NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase.

BACKGROUND: HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. RESULTS: We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. CONCLUSIONS: Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding-a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

Function of the bacteriophage P2 baseplate central spike Apex domain in the infection process.

The contractile tail of bacteriophage P2 functions to drive the tail tube across the outer membrane of its host bacterium, a prerequisite event for subsequent translocation of phage genomic DNA into the host cell. The tube is equipped with a spike-shaped protein (product of P2 gene V , gpV or Spike) that contains a membrane-attacking Apex domain carrying a centrally positioned Fe ion. The ion is enclosed in a histidine cage that is formed by three symmetry-related copies of a conserved HxH (histidine, any residue, histidine) sequence motif. Here, we used solution biophysics and X-ray crystallography to characterize the structure and properties of Spike mutants in which the Apex domain was either deleted or its histidine cage was either destroyed or replaced with a hydrophobic core. We found that the Apex domain is not required for the folding of full-length gpV or its middle intertwined ß-helical domain. Furthermore, despite its high conservation, the Apex domain is dispensable for infection in laboratory conditions. Collectively, our results show that the diameter of the Spike but not the nature of its Apex domain determines the efficiency of infection, which further strengthens the earlier hypothesis of a drill bit-like function of the Spike in host envelope disruption.

Flavivirus enzymes and their inhibitors.

Flaviviruses such as dengue, Japanese encephalitis, West Nile, Yellow Fever and Zika virus, cause viral hemorrhagic fever and encephalitis in humans. However, antiviral therapeutics to treat or prevent flavivirus infections are not yet available. Thus, there is pressing need to develop therapeutics and vaccines that target flavivirus infections. All flaviviruses carry a positive-sense single-stranded RNA genome, which encodes ten proteins; three structural proteins form the virus shell, and seven nonstructural (NS) proteins are involved in replication of the viral genome. While all NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are part of a functional membrane-bound replication complex, enzymatic activities required for flaviviral replication reside in only two NS proteins, NS3 and NS5. NS3 functions as a protease, helicase, and triphosphatase, and NS5 as a capping enzyme, methyltransferase, and RNA-dependent RNA polymerase. In this chapter, we provide an overview of viral replication focusing on the structure and function of NS3 and NS5 replicases. We further describe strategies and examples of current efforts to identify potential flavivirus inhibitors against NS3 and NS5 enzymatic activities that can be developed as therapeutic agents to combat flavivirus infections.

Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags.

We describe Surface Oligopeptide knock-in for Rapid Target Selection (SORTS), a novel method to select mammalian cells with precise genome modifications that does not rely on cell cloning. SORTS is designed to disrupt the target gene with an expression cassette encoding an epitope tag embedded into human glycophosphatidylinositol (GPI)-anchored protein CD52. The cassette is very short, usually less than 250 nucleotides, which simplifies donor DNA construction and facilitates transgene integration into the target locus. The chimeric protein is then expressed from the target promoter, processed and exposed on the plasma membrane where it serves as a marker for FACS sorting with tag-specific antibodies. Simultaneous use of two different epitope tags enables rapid isolation of cells with biallelic knock-ins. SORTS can be easily and reliably applied to a number of genome-editing problems such as knocking out genes encoding intracellular or secreted proteins, protein tagging and inactivation of HIV-1 provirus.

A qPCR assay for measuring the post-integrational DNA repair in HIV-1 replication.

The post-integrational gap repair is a critical and poorly studied stage of the lentiviral life cycle. It might be performed by various cellular DNA repair pathways but the exact mechanism of the repair process has not yet been described. One of the reasons for that is the lack of a functional quantitative assay that could precisely measure the amount of integrated viral DNA that has completed the post-integrational gap repair stage. Here, we present an approach that is based on a widely used Alu-specific PCR for the estimation of integrated viral DNA but includes several steps that allow discrimination between integrated-repaired and integrated-unrepaired viral DNA forms. We used the approach for the estimation of the kinetics of gap repair in a viral vector system and showed that the gap repair process starts at 17 h post infection and lasts 10 more hours. We also showed that the addition of Nu7441 - a small molecule inhibitor of DNA-breaks sensor kinase in the non-homologous end joining DNA repair pathway - specifically inhibits the gap repair process while having no influence on the integration itself.

Human Ku70 protein binds hairpin RNA and double stranded DNA through two different sites.

Human protein Ku usually functions in the cell as a complex of two subunits, Ku70 and Ku80. The Ku heterodimer plays a key role in the non-homologous end joining DNA repair pathway by specifically recognizing the DNA ends at the site of the lesion. The binding of the Ku heterodimer to DNA has been well-studied, and its interactions with RNA have been also described. However, Ku70 subunit is known to have independent DNA binding capability, which is less characterized. RNA binding properties of Ku70 have not been yet specially studied. We have prepared recombinant full-length Ku70 and a set of its truncated mutants in E. coli, and studied their interactions with nucleic acids of various structures: linear single- and double-stranded DNA and RNA, as well as closed circular DNA and hairpin RNA. Ku70 has demonstrated a high affinity binding to double stranded DNA and hairpin RNA with a certain structure only. Interestingly, in contrast to the Ku heterodimer, Ku70 is found to interact with closed circular DNA. We also show for the first time that Ku70 employs two different sites for DNA and RNA binding. The double-stranded DNA is recognized by the C-terminal part of Ku70 including SAP domain as it has been earlier demonstrated, whereas hairpin RNA binding is provided by amino acids 251-438.

Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting.

Human Ku70/Ku80 protein is known to influence HIV-1 replication. One of the possible reasons may be the protection of integrase from proteasomal degradation by Ku70 subunit. We demonstrated that recombinant HIV-1 integrase and Ku70 form a stable complex, while no interaction of Ku70 with integrase from prototype foamy virus was observed. By analyzing protein subdomains we determined two binding sites in the structure of both Ku70 and integrase: the 51-160 a.a. region of integrase interacts with residues 251-438 of Ku70, whereas Ku70 N-terminal domain (1-250 a.a.) contacts an α6-helix in the 200-220 a.a. integrase region. Single substitutions within integrase (E212A or L213A) block the interaction with Ku70 thus indicating that the binding site formed by the 200-220 a.a. integrase region is crucial for complex formation. E212A/L213A substitutions decreased the integrase capacity to bind Ku70 in HEK293T cells. A conjugate of 2'-ОMe-GGUUUUUGUGU oligonucleotide with eosin is shown by molecular modeling to shield integrase residues E212/L213 and is effective in blocking complex formation of Ku70 with integrase what makes the complex between α6-helix and Ku70(1-250) a possible target for drug development.

Consensus HIV-1 subtype A integrase and its raltegravir-resistant variants: design and characterization of the enzymatic properties.

Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded.

Structure-Activity Relationship Studies of HIV-1 Integrase Oligonucleotide Inhibitors.

Integration of human immunodeficiency virus type 1 DNA into an infected cell genome is one of the key steps of the viral replication cycle. Therefore, viral enzyme integrase, which realizes the integration, represents an attractive and validated target for the development of new antiviral drugs. In this paper, the anti-integrase activity of a series of conjugates of single-stranded oligonucleotides with hydrophobic molecules was tested, and the structure-activity relationships were also analyzed. Both oligonucleotide and hydrophobic parts of the conjugates influenced the inhibitory potency. Conjugates of 11-mer phosphorothioate oligonucleotides with 6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein (HEX) were found to be the most efficient inhibitors (IC50 = 20 nM) and might be considered as lead compounds for further development of integrase inhibitors.

Modulation of HIV-1 integrase activity by single-stranded oligonucleotides and their conjugates with eosin.

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.