Assessment of the Role of the Central DNA Flap in Human Immunodeficiency Virus Type 1 Replication by Using a Single-Cycle Replication System

Ao, Zhujun; Yao, Xiaojian; Cohen, Éric A.

doi:10.1128/jvi.78.6.3170-3177.2004

Cited by 47 publications

(65 citation statements)

References 46 publications

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“…Lentiviral HIV-1 and FIV INs depend on LEDGF for nuclear localization and protection from ubiquitin-proteasome, while the IN of the oncoretrovirus MoMLV does not, although all of them can be co-immunoprecipitated with LEDGF (Llano et al 2004a). Although HIV-1 IN contains a basic bipartite-type NLS at residues 186-188 and 211-219 and an atypical NLS in the CCD at 161-173 (Tsurutani et al 2000;Bouyac-Bertoia et al 2001;Ao et al 2004), its dispensable role in nuclear trafficking might be attributed . Similarly, NLS properties were also found in the arginine-and lysine-rich C-terminus of foamy virus IN (Hossain et al 2014).…”

Section: Integrasementioning

confidence: 99%

“…The import mediated by the IN CTD-importin 7 interaction is specific for digitonin-permeabilized cell systems. A reduction in HIV-1 infectivity was observed in HeLa and glioma cells (Fassati et al 2003;Bukrinsky 2004;Ao et al 2004). While slipping through the NPC, IN can also bind with NUP153 (Woodward et al 2009) and NUP62 (Ao et al 2012).…”

Section: Integrasementioning

confidence: 99%

“…However DNA flapdefective viral genomes accumulate on the cytoplasmic side of the nuclear membrane (Arhel et al 2006). Flap may have a protective role in viral cDNA stability, based on the observation that PIC collected at 10 h, but not that from earlier samples, is resistant to DNase I digestion (Ao et al 2004). However, both cPPT and CTS are essential for a functional central DNA flap, as viral infectivity and gene transfer are impaired in mutants.…”

Section: Central Dna Flapmentioning

confidence: 99%

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Cellular and viral determinants of retroviral nuclear entry

2016

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Retroviruses must integrate their cDNA into the host genome to generate proviruses. Viral DNA-protein complexes interact with cellular proteins and produce pre-integration complexes, which carry the viral genome and cross the nuclear pore channel to enter the nucleus and integrate viral DNA into host chromosomal DNA. If the reverse transcripts fail to integrate, linear or circular DNA species such as 1-and 2-long terminal repeats are generated. Such complexes encounter numerous cellular proteins in the cytoplasm, which restrict viral infection and protect the nucleus. To overcome host cell defenses, the pathogens have evolved several evasion strategies. Viral proteins often contain nuclear localization signals, allowing entry into the nucleus. Among more than 1000 proteins identified as required for HIV infection by RNA interference screening, karyopherins, cleavage and polyadenylation specific factor 6, and nucleoporins have been predominantly studied. This review discusses current opinions about the synergistic relationship between the viral and cellular factors involved in nuclear import, with focus on the unveiled mysteries of the host-pathogen interaction, and highlights novel approaches to pinpoint therapeutic targets.

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Section: Integrasementioning

confidence: 99%

Section: Integrasementioning

confidence: 99%

Section: Central Dna Flapmentioning

confidence: 99%

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Cellular and viral determinants of retroviral nuclear entry

2016

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“…1) (4 -7). Although central flap function remains controversial (8,9), its mutation or deletion has been shown to impair virus replication (4,10,11), and it improves transduction efficiency when incorporated into lentiviral vectors (12)(13)(14)(15)(16).…”

Section: Hiv-1 Dna Synthesismentioning

confidence: 99%

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Grice

2012

Journal of Biological Chemistry

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Synthesis of integration-competent, double-stranded DNA from the (؉)-RNA strand genome of retroviruses and long terminal repeat-containing retrotransposons reflects a multistep process catalyzed by the virus-encoded reverse transcriptase (RT). In conjunction with RNA-and DNA-templated DNA synthesis, a hydrolytic activity of the same enzyme (RNase H) is required to remove genomic RNA of the RNA/DNA replication intermediate. Together, these combined synthetic and degradative functions ensure correct selection, extension, and removal of the RNA primers of (؊)-and (؉)-strand DNA synthesis (tRNA and the polypurine tract, respectively). For HIV-1 RT, a quarter century of research has not only illuminated the biochemical properties, structure, and conformational dynamics of this highly versatile enzyme but has also witnessed drug discovery advances from the first Food and Drug Administration-approved anti-RT drug to recent use of RT inhibitors as potential colorectal microbicides. Salient features of HIV-1 RT and extension of these findings into programs of drug discovery are reviewed here. HIV-1 DNA SynthesisThe individual steps of HIV-1 DNA synthesis, catalyzed by the multifunctional reverse transcriptase (RT), 2 are summarized schematically in Fig. 1. (Ϫ)-Strand DNA synthesis, initiated from a cellular tRNA (tRNA 3 Lys ) hybridized to the genomeencoded primer-binding site, continues to the 5Ј terminus, creating (Ϫ)-strong-stop DNA. RNase H-mediated degradation of the resulting RNA/DNA hybrid promotes relocation of nascent (Ϫ)-DNA to the genome 3Ј terminus by a strand transfer event that exploits sequence homology between the 5Ј and 3Ј termini. RNA-templated DNA synthesis continues, accompanied by RNase H-mediated degradation of the RNA genome, the exception to which are two short purine-rich segments (the 3Ј-and central polypurine tracts (PPTs)) from which (ϩ)-strand DNA-dependent DNA synthesis is initiated. Newly synthesized (Ϫ)-strand DNA and 18 nucleotides of the covalently attached tRNA 3 Lys primer provide the template for 3Ј-PPTprimed (ϩ)-strand DNA synthesis until the replication complex stalls at a position corresponding to the first modified tRNA base (A57). As a consequence, the C-terminal RNase H domain is positioned at the (Ϫ)-DNA/tRNA junction, and degradation of the tRNA "template" promotes a second or (ϩ)-strand transfer event supported by homology between (Ϫ)-and (ϩ)-strand DNA primer-binding sites. Although bidirectional DNA synthesis would be sufficient to complete DNA synthesis, HIV utilizes a second, central PPT primer, thereby producing a (ϩ)-strand discontinuity (1). Following (ϩ)-strand transfer, 3Ј-PPT-mediated DNA synthesis continues, displacing ϳ100 nucleotides of central PPT-primed (ϩ)-DNA, but abruptly ceases at the central termination sequence, a prominent feature of which is phased A-tracts that induce minor groove compression (1-3), creating the "central flap" (Fig. 1) (4 -7). Although central flap function remains controversial (8, 9), its mutation or deletion has been shown to impair...

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“…Mutations in the cPPT have been reported to decrease the efficiency of HIV-1 replication or Ty1 retrotransposition, though defects in HIV-1 replication are not always observed (48). There are also reports that the central DNA flap of HIV-1 is important for nuclear import of the preintegration complex in nondividing cells (2,87). In addition, inclusion of the cPPT and CTS usually stimulates the transduction efficiency of lentiviral vectors severalfold (4,18,69,88).…”

mentioning

confidence: 99%

Retrotransposon Suicide: Formation of Ty1 Circles and Autointegration via a Central DNA Flap

Garfinkel

Stefanisko

Nyswaner

et al. 2006

J Virol

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Despite their evolutionary distance, the Saccharomyces cerevisiae retrotransposon Ty1 and retroviruses use similar strategies for replication, integration, and interactions with their hosts. Here we examine the formation of circular Ty1 DNA, which is comparable to the dead-end circular products that arise during retroviral infection. Appreciable levels of circular Ty1 DNA are present with one-long terminal repeat (LTR) circles and deleted circles comprising major classes, while two-LTR circles are enriched when integration is defective. One-LTR circles persist when homologous recombination pathways are blocked by mutation, suggesting that they result from reverse transcription. Ty1 autointegration events readily occur, and many are coincident with and dependent upon DNA flap structures that result from DNA synthesis initiated at the central polypurine tract. These results suggest that Ty1-specific mechanisms minimize copy number and raise the possibility that special DNA structures are a targeting determinant.In addition to undergoing transpositional integration, the linear DNA synthesized by reverse transcription of the long terminal repeat (LTR) retrotransposon Ty1 and retrovirus RNA has multiple fates which can modulate the efficiency of integration into the host genome (75,76,80,82). Reverse transcription is a sequential process with specific requirements for priming and strand transfer steps. Specific host tRNAs prime minus-strand reverse transcription, and purine-rich fragments of retroelement RNA (polypurine tracts [PPTs]), which are relatively resistant to the RNase H activity of reverse transcriptase (RT), prime plus-strand synthesis. The minus strand is synthesized first, followed by plus-strand synthesis using the minus strand as a template. Reverse transcription generates a linear double-stranded cDNA that is a substrate for the element-encoded integrase (IN) protein, which inserts the linear DNA into the host genome and creates a target site duplication (TSD) in the process. Both reverse transcription and integration are essential steps in the life cycle of retroelements.Several lentiviruses, including human immunodeficiency virus type 1 (HIV-1) (13-15) and equine infectious anemia virus (71), as well as the retrotransposon Ty1 (30, 31), have two PPTs, one located just upstream of the 3Ј LTR boundary (PPT) and the second near the center of the element (cPPT) (Fig. 1A). A central DNA flap is created when the 5Ј end of the plus strand initiated at the cPPT is displaced by the 3Ј end of plus-strand DNA after strand transfer (Fig. 1B) The essential role of the PPT in reverse transcription is well established; however, the role of the cPPT is somewhat controversial. Mutations in the cPPT have been reported to decrease the efficiency of HIV-1 replication or Ty1 retrotransposition, though defects in HIV-1 replication are not always observed (48). There are also reports that the central DNA flap of HIV-1 is important for nuclear import of the preintegration complex in nondividing cells (2, 87). In addition,...

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Assessment of the Role of the Central DNA Flap in Human Immunodeficiency Virus Type 1 Replication by Using a Single-Cycle Replication System

Abstract: In this study, reverse transcriptase (RT)-and integrase (IN)-

Cited by 47 publications

References 46 publications

Cellular and viral determinants of retroviral nuclear entry

Cellular and viral determinants of retroviral nuclear entry

Human Immunodeficiency Virus Reverse Transcriptase: 25 Years of Research, Drug Discovery, and Promise

Retrotransposon Suicide: Formation of Ty1 Circles and Autointegration via a Central DNA Flap

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