RESUMEN
The retrovirus human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS. Although treatment of HIV/AIDS with antiretroviral therapy provides suppression of viremia, latent reservoirs of integrated proviruses preclude cure by current antiviral treatments. Understanding the mechanisms of host-viral interactions may elucidate new treatment strategies. Here, we performed a CRISPR/Cas9 transcriptional activation screen using a high-complexity, genome-wide sgRNA library to identify cellular factors that inhibit HIV-1 infection of human CD4+ T cells. MT4 cells were transduced with a CRISPR/Cas9 sgRNA library and infected with nef-deficient HIV-1NL4-3 expressing ganciclovir-sensitive thymidine kinase, thus enabling selection of HIV-1-resistant cells for analysis of enriched sgRNAs. After validation of screen hits, multiple host factors essential for HIV-1 infection were identified, including SET (SET nuclear proto-oncogene) and ANP32A (acidic nuclear phosphoprotein 32A, PP32A), which together form a histone acetylase inhibitor complex. Using multiple human cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-1-infected individuals, we demonstrate that SET depletion increased HIV-1 infectivity by augmenting DNA integration without significantly changing sites of integration. Conversely, SET overexpression decreased HIV-1 integration and infectivity. SET protein expression was significantly reduced in PBMCs from HIV-1-infected individuals and was downregulated by HIV-1 infection of healthy donor cells in vitro. Notably, HIV-1-induced downregulation of SET could be alleviated by inhibition of the protease granzyme A. Altogether, we have identified cellular inhibitors of HIV-1 infection on a genome-wide scale, which affords new insight into host-virus interactions and may provide new strategies for HIV-1 treatment.
Asunto(s)
VIH-1 , Humanos , Sistemas CRISPR-Cas , Histona Acetiltransferasas , VIH-1/genética , Leucocitos Mononucleares , Proteínas Nucleares , Proteínas de Unión al ARN , Activación Transcripcional , Integración ViralRESUMEN
Reverse transcriptases (RTs) are typically assayed using optimized Mg2+ concentrations (~5-10 mM) several-fold higher than physiological cellular free Mg2+ (~0.5 mM). Recent analyses demonstrated that HIV-1, but not Moloney murine leukaemia (MuLV) or avain myeloblastosis (AMV) virus RTs has higher fidelity in low Mg2+. In the current report, lacZα-based α-complementation assays were used to measure the fidelity of several RTs including HIV-1 (subtype B and A/E), several drug-resistant HIV-1 derivatives, HIV-2, and prototype foamy virus (PFV), all which showed higher fidelity using physiological Mg2+, while MuLV and AMV RTs demonstrated equivalent fidelity in low and high Mg2+. In 0.5 mM Mg2+, all RTs demonstrated approximately equal fidelity, except for PFV which showed higher fidelity. A Next Generation Sequencing (NGS) approach that used barcoding to determine mutation profiles was used to examine the types of mutations made by HIV-1 RT (type B) in low (0.5 mM) and high (6 mM) Mg2+ on a lacZα template. Unlike α-complementation assays which are dependent on LacZα activity, the NGS assay scores mutations at all positions and of every type. Consistent with α-complementation assays, a ~four-fold increase in mutations was observed in high Mg2+. These findings help explain why HIV-1 RT displays lower fidelity in vitro (with high Mg2+ concentrations) than other RTs (e.g. MuLV and AMV), yet cellular fidelity for these viruses is comparable. Establishing in vitro conditions that accurately represent RT's activity in cells is pivotal to determining the contribution of RT and other factors to the mutation profile observed with HIV-1.
Asunto(s)
Magnesio/metabolismo , ADN Polimerasa Dirigida por ARN/genética , Retroviridae/genética , ADN Viral/biosíntesis , ADN Viral/genética , Farmacorresistencia Viral/genética , Magnesio/análisis , Mutación , Tasa de Mutación , ADN Polimerasa Dirigida por ARN/metabolismo , Retroviridae/clasificación , Retroviridae/enzimologíaRESUMEN
Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP-) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP-16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP-16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TP- genotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TP- within chromatin profile states associated with weakly transcribed heterochromatin with fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TP- showed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein.
Asunto(s)
Carcinogénesis , Vectores Genéticos/toxicidad , Leucemia Experimental , Infecciones por Retroviridae , Infecciones Tumorales por Virus , Animales , Cromatina , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Genes myc , Humanos , Integrasas/metabolismo , Células K562 , Virus de la Leucemia Murina/genética , Ratones , Ratones Transgénicos , Integración ViralRESUMEN
DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified systematic evolution of ligands by exponential enrichment (SELEX) approach. Two Taq-specific primers that bound â¼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. TaqI contained 8 nucleotides (5'-CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCRs. Similarly, exonuclease- Klenow polymerase also selected a high-affinity primer that contained a related core promoter sequence from phage T7 RNAP (5'-ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity, suggesting that binding was highly sequence specific. The results are discussed in the context of possible effects on multiprimer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs.IMPORTANCE This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function or be a consequence of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered, and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promoters. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Virales/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Evolución Molecular , Cinética , Ligandos , Reacción en Cadena de la Polimerasa , Técnica SELEX de Producción de Aptámeros , Especificidad por SustratoRESUMEN
Reverse transcriptases (RTs) are typically assayed in vitro with 5-10 mM Mg2+, whereas the free Mg2+ concentration in cells is much lower. Artificially high Mg2+ concentrations used in vitro can misrepresent different properties of human immunodeficiency virus (HIV) RT, including fidelity, catalysis, pausing, and RNase H activity. Here, we analyzed nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in primer extension assays at different concentrations of free Mg2+. At low concentrations of Mg2+, NRTIs and dideoxynucleotides (AZTTP, ddCTP, ddGTP, and 3TCTP) inhibited HIV-1 and HIV-2 RT synthesis less efficiently than they did with large amounts of Mg2+, whereas inhibition by the "translocation-defective RT inhibitor" EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) was unaffected by Mg2+ concentrations. Steady-state kinetic analyses revealed that the reduced level of inhibition at low Mg2+ concentrations resulted from a 3-9-fold (depending on the particular nucleotide and inhibitor) less efficient incorporation (based on kcat/Km) of these NRTIs under this condition compared to incorporation of natural dNTPs. In contrast, EFdATP was incorporated with an efficiency similar to that of its analogue dATP at low Mg2+ concentrations. Unlike NRTIs, NNRTIs (nevirapine, efavirenz, and rilviripine), were approximately 4-fold (based on IC50 values) more effective at low than at high Mg2+ concentrations. Drug-resistant HIV-1 RT mutants also displayed the Mg2+-dependent difference in susceptibility to NRTIs and NNRTIs. In summary, analyzing the efficiency of inhibitors under more physiologically relevant low-Mg2+ conditions yielded results dramatically different from those from measurements using commonly employed high-Mg2+ in vitro conditions. These results also emphasize differences in Mg2+ sensitivity between the translocation inhibitor EFdATP and other NRTIs.
Asunto(s)
Didesoxinucleótidos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Magnesio/farmacología , Nucleósidos/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Nucleótidos de Desoxicitosina/farmacología , Nucleótidos de Desoxiguanina/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroforesis en Gel de Poliacrilamida , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Humanos , Cinética , Mutación , Nucleótidos de Timina/farmacología , Zalcitabina/farmacología , Zidovudina/análogos & derivados , Zidovudina/farmacologíaRESUMEN
UNLABELLED: The fidelity of human immunodeficiency virus (HIV) reverse transcriptase (RT) has been a subject of intensive investigation. The mutation frequencies for the purified enzyme in vitro vary widely but are typically in the 10(-4) range (per nucleotide addition), making the enzyme severalfold less accurate than most polymerases, including other RTs. This has often been cited as a factor in HIV's accelerated generation of genetic diversity. However, cellular experiments suggest that HIV does not have significantly lower fidelity than other retroviruses and shows a mutation frequency in the 10(-5) range. In this report, we reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results from experiments conducted with physiological (for lymphocytes) concentrations of free Mg(2+) (~0.25 mM) and is comparable to Moloney murine leukemia virus (MuLV) RT fidelity. The physiological conditions produced mutation rates that were 5 to 10 times lower than those obtained under typically employed in vitro conditions optimized for RT activity (5 to 10 mM Mg(2+)). These results were consistent in both commonly used lacZα complementation and steady-state fidelity assays. Interestingly, although HIV RT showed severalfold-lower fidelity under high-Mg(2+) (6 mM) conditions, MuLV RT fidelity was insensitive to Mg(2+). Overall, the results indicate that the fidelity of HIV replication in cells is compatible with findings of experiments carried out in vitro with purified HIV RT, providing more physiological conditions are used. IMPORTANCE: Human immunodeficiency virus rapidly evolves through the generation and subsequent selection of mutants that can circumvent the immune response and escape drug therapy. This process is fueled, in part, by the presumably highly error-prone HIV polymerase reverse transcriptase (RT). Paradoxically, results of studies examining HIV replication in cells indicate an error frequency that is ~10 times lower than the rate for RT in the test tube, which invokes the possibility of factors that make RT more accurate in cells. This study brings the cellular and test tube results in closer agreement by showing that HIV RT is not more error prone than other RTs and, when assayed under physiological magnesium conditions, has a much lower error rate than in typical assays conducted using conditions optimized for enzyme activity.
Asunto(s)
Cationes Bivalentes/metabolismo , Coenzimas/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Magnesio/metabolismo , Transcripción ReversaRESUMEN
BACKGROUND: Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg(2+) is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn(2+), Co(2+), and Zn(2+) can also support catalysis. Although Zn(2+) supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn(2+) is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn(2+). METHODS: We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn(2+), Co(2+), and Zn(2+). RESULTS: The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn(2+) when compared to Mg(2+) at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3' nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn(2+) than Mg(2+). In agreement with previous literature, we observed that Mn(2+) and Co(2+) dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn(2+) and Co(2+) remained similar to Mg(2+) at lower concentrations that are optimal for catalysis. CONCLUSION: This study shows that Zn(2+), at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.
Asunto(s)
Cationes Bivalentes/farmacología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cobalto/farmacología , Transcriptasa Inversa del VIH/química , Cinética , Operón Lac/genética , Manganeso/farmacología , Datos de Secuencia Molecular , Mutagénesis/efectos de los fármacos , Mutación , Zinc/farmacologíaRESUMEN
HIV-1 entry into host cells leads to one of the following three alternative fates: (1) HIV-1 elimination by restriction factors, (2) establishment of HIV-1 latency, or (3) active viral replication in target cells. Here, we report the development of an improved system for monitoring HIV-1 fate at single-cell and population levels and show the diverse applications of this system to study specific aspects of HIV-1 fate in different cell types and under different environments. An analysis of the transcriptome of infected, primary CD4+ T cells that support alternative fates of HIV-1 identifies differential gene expression signatures in these cells. Small molecules are able to selectively target cells that support viral replication with no significant effect on viral latency. In addition, HIV-1 fate varies in different tissues following infection of humanized mice in vivo. Altogether, these studies indicate that intra- and extra-cellular environments contribute to the fate of HIV-1 infection.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Microambiente Celular , Infecciones por VIH/virología , VIH-1/patogenicidad , Animales , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/inmunología , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , VIH-1/inmunología , Interacciones Huésped-Patógeno , Humanos , Ratones Endogámicos NOD , Ratones SCID , Células THP-1 , Transcriptoma , Internalización del Virus , Latencia del Virus , Replicación ViralRESUMEN
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20152-w.
RESUMEN
The early steps of HIV-1 infection, such as uncoating, reverse transcription, nuclear import, and transport to integration sites are incompletely understood. Here, we imaged nuclear entry and transport of HIV-1 replication complexes in cell lines, primary monocyte-derived macrophages (MDMs) and CD4+ T cells. We show that viral replication complexes traffic to and accumulate within nuclear speckles and that these steps precede the completion of viral DNA synthesis. HIV-1 transport to nuclear speckles is dependent on the interaction of the capsid proteins with host cleavage and polyadenylation specificity factor 6 (CPSF6), which is also required to stabilize the association of the viral replication complexes with nuclear speckles. Importantly, integration site analyses reveal a strong preference for HIV-1 to integrate into speckle-associated genomic domains. Collectively, our results demonstrate that nuclear speckles provide an architectural basis for nuclear homing of HIV-1 replication complexes and subsequent integration into associated genomic loci.
Asunto(s)
Infecciones por VIH/virología , VIH-1/patogenicidad , Linfocitos T CD4-Positivos/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Genoma Viral/genética , Células HEK293 , Infecciones por VIH/genética , VIH-1/genética , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , Microscopía Fluorescente , Virología , Integración Viral/genética , Integración Viral/fisiología , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
Lentiviral DNA integration favors transcriptionally active chromatin. We previously showed that the interaction of human immunodeficiency virus type 1 (HIV-1) capsid with cleavage and polyadenylation specificity factor 6 (CPSF6) localizes viral preintegration complexes (PICs) to nuclear speckles for integration into transcriptionally active speckle-associated domains (SPADs). In the absence of the capsid-CPSF6 interaction, PICs uncharacteristically accumulate at the nuclear periphery and target heterochromatic lamina-associated domains (LADs) for integration. The integrase-binding protein lens epithelium-derived growth factor (LEDGF)/p75 in contrast to CPSF6 predominantly functions to direct HIV-1 integration to interior regions of transcription units. Though CPSF6 and LEDGF/p75 can reportedly interact with the capsid and integrase proteins of both primate and nonprimate lentiviruses, the extents to which these different viruses target SPADs versus LADs, as well as their dependencies on CPSF6 and LEDGF/p75 for integration targeting, are largely unknown. Here, we mapped 5,489,157 primate and nonprimate lentiviral integration sites in HEK293T and Jurkat T cells as well as derivative cells that were knocked out or knocked down for host factor expression. Despite marked preferences of all lentiviruses to target genes for integration, nonprimate lentiviruses only marginally favored SPADs, with corresponding upticks in LAD-proximal integration. While LEDGF/p75 knockout disrupted the intragenic integration profiles of all lentiviruses similarly, CPSF6 depletion specifically counteracted SPAD integration targeting by primate lentiviruses. CPSF6 correspondingly failed to appreciably interact with nonprimate lentiviral capsids. We conclude that primate lentiviral capsid proteins evolved to interact with CPSF6 to optimize PIC localization for integration into transcriptionally active SPADs.IMPORTANCE Integration is the defining step of the retroviral life cycle and underlies the inability to cure HIV/AIDS through the use of intensified antiviral therapy. The reservoir of latent, replication-competent proviruses that forms early during HIV infection reseeds viremia when patients discontinue medication. HIV cure research is accordingly focused on the factors that guide provirus formation and associated chromatin environments that regulate transcriptional reactivation, and studies of orthologous infectious agents such as nonprimate lentiviruses can inform basic principles of HIV biology. HIV-1 utilizes the integrase-binding protein LEDGF/p75 and the capsid interactor CPSF6 to target speckle-associated domains (SPADs) for integration. However, the extent to which these two host proteins regulate integration of other lentiviruses is largely unknown. Here, we mapped millions of retroviral integration sites in cell lines that were depleted for LEDGF/p75 and/or CPSF6. Our results reveal that primate lentiviruses uniquely target SPADs for integration in a CPSF6-dependent manner.
Asunto(s)
Lentivirus/genética , Primates/genética , Integración Viral/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Animales , Gatos/genética , Gatos/virología , Bovinos/genética , Bovinos/virología , Línea Celular , Evolución Molecular , Células HEK293 , Caballos/genética , Caballos/virología , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Células Jurkat , Macaca mulatta/genética , Macaca mulatta/virología , Ratones/genética , Ratones/virología , Primates/virología , Replicación ViralRESUMEN
HIV-1 integration favors active chromatin, which is primarily mediated through interactions between the viral capsid and integrase proteins with host factors cleavage and polyadenylation specificity factor 6 (CPSF6) and lens epithelium-derived growth factor/p75, respectively. Previously published image-based studies had suggested that HIV-1 prefers to integrate into chromatin that associates spatially with the nuclear periphery. Here, we re-evaluated previously reported HIV-1 nuclear distance measures across studies and show that HIV-1 prefers peri-nuclear and mid-nuclear zones similarly, with a common preference between studies mapping to the boundary between these two radial areas. We also discuss emerging roles for the capsid-CPSF6 interaction in facilitating HIV-1 pre-integration complex nuclear import and subsequent intranuclear trafficking to preferred sites of viral DNA integration.
RESUMEN
HIV-1 integration into the host genome favors actively transcribed genes. Prior work indicated that the nuclear periphery provides the architectural basis for integration site selection, with viral capsid-binding host cofactor CPSF6 and viral integrase-binding cofactor LEDGF/p75 contributing to selection of individual sites. Here, by investigating the early phase of infection, we determine that HIV-1 traffics throughout the nucleus for integration. CPSF6-capsid interactions allow the virus to bypass peripheral heterochromatin and penetrate the nuclear structure for integration. Loss of interaction with CPSF6 dramatically alters virus localization toward the nuclear periphery and integration into transcriptionally repressed lamina-associated heterochromatin, while loss of LEDGF/p75 does not significantly affect intranuclear HIV-1 localization. Thus, CPSF6 serves as a master regulator of HIV-1 intranuclear localization by trafficking viral preintegration complexes away from heterochromatin at the periphery toward gene-dense chromosomal regions within the nuclear interior.
Asunto(s)
Cápside/metabolismo , Núcleo Celular/virología , ADN Viral/genética , Infecciones por VIH/metabolismo , VIH-1/fisiología , Integración Viral , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , ADN Viral/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Unión Proteica , Replicación Viral , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.
RESUMEN
Steady state kinetic assays have been a reliable way to estimate fidelity of several polymerases (Menendez-Arias, 2009; Rezende and Prasad, 2004; Svarovskaia et al., 2003). The ability to analyze the extension of primers with specific mismatches at the 3' end is a major strength of the mismatched primer extension assays. Recently, we used the mismatched primer extension assays to show that the fidelity of HIV RT increases dramatically when concentration of Mg2+ is reduced to a physiologically relevant concentration (~0.25 mM) (Achuthan et al., 2014). Here, we describe in detail how to perform the mismatched primer extension assay to measure the standard extension efficiency using human immunodeficiency virus reverse transcriptase (HIV RT) at 2 mM Mg2+. The relative fidelity of the polymerase can then be estimated using the standard extension efficiency. The assay described here is based on the method published in Mendelman et al. (1990).