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Classical DNA and RNA polymerase (pol) enzymes have defined roles with their respective substrates, but several pols have been found to have multiple functions. We reported previously that purified human DNA pol η (hpol η) can incorporate both deoxyribonucleoside triphosphates (dNTPs) and ribonucleoside triphosphates (rNTPs) and can use both DNA and RNA as substrates. X-ray crystal structures revealed that two pol η residues, Phe-18 and Tyr-92, behave as steric gates to influence sugar selectivity. However, the physiological relevance of these phenomena has not been established. Here, we show that purified hpol η adds rNTPs to DNA primers at physiological rNTP concentrations and in the presence of competing dNTPs. When two rATPs were inserted opposite a cyclobutane pyrimidine dimer, the substrate was less efficiently cleaved by human RNase H2. Human XP-V fibroblast extracts, devoid of hpol η, could not add rNTPs to a DNA primer, but the expression of transfected hpol η in the cells restored this ability. XP-V cell extracts did not add dNTPs to DNA primers hybridized to RNA, but could when hpol η was expressed in the cells. HEK293T cell extracts could add dNTPs to DNA primers hybridized to RNA, but lost this ability if hpol η was deleted. Interestingly, a similar phenomenon was not observed when other translesion synthesis (TLS) DNA polymerases-hpol ι, κ, or ζ-were individually deleted. These results suggest that hpol η is one of the major reverse transcriptases involved in physiological processes in human cells.
© 2019 Su et al.
Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η .
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
The RNA genome of human immunodeficiency virus type 1 (HIV-1) is enclosed by a capsid shell that dissociates within the cell in a multistep process known as uncoating, which influences completion of reverse transcription of the viral genome. Double-stranded viral DNA is imported into the nucleus for integration into the host genome, a hallmark of retroviral infection. Reverse transcription, nuclear entry, and integration are coordinated by a capsid uncoating process that is regulated by cellular proteins. Although uncoating is not well understood, recent studies have revealed insights into the process, particularly with respect to nuclear import pathways and protection of the viral genome from DNA sensors. Understanding uncoating will be valuable toward developing novel antiretroviral therapies for HIV-infected individuals.
Copyright © 2014 Elsevier Inc. All rights reserved.
BACKGROUND - Retroviral integrase catalyzes integration of viral DNA into the host genome. Integrase interactor (INI)1/hSNF5 is a host factor that binds to HIV-1 IN within the context of Gag-Pol and is specifically incorporated into HIV-1 virions during assembly. Previous studies have indicated that INI1/hSNF5 is required for late events in vivo and for integration in vitro. To determine the effects of disrupting the IN-INI1 interaction on the assembly and infectivity of HIV-1 particles, we isolated mutants of IN that are defective for binding to INI1/hSNF5 and tested their effects on HIV-1 replication.
RESULTS - A reverse yeast two-hybrid system was used to identify INI1-interaction defective IN mutants (IID-IN). Since protein-protein interactions depend on the surface residues, the IID-IN mutants that showed high surface accessibility on IN crystal structures (K71R, K111E, Q137R, D202G, and S147G) were selected for further study. In vitro interaction studies demonstrated that IID-IN mutants exhibit variable degrees of interaction with INI1. The mutations were engineered into HIV-1(NL4-3) and HIV-Luc viruses and tested for their effects on virus replication. HIV-1 harboring IID-IN mutations were defective for replication in both multi- and single-round infection assays. The infectivity defects were correlated to the degree of INI1 interaction of the IID-IN mutants. Highly defective IID-IN mutants were blocked at early and late reverse transcription, whereas partially defective IID-IN mutants proceeded through reverse transcription and nuclear localization, but were partially impaired for integration. Electron microscopic analysis of mutant particles indicated that highly interaction-defective IID-IN mutants produced morphologically aberrant virions, whereas the partially defective mutants produced normal virions. All of the IID-IN mutant particles exhibited normal capsid stability and reverse transcriptase activity in vitro.
CONCLUSIONS - Our results demonstrate that a severe defect in IN-INI1 interaction is associated with production of defective particles and a subsequent defect in post-entry events. A partial defect in IN-INI1 interaction leads to production of normal virions that are partially impaired for early events including integration. Our studies suggest that proper interaction of INI1 with IN within Gag-Pol is necessary for proper HIV-1 morphogenesis and integration.
Human immunodeficiency virus type 1 (HIV-1) infection is dependent on the proper disassembly of the viral capsid, or "uncoating," in target cells. The HIV-1 capsid consists of a conical multimeric complex of the viral capsid protein (CA) arranged in a hexagonal lattice. Mutations in CA that destabilize the viral capsid result in impaired infection owing to defects in reverse transcription in target cells. We describe here the mechanism of action of a small molecule HIV-1 inhibitor, PF-3450074 (PF74), which targets CA. PF74 acts at an early stage of HIV-1 infection and inhibits reverse transcription in target cells. We show that PF74 binds specifically to HIV-1 particles, and substitutions in CA that confer resistance to the compound prevent binding. A single point mutation in CA that stabilizes the HIV-1 core also conferred strong resistance to the virus without inhibiting compound binding. Treatment of HIV-1 particles or purified cores with PF74 destabilized the viral capsid in vitro. Furthermore, the compound induced the rapid dissolution of the HIV-1 capsid in target cells. PF74 antiviral activity was promoted by binding of the host protein cyclophilin A to the HIV-1 capsid, and PF74 and cyclosporine exhibited mutual antagonism. Our data suggest that PF74 triggers premature HIV-1 uncoating in target cells, thereby mimicking the activity of the retrovirus restriction factor TRIM5α. This study highlights uncoating as a step in the HIV-1 life cycle that is susceptible to small molecule intervention.
BACKGROUND - Complementary approaches to assaying global gene expression are needed to assess gene expression in regions that are poorly assayed by current methodologies. A key component of nearly all gene expression assays is the reverse transcription of transcribed sequences that has traditionally been performed by priming the poly-A tails on many of the transcribed genes in eukaryotes with oligo-dT, or by priming RNA indiscriminately with random hexamers. We designed an algorithm to find common sequence motifs that were present within most protein-coding genes of Saccharomyces cerevisiae and of Neurospora crassa, but that were not present within their ribosomal RNA or transfer RNA genes. We then experimentally tested whether degenerately priming these motifs with multi-targeted primers improved the accuracy and completeness of transcriptomic assays.
RESULTS - We discovered two multi-targeted primers that would prime a preponderance of genes in the genomes of Saccharomyces cerevisiae and Neurospora crassa while avoiding priming ribosomal RNA or transfer RNA. Examining the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development, we demonstrated that using multi-targeted primers in reverse transcription led to superior performance of microarray profiling and next-generation RNA tag sequencing. Priming with multi-targeted primers in addition to oligo-dT resulted in higher sensitivity, a larger number of well-measured genes and greater power to detect differences in gene expression.
CONCLUSIONS - Our results provide the most complete and detailed expression profiles of the yeast nitrogen starvation response and N. crassa early sexual development to date. Furthermore, our multi-targeting priming methodology for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences, facilitating a more complete and precise assay of the transcribed sequences within the genome.
The host protein TRIM5alpha inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5alpha. Here, we show that TRIM5alpha is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5alpha-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5alpha protein but not the nonrestrictive human TRIM5alpha protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5alpha was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV) but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5alpha and TRIMCyp proteins. We also detected degradation of endogenous TRIM5alpha in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5alpha degradation by a proteasome-dependent mechanism.