Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginineserine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran-and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.T he transport of macromolecules between cytoplasm and nucleus is orchestrated by a family of nuclear import and export receptors (1). Referred to as importins and exportins, these proteins bind their specific cargoes and translocate them across the nuclear pore complex. The process is regulated by the small GTPase Ran that partitions between cytoplasm and nucleus in the predominantly GDP-and GTP-bound form, respectively. Importins associate with their cargoes in the cytoplasm, and the competitive binding of RanGTP induces them to release their cargoes in the nucleus (2). Most nuclear import/export receptors belong to the β-karyopherin family of proteins, with 22 members encoded in the human genome (3). The majority of β-karyopherins bind their cargoes directly, recognizing a linear nuclear localization or export signal and/or a specific tertiary/quaternary structural feature (4, 5).A fundamentally important type of a nuclear localization signal (NLS), comprising sequences rich in Arg-Ser and/or ArgAsp/Glu/Gly dipeptides (referred to as RS or RS-like domains), belongs to the family of Ser/Arg-rich (SR) proteins. These nuclear proteins also contain RNA recognition motif (RRM) domains and play essential roles in pre-mRNA splicing and 3′ processing and participate in transcription regulation, mRNA transport, translation, and nonsense-mediated mRNA decay (6). The splicing factors alternative splicing factor/splicing factor 2 (ASF/SF2) and SC35 along with the cleavage and polyadenylation specificity factor 6 (CPSF6, also known as CF-Im-68) are among the best-characterized metazoan SR proteins (7-10). The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12-16). Phosphorylation of SR proteins is thought to...

BackgroundCorrect disassembly of the HIV-1 capsid shell, called uncoating, is increasingly recognised as central for multiple steps during retroviral replication. However, the timing, localisation and mechanism of uncoating are poorly understood and progress in this area is hampered by difficulties in measuring the process. Previous work suggested that uncoating occurs soon after entry of the viral core into the cell, but recent studies report later uncoating, at or in the nucleus. Furthermore, inhibiting reverse transcription delays uncoating, linking these processes. ResultsHere, we have used a combined approach of experimental interrogation of viral mutants and mathematical modelling to investigate the timing of uncoating with respect to reverse transcription. By developing a minimal, testable, model and employing multiple uncoating assays to overcome the disadvantages of each single assay, we find that uncoating is not concomitant with the initiation of reverse transcription. Instead, uncoating appears to be triggered once reverse transcription reaches a certain stage, namely shortly after first strand transfer.ConclusionsUsing multiple approaches, we have identified a point during reverse transcription that induces uncoating of the HIV-1 CA shell. We propose that uncoating initiates after the first strand transfer of reverse transcription.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0292-7) contains supplementary material, which is available to authorized users.

]. DNA-damaging agents, such as ionizing radiation and chemical compounds generating replication-blocking lesions like cis-platinum, also cause DSBs. Retroviral integration involving a cut of the genomic DNA can also be considered a potentially mutagenic event. The cellular DNA repair machinery could thus be required either for cell defense against viral infection or, inversely, for the stable integration of the viral genome. Since these machineries are not completely defined at present, they require better characterization. Retroviral integration is catalyzed by the virally encoded integrase (IN) in concert with other viral and cellular factors forming the preintegration complex (PIC). As a final step, stable integration of retroviral DNA requires the repair of the DNA gaps remaining after the reaction catalyzed by IN. Several cellular repair mechanisms, including base excision repair (3, 14, 43, 44), homologous recombination (HR) (19), and nonhomologous end-joining (NHEJ) (9-11, 26), are thought to be involved in the integration step. Direct interactions between HIV-1 IN and human RAD18 have been reported (31), and cell-based studies have indicated that RAD18 and RAD52 can inhibit HIV-1 infection (22,27). These data suggest that cellular RAD DNA repair machineries can play a dual role by preventing or restricting retroviral viral replication/parasitism and/or participating directly in the stability of the integrated viral DNA, a crucial step of the infection process.Human RAD51 (hRAD51), belonging to the HR DNA repair RAD52 epistasis group, has been previously shown to interact functionally with HIV-1 IN both in vitro and in a yeast integration model (12). A downregulation of HIV-1 IN activity by hRAD51 was also reported in in vitro integration assays, but the inhibition mechanism remained unsolved. Since the enhancement of this inhibition could constitute a new antiviral approach as well as signify an original restriction pathway, we focused our work here on the molecular mechanism underlying this process both in vitro and in vivo. We especially studied whether this hRAD51-mediated IN inhibition could be promoted in HIV-1-infected cells.Using standard in vitro integration assays under conditions promoting or inhibiting hRAD51 activity (hRAD51 mutants, presence or absence of ATP, addition of a hRAD51 stimulatory compound), we show that the formation of an active hRAD51 presynaptic nucleofilament corresponding to the nucleocomplex formed on the two homologous DNA strands during HR is required for inhibition. An IN-DNA dissociation system was also set up in order to better determine the effect of the hRAD51 nucleofilament on the active integration complex. Using this system we show that this nucleofilament can dissociate IN from its substrate. Finally, the availability of RS-1, a chemical compound able to stimulate hRAD51 activity, allowed us to demonstrate that the stimulation of the hRAD51-mediated DNA repair process in HIV-1-infected cells can lead to the inhibition of retroviral replication by decreasing int...

We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.Hepatitis C virus (HCV) infection causes serious liver diseases, such as chronic hepatitis, which can evolve into cirrhosis and hepatocellular carcinoma (20,39). The most effective therapy, a combination of pegylated interferon and ribavirin, is efficient in only 50% of patients (32). Therefore, new treatments based on specific and well-tolerated compounds need to be developed.HCV RNA replication is catalyzed by the viral polymerase NS5B. This RNA-dependent RNA polymerase (RdRp) synthesizes a negative-strand RNA that serves as a template for the synthesis of new positive RNA strands. Viral RNA synthesis can be divided into two major steps: (i) initiation, which corresponds to the formation of a 2-or 3-nucleotide (nt) product and which occurs by a de novo mechanism in vitro as well as in the cellular replicon system that mimics some steps of the in vivo viral cycle (9, 31, 49), and (ii) elongation, which yields a full copy of the template. The transition between these two steps, in which 3-nt to 5-nt products are synthesized, may involve conformational changes of the viral polymerase that are not yet understood (14). The structure of the enzyme, as determined by X-ray crystallography, revealed, like for many other RNA and DNA polymerases, a right-handed-like structure with finger, palm, and thumb domains (1,7,28). It has been shown that the NS5B protein may adopt a closed and active conformation. The contact between the two loops extending from the fingers and the thumb links these two domains and closes the back of the enzyme to form a tunnel that constitutes the entry site for ribonucleotides. This suggests that a concerted movement of the thumb and fingertips occurs during the polymerization steps. The...