtype B and C clinical isolates.

The M184V substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), encoding high-level resistance to lamivudine (3TC), results in decreased HIV-1 replicative capacity, diminished RT processivity, and increased RT fidelity in biochemical assays. We assessed the effect of M184V on the development of resistance to the nonnucleoside RT inhibitors efavirenz (EFV) and nevirapine, and to the protease inhibitor amprenavir (APV) in tissue culture. Genotypic analysis revealed differences in EFV resistance-conferring mutations in subtype B (K103N) versus subtype C (V106 M), and the appearance of both was significantly delayed in the M184V-containing variants compared with the wild type (WT). Similarly, there was a marked delay in the emergence of mutations associated with APV resistance (I54 M/L/V) in subtype B viruses harboring M184V compared with paired WT viral isolates.

Length distribution of long interspersed nucleotide elements (LINEs) and processed pseudogenes of human endogenous retroviruses: implications for retrotransposition and pseudogene detection

Deciphering the human genome includes reliable identification and structural characterization of individual retrotransposon elements. The most active group of autonomous transposable elements, the long interspersed nuclear elements (LINE), transpose themselves as well as other RNAs, including those of human endogenous retroviruses (HERV). During this transposition, however, the LINE-encoded reverse transcriptase (RT) often abortively dissociates from the RNA template, leaving a prematurely terminated, 5′ truncated copy. We have analyzed the length distributions of LINEs and of processed pseudogenes derived from HERV-W. As expected, we have found that the majority of 5′ truncated LINEs and HERV-W processed pseudogenes show a prevalence of very short elements terminated close to the 3′ end. On the other hand, the number of complete elements is far above the expectation. The characteristic distribution in both cases indicates two important conclusions: (i) dissociation of LINE RT from the template cannot be fully explained by low processivity of RT modelled as a stochastic, Poisson-type process. (ii) Currently cited numbers of pseudogenes within the human genome are underestimated, since a large percentage of pseudogenes are terminated in the 3′ untranslated region and remain undetectable in translated homology searches of protein databases against the human genome.

High Processivity of the Reverse Transcriptase from a Non-long Terminal Repeat Retrotransposon

R2 is a retrotransposable element that specifically inserts into the 28 S rRNA genes of arthropods. The element encodes a single protein with endonuclease activity that cleaves the 28 S gene target site and reverse transcriptase (RT) activity that uses the cleaved DNA to prime reverse transcription. Here we compare various properties of the R2 RT activity with those of the well characterized retroviral RT, avian myeloblastosis virus (AMV). In processivity assays using heterogeneous RNA templates, R2 RT can synthesize cDNA over twice the length of that synthesized by AMV RT and can synthesize cDNA over 4 times longer than AMV RT in assays with poly(rA) templates. The higher processivity of R2 RT compared with retroviral RTs is a result of the slower rate of dissociation of the enzyme from RNA templates. The elongation rates of the two enzymes are similar. Finally, a highly distinct property of the R2 RT, compared with retroviral enzymes, is its ability to displace RNA strands annealed to RNA templates during cDNA synthesis. We suggest that both the higher processivity and displacement properties of R2 RT compared with retroviral RT result from the greater affinity of the R2 protein for the RNA template upstream of its active site.

Mutation of the catalytic domain of the foamy virus reverse transcriptase leads to loss of processivity and infectivity.

Foamy virus (FV) replication is resistant to most nucleoside analog reverse transcriptase (RT) inhibitors. In an attempt to create a 2',3'-dideoxy-3'-thiacytidine (3TC)-sensitive virus, the second residue in the highly conserved YXDD motif of simian foamy virus-chimpanzee (human isolate) [SFVcpz(hu)] RT was changed from Val (V) to Met (M). Unexpectedly, the resultant virus, SFVcpz(hu) RT-V313M, replicated poorly, and Met rapidly reverted to Val. Despite the presence of approximately 50% of wild-type RT activity in RT-V313M virions, full-length DNA products were not detected in transfected cells. Using purified recombinant enzymes, we found that the wild-type FV RT is significantly more processive than human immunodeficiency virus type 1 RT. However, the V313M mutant has about 40% of the wild-type level of FV RT activity and has a lower processivity than the wild-type FV enzyme. The V313M mutant RT is also relatively resistant to 3TC. These results suggest that the decrease in RT activity and processivity of FV RT-V313M prevents completion of reverse transcription and greatly diminishes viral replication.