Bronchial epithelia from adults and children: SARS-CoV-2 spread via syncytia formation and type III interferon infectivity restriction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract, where infections can cause an acute respiratory distress syndrome with a high degree of mortality in elderly patients. We used reconstituted primary bronchial epithelia from adult and child donors to follow the SARS-CoV-2 infection dynamics. We show that, in epithelia from adult donors, infections initiate in multiciliated cells and spread within 24 to 48 h throughout the whole epithelia. Syncytia formed of ciliated and basal cells appeared at the apical side of the epithelia within 3 to 4 d and were released into the apical lumen, where they contributed to the transmittable virus dose. A small number of reconstituted epithelia were intrinsically more resistant to virus infection, limiting virus spread to different degrees. This phenotype was more frequent in epithelia derived from children versus adults and correlated with an accelerated release of type III interferon. Treatment of permissive adult epithelia with exogenous type III interferon restricted infection, while type III interferon gene knockout promoted infection. Furthermore, a transcript analysis revealed that the inflammatory response was specifically attenuated in children. Taken together, our findings suggest that apical syncytia formation is an underappreciated source of virus propagation for tissue or environmental dissemination, whereas a robust type III interferon response such as commonly seen in young donors restricted SARS-CoV-2 infection. Thus, the combination of interferon restriction and attenuated inflammatory response in children might explain the epidemiological observation of age-related susceptibility to COVID-19.

Modulation of the intrinsic chromatin binding property of HIV-1 integrase by LEDGF/p75.

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular cofactor LEDGF/p75. To investigate this and better understand the LEDGF/p75-mediated chromatin tethering of HIV-1 integrase, we used a combination of biochemical and chromosome-binding assays. Our study revealed that retroviral integrase has an intrinsic ability to bind and recognize specific chromatin regions in metaphase even in the absence of its cofactor. Furthermore, this integrase chromatin-binding property was modulated by the interaction with its cofactor LEDGF/p75, which redirected the enzyme to alternative chromosome regions. We also better determined the chromatin features recognized by each partner alone or within the functional intasome, as well as the chronology of efficient LEDGF/p75-mediated targeting of HIV-1 integrase to chromatin. Our data support a new chromatin-binding function of integrase acting in concert with LEDGF/p75 for the optimal association with the nucleosomal substrate. This work also provides additional information about the behavior of retroviral integration complexes in metaphase chromatin and the mechanism of action of LEDGF/p75 in this specific context.

Targeting the Integrated Stress Response Kinase GCN2 to Modulate Retroviral Integration.

Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.

Discovery of Novel Integrase Inhibitors Acting outside the Active Site Through High-Throughput Screening.

Currently, an increasing number of drugs are becoming available to clinics for the treatment of HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients, due to resistance with or without treatment adherence concerns. Accordingly, it is important to continue to discover small molecules that have a novel mechanism of inhibition. In this work, HIV integrase inhibitors were selected by high-throughput screening. Chemical structure comparisons enabled the identification of stilbene disulfonic acids as a potential new chemotype. Biochemical characterization of the lead compound stilbenavir (NSC34931) and a few derivatives was performed. Stilbene disulfonic acid derivatives exhibit low to sub-micromolar antiviral activity, and they inhibit integrase through DNA-binding inhibition. They probably bind to the C-terminal domain of integrase, in the cavity normally occupied by the noncleaved strand of the viral DNA substrate. Because of this original mode of action compared to active site strand transfer inhibitors, they do not exhibit cross-resistance to the three main resistance pathways to integrase inhibitors (G140S-Q148H, N155H, and Y143R). Further structure-activity optimization should enable the development of more active and less toxic derivatives with potential clinical relevance.

Selectivity for strand-transfer over 3'-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme-DNA interactions in the active site.

Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed.

HIV-1 Integrase-Targeted Short Peptides Derived from a Viral Protein R Sequence.

HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69⁻75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.

G-quadruplexes in viruses: function and potential therapeutic applications.

G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of ∼300,000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.

Molecular dynamics approaches estimate the binding energy of HIV-1 integrase inhibitors and correlate with in vitro activity.

The design of novel integrase (IN) inhibitors has been aided by recent crystal structures revealing the binding mode of these compounds with a full-length prototype foamy virus (PFV) IN and synthetic viral DNA ends. Earlier docking studies relied on incomplete structures and did not include the contribution of the viral DNA to inhibitor binding. Using the structure of PFV IN as the starting point, we generated a model of the corresponding HIV-1 complex and developed a molecular dynamics (MD)-based approach that correlates with the in vitro activities of novel compounds. Four well-characterized compounds (raltegravir, elvitegravir, MK-0536, and dolutegravir) were used as a training set, and the data for their in vitro activity against the Y143R, N155H, and G140S/Q148H mutants were used in addition to the wild-type (WT) IN data. Three additional compounds were docked into the IN-DNA complex model and subjected to MD simulations. All three gave interaction potentials within 1 standard deviation of values estimated from the training set, and the most active compound was identified. Additional MD analysis of the raltegravir- and dolutegravir-bound complexes gave internal and interaction energy values that closely match the experimental binding energy of a compound related to raltegravir that has similar activity. These approaches can be used to gain a deeper understanding of the interactions of the inhibitors with the HIV-1 intasome and to identify promising scaffolds for novel integrase inhibitors, in particular, compounds that retain activity against a range of drug-resistant mutants, making it possible to streamline synthesis and testing.

6,7-Dihydroxy-1-oxoisoindoline-4-sulfonamide-containing HIV-1 integrase inhibitors.

Although an extensive body of scientific and patent literature exists describing the development of HIV-1 integrase (IN) inhibitors, Merck's raltegravir and Gilead's elvitegravir remain the only IN inhibitors FDA-approved for the treatment of AIDS. The emergence of raltegravir-resistant strains of HIV-1 containing mutated forms of IN underlies the need for continued efforts to enhance the efficacy of IN inhibitors against resistant mutants. We have previously described bicyclic 6,7-dihydroxyoxoisoindolin-1-ones that show good IN inhibitory potency. This report describes the effects of introducing substituents into the 4- and 5-positions of the parent 6,7-dihydroxyoxoisoindolin-1-one platform. We have developed several sulfonamide-containing analogs that enhance potency in cell-based HIV assays by more than two orders-of-magnitude and we describe several compounds that are more potent than raltegravir against the clinically relevant Y143R IN mutant.

Template requirements of Zika RNA polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA.

As ZIKV continues to spread, many "unknowns" remain and research is needed to advance the understanding of this important pathogen. Viral RNA dependent-RNA polymerases (RdRp) are validated targets for inhibitors of the replication of several viruses. Several studies have set up in vitro enzymatic assays of the RdRp of the Zika virus for testing of candidate inhibitors. While most of these studies use short synthetic polymers, we have shown in a previous work that the Zika polymerase domain is capable of a de novo synthesis of the viral genome using the natural viral RNA as template. Here we have studied the role of the sequences at the 3'end of the minus-strand RNA in the initiation of the RNA synthesis by the Zika isolated RdRp. Our results strongly suggest that the region containing the 105 first nucleotides from the 3' end of the minus-strand RNA is important for initiation of the positive RNA synthesis. This indicates that this region displays all the primary and secondary structures to be efficiently recognized by the recombinant RdRp in vitro. Moreover, we show that the 46 nucleotides are sufficient to initiate RNA synthesis. In addition, the ZIKV polymerase domain poorly replicated the RNA of other RNA viruses and appeared highly selective for its own RNA.

Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572).

Raltegravir (RAL) and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) efficiently block viral replication in vitro and suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3' end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV-1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL-resistant INs, we determined crystal structures of wild-type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the ß4-α2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared with those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.

MK-0536 inhibits HIV-1 integrases resistant to raltegravir.

With the U.S. Food and Drug Administration approval of raltegravir (RAL; MK-0518; Merck & Co.), HIV-1 integrase (IN) is the newest therapeutic target for AIDS and HIV infections. Recent structural analyses show that IN strand transfer inhibitors (INSTIs) share a common binding mode in the enzyme active site. While RAL represents a therapeutic breakthrough, the emergence of IN resistance mutations imposes the development of new INSTIs. We report here the biochemical and antiviral activities of MK-0536, a new IN inhibitor. We demonstrate that, like RAL, MK-0536 is highly potent against recombinant IN and viral replication. It is also effective against INs that carry the three main RAL resistance mutations (Y143R, N155H, and to a lesser extent G140S-Q148H) and against the G118R mutant. Modeling of IN developed from recent prototype foamy virus structures is presented to account for the differences in the drug activities of MK-0536 and RAL against the IN mutants.

Development of tricyclic hydroxy-1H-pyrrolopyridine-trione containing HIV-1 integrase inhibitors.

New tricyclic HIV-1 integrase (IN) inhibitors were prepared that combined structural features of bicyclic pyrimidinones with recently disclosed 4,5-dihydroxy-1H-isoindole-1,3(2H)-diones. This combination resulted in the introduction of a nitrogen into the aryl ring and the addition of a fused third ring to our previously described inhibitors. The resulting analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays, with good selectivity for strand transfer relative to 3'-processing.

Absence of Resistance Mutations in the Integrase Coding Region among ART-Experienced Patients in the Republic of the Congo.

BACKGROUND: HIV infects around one hundred thousand patients in the Republic of the Congo. Approximately 25% of them receive an antiretroviral treatment; current first-line regimens include two NRTIs and one NNRTI, reverse transcriptase inhibitors. Recently, protease inhibitors (PIs) were also introduced as second-line therapy upon clinical signs of treatment failure. Due to the limited number of molecular characterizations and amount of drug resistance data available in the Republic of the Congo, this study aims to evaluate the prevalence of circulating resistance mutations within the pol region. METHODS: HIV-positive, ART-experienced patients have been enrolled in four semi-urban localities in the Republic of the Congo. Plasma samples were collected, and viral RNA was extracted. The viral load for each patient was evaluated by RT-qPCR, following the general diagnostic procedures of the University Hospital of Bordeaux. Finally, drug resistance genotyping and phylogenetic analysis were conducted following Sanger sequencing of the pol region. RESULTS: A high diversity of HIV-1 strains was observed with many recombinant forms. Drug resistance mutations in RT and PR genes were determined and correlated to HAART. Because integrase inhibitors are rarely included in treatments in the Republic of the Congo, the prevalence of integrase drug resistance mutations before treatment was also determined. Interestingly, very few mutations were observed. CONCLUSIONS: We confirmed a high diversity of HIV-1 in the Republic of the Congo. Most patients presented an accumulation of mutations conferring resistance against NRTIs, NNRTIs and PIs. Nonetheless, the absence of integrase mutations associated with drug resistance suggests that the introduction of integrase inhibitors into therapy will be highly beneficial to patients in the Republic of the Congo.

Quinolinonyl Non-Diketo Acid Derivatives as Inhibitors of HIV-1 Ribonuclease H and Polymerase Functions of Reverse Transcriptase.

Novel anti-HIV agents are still needed to overcome resistance issues, in particular inhibitors acting against novel viral targets. The ribonuclease H (RNase H) function of the reverse transcriptase (RT) represents a validated and promising target, and no inhibitor has reached the clinical pipeline yet. Here, we present rationally designed non-diketo acid selective RNase H inhibitors (RHIs) based on the quinolinone scaffold starting from former dual integrase (IN)/RNase H quinolinonyl diketo acids. Several derivatives were synthesized and tested against RNase H and viral replication and found active at micromolar concentrations. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, and Mg2+ titration experiments demonstrated that our compounds coordinate the Mg2+ cofactor and interact with amino acids of the RNase H domain that are highly conserved among naïve and treatment-experienced patients. In general, the new inhibitors influenced also the polymerase activity of RT but were selective against RNase H vs the IN enzyme.

Biochemical and pharmacological analyses of HIV-1 integrase flexible loop mutants resistant to raltegravir.

Resistance to raltegravir (RAL), the first HIV-1 integrase (IN) inhibitor approved by the FDA, involves three genetic pathways: IN mutations N155H, Q148H/R/K, and Y143H/R/C. Those mutations are generally associated with secondary point mutations. The resulting mutant viruses show a high degree of resistance against RAL but somehow are affected in their replication capacity. Clinical and virological data indicate the high relevance of the combination G140S + Q148H because of its limited impact on HIV replication and very high resistance to RAL. Here, we report how mutations at the amino acid residues 140, 148, and 155 affect IN enzymatic activity and RAL resistance. We show that single mutations at position 140 have limited impact on 3'-processing (3'-P) but severely inactivate strand transfer (ST). On the other hand, single mutations at position 148 have a more profound effect and inactivate both 3'-P and ST. By examining systematically all of the double mutants at the 140 and 148 positions, we demonstrate that only the combination G140S + Q148H is able to restore the catalytic properties of IN. This rescue only operates in cis when both the 140S and 148H mutations are in the same IN polypeptide flexible loop. Finally, we show that the G140S-Q148H double mutant exhibits the highest resistance to RAL. It also confers cross-resistance to elvitegravir but less to G-quadraduplex inhibitors such as zintevir. Our results demonstrate that IN mutations at positions 140 and 148 in the IN flexible loop can account for the phenotype of RAL-resistant viruses.

Pyrrolyl Pyrazoles as Non-Diketo Acid Inhibitors of the HIV-1 Ribonuclease H Function of Reverse Transcriptase.

Due to the biological liability of diketo acid (DKA) chain, we transferred this element of our previously reported anti-HIV-1 pyrrolyl derivatives to a non-DKA scaffold, obtaining a series of pyrrolyl-pyrazole carboxylic acids as new RNase H inhibitors. Among the newly synthesized derivatives, oxyphenylpyrrolyl-pyrazoles demonstrated inhibitory activities within the low micromolar/submicromolar range with compound 11b being the most potent. Interestingly, all tested compounds showed up to 2 orders of magnitude of selectivity for RNase H vs integrase. Docking studies within the RNase H catalytic site, coupled with site-directed mutagenesis, showed the key structural features that could confer the ability to establish specific interactions within RNase H. Furthermore, they proved the ability of our compounds to interact with amino acids highly conserved among HIV-1 subspecies isolated among patients carrying drug-resistant variants. In the end, the newly discovered pyrazole carboxylic acid derivatives feature promising serum stability with respect to their corresponding DKAs.

Cellular uptake of ODNs in HIV-1 human-infected cells: a role for viral particles in DNA delivery?

We have previously described how a 16 nucleotides ODN (termed 93del) is capable of inhibiting the activity of recombinant integrase in a cell-free system as well as HIV-1 replication in human-infected cells with IC(50) in the low nanomolar range. Intracellular HIV-1 replication was inhibited when the ODN was added at the onset of infection. These results raise several questions. Is a naked ODN able to enter the cell? Does the virus play a role in ODN entry? The uptake of several ODNs (93del, 60del(sc), TBA, T30923) was evaluated and then tracked by labeling the ODN with a fluorescent dye and assessing its intracellular localization by confocal microscopy. A significant level of cellular uptake of free ODN was observed in several cell lines: HeLa epithelial cells, Huh7 hepatic cells, and H9 lymphocytes, and was detected for all ODNs tested except for TBA. Striking differences were observed when naked ODNs were added to cell in the presence or absence of the virus. When HIV-1 virions were present a sharp increase in cellular fluorescence was observed. These results strongly suggest a role for HIV-1 virions in the uptake of certain ODNs.

De novo RNA synthesis catalyzed by the Zika Virus RNA polymerase domain.

Mosquito- and tick-borne pathogens including Chikungunya, Dengue, Japanese encephalitis, West Nile, Yellow fever and Zika virus, represent a new economic and public health challenge. In the absence of effective vaccines and specific therapies, only supportive regimens are administrated for most of these infections. Thus, the development of a targeted therapy is mandatory to stop the rapid progression of these pathogens and preoccupant associated burdens such as Guillain-Barre syndrome, microcephaly. For this, it is essential to develop biochemical tools to help study and target key viral enzymes involved in replication such as helicase complexes, methyl-transferases and RNA-dependent RNA polymerases. Here, we show that a highly purified ZIKV polymerase domain is active in vitro. Importantly, we show that this isolated domain is capable of de novo synthesis of the viral genome and efficient elongation without terminal nucleotide transferase activity. Altogether, this isolated polymerase domain will be a precious tool to screen and optimize specific nucleoside and non-nucleoside inhibitors to fight against Zika infections.

Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors.

Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.