Montermoso, S. | Search bioRxiv

bioRxiv2025

Structures of Nucleotide-Bound Redondovirus Rep Protein Link Conformation and Function

Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3 end for polymerase extension, provide an NTP-dependent helicase activity for DNA unwinding, and join nicked ends to form circular viral genomes. Here, we present the first structures of a Rep protein from the Redondoviridae family, a newly discovered family of human-associated CRESS-DNA viruses that replicates within the oral protozoan Entamoeba gingivalis. Using cryo-EM, we characterized the hexameric structures of a Redondovirus Rep helicase bound with ATP{gamma}S, representing the initial ATP-bound state, and with ADP, reflecting the protein state after hydrolysis. The ADP state, but not the ATP state of Rep shows a staircase arrangement of DNA-binding loops that plays a central role in current models for SF3 helicase function. Additionally, we determined a head- to-tail dodecameric structure of ATP{gamma}S-bound Rep, in which both the helicase and endonuclease domains are ordered. Conservation of residues involved in stabilizing the dodecamer suggest that this assembly may be functionally relevant for many CRESS-DNA viruses. The positioning of endonuclease domains in the Rep hexamer, combined with our biophysical analyses of Rep oligomerization, provide new insights into Rep function during viral replication.

microbiology↗

bioRxiv2024

Comprehensive proteolytic profiling of Aedes aegypti mosquito midgut extracts: Unraveling the blood meal protein digestion system

To sustain the gonotrophic cycle, the Aedes aegypti mosquito must acquire a blood meal from a human or other vertebrate host. However, in the process of blood feeding, the mosquito may facilitate the transmission of several bloodborne viral pathogens (e.g., dengue, Zika, and chikungunya). The blood meal is essential as it contains proteins that are digested into polypeptides and amino acid nutrients that are eventually used for egg production. These proteins are digested by several midgut proteolytic enzymes. As such, the female mosquitos reliance on blood may serve as a potential target for vector and viral transmission control. However, this strategy may prove to be challenging since midgut proteolytic activity is a complex process dependent on several exo- and endo-proteases. Therefore, to understand the complexity of Ae. aegypti blood meal digestion, we used Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) to generate global proteolytic profiles of sugar- and blood-fed midgut tissue extracts, along with substrate profiles of recombinantly expressed midgut proteases. Our results reveal a shift from high exoproteolytic activity in sugar-fed mosquitoes to an expressive increase in endoproteolytic activity in blood-fed mosquitoes. This approach allowed for the identification of 146 cleaved peptide bonds (by the combined 6 h and 24 h blood-fed samples) in the MSP-MS substrate library, and of these 146, 99 (68%) were cleaved by the five recombinant proteases evaluated. These reveal the individual contribution of each recombinant midgut protease to the overall blood meal digestion process of the Ae. aegypti mosquito. Further, our molecular docking simulations support the substrate specificity of each recombinant protease. Therefore, the present study provides key information of midgut proteases and the blood meal digestion process in mosquitoes, which may be exploited for the development of potential inhibitor targets for vector and viral transmission control strategies. Author SummaryThe Aedes aegypti mosquito is a vector of viral pathogens that can be transmitted directly to humans. For instance, the transmission of dengue, Zika, or chikungunya viruses may happen during the Ae. aegypti acquisition of an infected blood meal. This blood meal is important for the anautogenous mosquito because without the digestion of blood proteins the mosquito will not obtain the necessary nutrients needed for egg production. After imbibing a blood meal, midgut digestive enzymes (proteases) are expressed and secreted into the lumen. To fully understand their roles in blood meal digestion, we used a special technique called Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS). This method allows us to generate global proteolytic activity profiles of Ae. aegypti midgut tissue extracts that were fed with sugar or blood. In addition, we generated substrate cleavage profiles of recombinantly expressed midgut proteases allowing us to understand the enzyme preferences for blood proteins. Therefore, utilizing this approach, we found the contribution of each individual recombinant protease tested relative to the global activity profile of blood-fed midgut tissue extracts. This may be a starting point for the validation of midgut protease inhibition and the development of a new potential vector control strategy.

biochemistry↗

bioRxiv2022

Structure of a HIV-1 IN-Allosteric Inhibitor Complex at 2.93 Angstrom Resolution: Routes to Inhibitor Optimization

HIV integrase (IN) inserts viral DNA into the host genome and is the target of the strand transfer inhibitors (STIs), a class of small molecules currently in clinical use. Another potent class of antivirals is the allosteric inhibitors of integrase, or ALLINIs. ALLINIs promote IN aggregation by stabilizing an interaction between the catalytic core domain (CCD) and carboxy -terminal domain (CTD) that undermines viral particle formation in late replication. Ongoing challenges with inhibitor potency, toxicity, and viral resistance motivate research to understand their mechanism. Here, we report a 2.93 [A] X-ray crystal structure of the minimal ternary complex between CCD, CTD, and the ALLINI BI-224436. This structure reveals an asymmetric ternary complex with a prominent network of {pi}-mediated interactions that suggest specific avenues for future ALLINI development and optimization.

biochemistry↗