Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown

Galvis, Alvaro E.; Fisher, Hugh E.; Nitta, Takayuki; Fan, Hung; Camerini, David

doi:10.1128/jvi.00704-14

Cited by 32 publications

(36 citation statements)

References 50 publications

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“…All structures except the apoenzyme are binuclear with a water/hydroxide bridging the Fe and Zn ions in optimal position to attack the scissile phosphate. The results resolve previous uncertainties regarding Dbr1 active site structure and the newly developed dark-quenched bRNA probe is facilitating the screening of large small molecule libraries for Dbr1 inhibitors that may hold promise as therapeutic agents for neurodegenerative diseases such as ALS (29) and for retroviral infections such as HIV (13,14,17).…”

Section: Significancementioning

confidence: 52%

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Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Intron lariats are circular, branched RNAs (bRNAs) produced during pre-mRNA splicing. Their unusual chemical and topological properties arise from branch-point nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages. The 2′,5′-bonds must be hydrolyzed by the RNA debranching enzyme Dbr1 before spliced introns can be degraded or processed into small nucleolar RNA and microRNA derived from intronic RNA. Here, we measure the activity of Dbr1 from Entamoeba histolytica by using a synthetic, dark-quenched bRNA substrate that fluoresces upon hydrolysis. Purified enzyme contains nearly stoichiometric equivalents of Fe and Zn per polypeptide and demonstrates turnover rates of ∼3 s −1. Similar rates are observed when apo-Dbr1 is reconstituted with Fe(II)+Zn(II) under aerobic conditions. Under anaerobic conditions, a rate of ∼4.0 s −1 is observed when apoenzyme is reconstituted with Fe(II). In contrast, apo-Dbr1 reconstituted with Mn(II) or Fe(II) under aerobic conditions is inactive. Diffraction data from crystals of purified enzyme using X-rays tuned to the Fe absorption edge show Fe partitions primarily to the β-pocket and Zn to the α-pocket. Structures of the catalytic mutant H91A in complex with 7-mer and 16-mer synthetic bRNAs reveal bona fide RNA branchpoints in the Dbr1 active site. A bridging hydroxide is in optimal position for nucleophilic attack of the scissile phosphate. The results clarify uncertainties regarding structure/function relationships in Dbr1 enzymes, and the fluorogenic probe permits high-throughput screening for inhibitors that may hold promise as treatments for retroviral infections and neurodegenerative disease.RNA debranching | intron lariat | enzyme kinetics | X-ray crystallography | Dbr1 T he enzymatic processing of diverse RNA molecules requires selective recognition of their unique physicochemical properties. The sequential trans-esterification reactions catalyzed by the spliceosome yield mature messenger RNA (mRNA) and excised intron lariats (1, 2), the latter of which contain internal branchpoint adenosine nucleotides harboring vicinal 2′,5′-and 3′,5′-phosphodiester linkages (3). Mature mRNA transcripts are exported to the cytosol for protein synthesis, but lariat introns must be linearized before they can be turned over or processed into the subset of small nucleolar RNAs and microRNAs that are derived from intronic RNA (4, 5). The lariat forms when the 2′-hydroxyl group of an adenosine nucleotide near the 3′-end of the intron acts as the nucleophile to attack the 5′-splice site, producing 5′-exon-3′-OH and intron lariat/ 3′-exon intermediates. The 3′-hydroxyl group of the 5′-exon-3′-OH intermediate subsequently acts as the nucleophile to attack the 3′-splice site, resulting in intron excision and exon ligation (6, 7) (Fig. 1A). The resulting vicinal 2′,5′-and 3′,5′-phosphodiester linkages confer unique topological and chemical features to the branchpoint and flanking nucleotides, and these lariats persist in yeast cells lacking active Dbr1 (RNA lariat debranching enzyme) ...

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Section: Significancementioning

confidence: 52%

“…gene expression was reported to significantly diminish retroviral replication in cells infected with the HIV (13,14). This observation is consistent with the evolutionarily conserved genetic structures and replication mechanisms of retrotransposons and retroviruses (15,16).…”

Section: Significancementioning

confidence: 99%

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Clark

Katolik

Roberts

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been reported that ssDNA is the predominant cytosolic DNA species during the first 4 h of HIV infection, while dsDNA is found later in the nuclear or perinuclear fractions 37 . Replication of HIV-1 is initiated by reverse transcription of the first ~181 nucleotides of the HIV-1 RNA genome, primed by lysyl-tRNA (tRNA Lys3 ), which generates sstDNA (Fig.…”

Section: Resultsmentioning

confidence: 99%

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

et al. 2015

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Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

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“…One possible explanation was that P70 env is found in the perinuclear region of the cytoplasm, which still may allow for interactions between nuclear Zfp111 and P70 env . A second cell fractionation procedure included washing of the nuclei with an ionic/nonionic detergent mixture, to give a perinuclear fraction separate from the rest of the cytoplasm (36,37). Western blot analysis revealed that P70 env was found exclusively in the nuclear fraction along with Zfp111 and not in the perinuclear fraction (Fig.…”

Section: P70mentioning

confidence: 99%

Role for a Zinc Finger Protein (Zfp111) in Transformation of 208F Rat Fibroblasts by Jaagsiekte Sheep Retrovirus Envelope Protein

Hsu

Phung

Choe

et al. 2015

J Virol

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The native envelope gene (env) of Jaagsiekte sheep retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation, we performed yeast 2-hybrid screening for cellular proteins that interact with Env. Among several candidates, we identified mouse or rat zinc finger protein 111 (zfp111). The interaction between Env and Zfp111 was confirmed through in vivo coimmunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Env, while overexpression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111. In addition, Zfp111 preferentially bound to a higher-mobility form of JSRV Env that has not been described previously. The higher-mobility form of Env (P70 env ) was found exclusively in the nuclear fraction, and size of its polypeptide backbone was the same as that of the cytoplasmic Env polyprotein (Pr80 env ). The differences in glycosylation between the two versions of Env were characterized. These results identify a novel cellular protein, Zfp111, that binds to the JSRV Env protein, and this binding plays a role in Env transformation. These results indicate that JSRV transformation also involves proteins and interactions in the nucleus. IMPORTANCEThe envelope protein (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncogene, but its mechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, zinc finger protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells and to be involved in JSRV transformation. Moreover, coexpression of JSRV Env and Zfp111 led to the identification of a novel nuclear form of the JSRV Env protein that binds Zfp111. Nuclear Env was found to differ by glycosylation from the cytoplasmic Env precursor to the virion envelope proteins. These results suggest that JSRV Env transformation may involve nuclear events such as an alteration in transcription mediated by Env-Zfp111 interactions. Jaagsiekte sheep retrovirus (JSRV) is a betaretrovirus that causes ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer in sheep that reflects malignant transformation of lung secretory epithelial cells (1, 2). OPA is morphologically similar to human lepidic adenocarcinoma, formerly known as bronchioloalveolar carcinoma (more recently designated adenocarcinoma in situ [AIS]) (3), a type of lung cancer that is less associated with cigarette smoke, and it is a good model for this type of human lung cancer (4-6).JSRV is a simple retrovirus that contains the standard retroviral genes gag, pro, pol, and env (7). While JSRV does not carry a transduced cellular oncogene, in experimental inoculation o...

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Impairment of HIV-1 cDNA Synthesis by DBR1 Knockdown

Cited by 32 publications

References 50 publications

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Metal dependence and branched RNA cocrystal structures of the RNA lariat debranching enzyme Dbr1

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

Role for a Zinc Finger Protein (Zfp111) in Transformation of 208F Rat Fibroblasts by Jaagsiekte Sheep Retrovirus Envelope Protein

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