Application of PolyPRep tools on HIV protease polyproteins using molecular docking

In recent years, the development of high-throughput technologies for obtaining sequence data leveraged the possibility of analysis of protein data in silico. However, when it comes to viral polyprotein interaction studies, there is a gap in the representation of those proteins, given their size and length. The prepare for studies using state-of-the-art techniques such as Machine Learning, a good representation of such proteins is a must. We present an alternative to this problem, implementing a fragmentation and modeling protocol to prepare those polyproteins in the form of peptide fragments. Such procedure is made by several scripts, implemented together on the workflow we call PolyPRep, a tool written in Python script and available in GitHub. This software is freely available only for noncommercial users.

Genetic Diversity and Detection of Atypical Porcine Pestivirus Infections

Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previously reported qPCR-based assays were unable to detect APPV in novel cases of congenital tremor originated from multiple farms from U.S. Midwest (MW). These assays targeted the viral polyprotein coding genes, which were shown to display substantial variation, with sequence identity ranging from 58.2 to 70.7% among 15 global APPV strains. In contrast, the 5’ UTR was found to have a much higher degree of sequence conservation. In order to obtain the complete 5’ UTR of the APPV strains originated from MW, the 5’ end of the viral cDNA was obtained by using template switching approach followed by amplification and dideoxy sequencing. Eighty one percent of the 5’UTR was identical across 14 global and 5 MW strains with complete, or relatively complete 5’ UTR. Notably, some of the most highly conserved 5’UTR segments overlapped with potentially important regions of an internal ribosome entry site (IRES), suggesting their functional role in viral protein translation. A newly designed single qPCR assay, targeting 100% conserved 5’UTR regions across 19 strains, was able to detect APPV in samples of well documented cases of congenital tremor which originated from five MW farm sites (1-18 samples/site). As these fully conserved 5’ UTR sequences may have functional importance, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

Drug repurposing screening identified tropifexor as a SARS-CoV-2 papain-like protease inhibitor

The global COVID-19 pandemic underscores the dire need of effective antivirals. Encouraging progress has been made in developing small molecule inhibitors targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro). However, the development of papain-like protease (PLpro) inhibitors faces several obstacles. Nevertheless, PLpro represents a high-profile drug target given its multifaceted roles in viral replication. PLpro is involved in not only the cleavage of viral polyprotein but also modulation of host immune response. In this study, we conducted a drug-repurposing screening of PLpro against the MedChemExpress bioactive compound library and identified three hits, EACC, KY-226, and tropifexor, as potent PLpro inhibitors with IC50 values ranging from 3.39 to 8.28 µM. The three hits showed dose-dependent binding to PLpro in the thermal shift assay. In addition, tropifexor inhibited the cellular PLpro activity in the FlipGFP assay with an IC50 of 10.6 µM. Gratifyingly, tropifexor showed antiviral activity against SARS-CoV-2 in Calu-3 cells with an EC50 of 4.03 µM, a 7.8-fold increase compared to GRL0617 (EC50 = 31.4 µM). Overall, tropifexor represents a novel PLpro inhibitor that can be further developed as SARS-CoV-2 antivirals.

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 Å resolution crystal structure of 3CLpro C145S bound to NEMO226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.

Protease cleavage of RNF20 facilitates coronavirus replication via stabilization of SREBP1

COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.

Prediction of coronavirus 3C-like protease cleavage sites using machine-learning algorithms

The coronavirus 3C-like (3CL) protease is a Cysteine protease. It plays an important role in viral infection and immune escape by not only cleaving the viral polyprotein ORF1ab at 11 sites, but also cleaving the host proteins. However, there is still a lack of effective tools for determining the cleavage sites of the 3CL protease. This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein, and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus, Betacoronavirus and Gammacoronavirus. Strong residue preferences were observed at the neighboring positions of the cleavage sites. A random forest (RF) model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues at cleavage site and neighboring positions by amino acid indexes, and the model achieved an AUC of 0.96 in cross-validations. The RF model was further tested on an independent test dataset composed of cleavage sites on host proteins, and achieved an AUC of 0.88 and a prediction precision of 0.80 when considering the accessibility of the cleavage site. Then, 1,079 human proteins were predicted to be cleaved by the 3CL protease by the RF model. These proteins were enriched in pathways related to neurodegenerative diseases and pathogen infection. Finally, a user-friendly online server named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model. Overall, the study not only provides an effective tool for identifying the cleavage sites of the 3CL protease, but also provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.

An Epitope Platform for Safe and Effective HTLV-1-Immunization: Potential Applications for mRNA and Peptide-Based Vaccines

Human T-cell lymphotropic virus type 1 (HTLV-1) infection affects millions of individuals worldwide and can lead to severe leukemia, myelopathy/tropical spastic paraparesis, and numerous other disorders. Pursuing a safe and effective immunotherapeutic approach, we compared the viral polyprotein and the human proteome with a sliding window approach in order to identify oligopeptide sequences unique to the virus. The immunological relevance of the viral unique oligopeptides was assessed by searching them in the immune epitope database (IEDB). We found that HTLV-1 has 15 peptide stretches each consisting of uniquely viral non-human pentapeptides which are ideal candidate for a safe and effective anti-HTLV-1 vaccine. Indeed, experimentally validated HTLV-1 epitopes, as retrieved from the IEDB, contain peptide sequences also present in a vast number of human proteins, thus potentially instituting the basis for cross-reactions. We found a potential for cross-reactivity between the virus and the human proteome and described an epitope platform to be used in order to avoid it, thus obtaining effective, specific, and safe immunization. Potential advantages for mRNA and peptide-based vaccine formulations are discussed.

The Erns Carboxyterminus: Much More Than a Membrane Anchor

Pestiviruses express the unique essential envelope protein Erns, which exhibits RNase activity, is attached to membranes by a long amphipathic helix, and is partially secreted from infected cells. The RNase activity of Erns is directly connected with pestivirus virulence. Formation of homodimers and secretion of the protein are hypothesized to be important for its role as a virulence factor, which impairs the host’s innate immune response to pestivirus infection. The unusual membrane anchor of Erns raises questions with regard to proteolytic processing of the viral polyprotein at the Erns carboxy-terminus. Moreover, the membrane anchor is crucial for establishing the critical equilibrium between retention and secretion and ensures intracellular accumulation of the protein at the site of virus budding so that it is available to serve both as structural component of the virion and factor controlling host immune reactions. In the present manuscript, we summarize published as well as new data on the molecular features of Erns including aspects of its interplay with the other two envelope proteins with a special focus on the biochemistry of the Erns membrane anchor.

Structural insights into the interaction of papain-like protease 2 from the alphacoronavirus porcine epidemic diarrhea virus and ubiquitin

Porcine epidemic diarrhea is a devastating porcine disease that is caused by the alphacoronavirus porcine epidemic diarrhea virus (PEDV). Like other members of the Coronaviridae family, PEDV encodes a multifunctional papain-like protease 2 (PLP2) that has the ability to process the coronavirus viral polyprotein to aid in RNA replication and antagonize the host innate immune response through cleavage of the regulatory proteins ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15) (deubiquitination and deISGylation, respectively). Because Betacoronavirus PLPs have been well characterized, it was sought to determine how PLP2 from the alphacoronavirus PEDV differentiates itself from its related counterparts. PEDV PLP2 was first biochemically characterized, and a 3.1 Å resolution crystal structure of PEDV PLP2 bound to Ub was then solved, providing insight into how Alphacoronavirus PLPs bind to their preferred substrate, Ub. It was found that PEDV PLP2 is a deubiquitinase and readily processes a variety of di-Ub linkages, in comparison with its Betacoronavirus counterparts, which have a narrower range of di-Ub activity but process both Ub and ISG15.

Structural basis of ribosomal frameshifting during translation of the SARS-CoV-2 RNA genome

Programmed ribosomal frameshifting is a key event during translation of the SARS-CoV-2 RNA genome allowing synthesis of the viral RNA-dependent RNA polymerase and downstream proteins. Here we present the cryo-electron microscopy structure of a translating mammalian ribosome primed for frameshifting on the viral RNA. The viral RNA adopts a pseudoknot structure that lodges at the entry to the ribosomal mRNA channel to generate tension in the mRNA and promote frameshifting, whereas the nascent viral polyprotein forms distinct interactions with the ribosomal tunnel. Biochemical experiments validate the structural observations and reveal mechanistic and regulatory features that influence frameshifting efficiency. Finally, we compare compounds previously shown to reduce frameshifting with respect to their ability to inhibit SARS-CoV-2 replication, establishing coronavirus frameshifting as a target for antiviral intervention.