SARS‐CoV‐2–host proteome interactions for antiviral drug discovery

Treatment options for COVID‐19, caused by SARS‐CoV‐2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have explored SARS‐CoV‐2–host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS‐CoV‐2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP‐MS) and the complementary proximity‐based labeling MS method (BioID‐MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS‐CoV‐2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image‐based drug screen with infectious SARS‐CoV‐2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein–protein interactions.     

From infection to cancer: how DNA tumour viruses alter host cell central carbon and lipid metabolism

Infections cause 13% of all cancers globally, and DNA tumour viruses account for almost 60% of these cancers. All viruses are obligate intracellular parasites and hijack host cell functions to replicate and complete their life cycles to produce progeny virions. While many aspects of viral manipulation of host cells have been studied, how DNA tumour viruses manipulate host cell metabolism and whether metabolic alterations in the virus life cycle contribute to carcinogenesis are not well understood. In this review, we compare the differences in central carbon and fatty acid metabolism in host cells following infection, oncogenic transformation, and virus-driven cancer of DNA tumour viruses including: Epstein–Barr virus, hepatitis B virus, human papillomavirus, Kaposi''s sarcoma-associated herpesvirus and Merkel cell polyomavirus.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

Molecular interactions of STAC proteins with skeletal muscle dihydropyridine receptor and excitation‐contraction coupling

Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca²⁺ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α_1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.     

Intrinsic disorder mediates hepatitis C virus core–host cell protein interactions

Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein–protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N‐terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two‐hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site‐directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.     

Coinfection with a virus constrains within‐host infection load but increases transmission potential of a highly virulent fungal plant pathogen

The trade‐off between within‐host infection rate and transmission to new hosts is predicted to constrain pathogen evolution, and to maintain polymorphism in pathogen populations. Pathogen life‐history stages and their correlations that underpin infection development may change under coinfection with other parasites as they compete for the same limited host resources. Cross‐kingdom interactions are common among pathogens in both natural and cultivated systems, yet their impacts on disease ecology and evolution are rarely studied. The host plant Plantago lanceolata is naturally infected by both Phomopsis subordinaria, a seed killing fungus, as well as Plantago lanceolata latent virus (PlLV) in the Åland Islands, SW Finland. We performed an inoculation assay to test whether coinfection with PlLV affects performance of two P. subordinaria strains, and the correlation between within‐host infection rate and transmission potential. The strains differed in the measured life‐history traits and their correlations. Moreover, we found that under virus coinfection, within‐host infection rate of P. subordinaria was smaller but transmission potential was higher compared to strains under single infection. The negative correlation between within‐host infection rate and transmission potential detected under single infection became positive under coinfection with PlLV. To understand whether within‐host and between‐host dynamics are correlated in wild populations, we surveyed 260 natural populations of P. lanceolata for P. subordinaria infection occurrence. When infections were found, we estimated between‐hosts dynamics by determining pathogen population size as the proportion of infected individuals, and within‐host dynamics by counting the proportion of infected flower stalks in 10 infected plants. In wild populations, the proportion of infected flower stalks was positively associated with pathogen population size. Jointly, our results suggest that the trade‐off between within‐host infection load and transmission may be strain specific, and that the pathogen life‐history that underpin epidemics may change depending on the diversity of infection, generating variation in disease dynamics.     

Resource limitation has a limited impact on the outcome of virus–fungus co‐infection in an insect host

Infection by pathogens is strongly affected by the diet or condition of the prospective host. Studies that examine the impact of diet have mainly focused on single pathogens; however, co‐infections within a single host are thought to be common. Different pathogen groups might respond differently to resource availability and diverse infections could increase the costs of host defense, meaning the outcome of mixed infections under varying dietary regimes is likely to be hard to predict. We used the generalist cabbage looper, Trichoplusia ni and two of its pathogens, the DNA virus T. ni nucleopolyhedrovirus (TniSNPV) and the entomopathogenic fungus, Beauveria bassiana to examine how nutrient reduction affected the outcome of mixed pathogen infection. We challenged insects with a low or high effective dose of virus, alone or combined with a single dose of fungus. We manipulated food availability after pathogen challenge by diluting artificial diet with cellulose, a non‐nutritious bulking agent, and examined its impact on host and pathogen fitness. Reducing diet quantity did not alter overall or pathogen‐specific mortality. In all cases, TniSNPV‐induced mortality was negatively affected by fungus challenge. Similarly, B. bassiana‐induced mortality was negatively affected by TniSNPV challenge, but only at the higher virus dose. Dietary dilution mainly affected B. bassiana speed of kill when mixed with a high dose of TniSNPV, with an increase in the duration of fungal infection when cellulose was low (high quantity). One pathogen dominated the production of transmission stages in the cadavers and co‐infection did not affect the yield of either pathogen. There was no evidence that co‐infections were more costly to the survivors of pathogen challenge. In conclusion, dietary dilution did not determine the outcome of mixed pathogen infection, but it had more subtle effects, that differed between the two pathogens and could potentially alter pathogen recycling and host–pathogen dynamics.     

Turnip crinkle virus‐encoded suppressor of RNA silencing interacts with Arabidopsis SGS3 to enhance virus infection

Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well‐characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant‐encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two‐hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co‐localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N‐terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP‐interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3‐overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans‐acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA‐binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing‐mediated antiviral defence.     

Non‐canonical autophagy functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus

Influenza A virus (IAV) and SARS‐CoV‐2 (COVID‐19) cause pandemic infections where cytokine storm syndrome and lung inflammation lead to high mortality. Given the high social and economic cost of respiratory viruses, there is an urgent need to understand how the airways defend against virus infection. Here we use mice lacking the WD and linker domains of ATG16L1 to demonstrate that ATG16L1‐dependent targeting of LC3 to single‐membrane, non‐autophagosome compartments – referred to as non‐canonical autophagy – protects mice from lethal IAV infection. Mice with systemic loss of non‐canonical autophagy are exquisitely sensitive to low‐pathogenicity IAV where extensive viral replication throughout the lungs, coupled with cytokine amplification mediated by plasmacytoid dendritic cells, leads to fulminant pneumonia, lung inflammation and high mortality. IAV was controlled within epithelial barriers where non‐canonical autophagy reduced IAV fusion with endosomes and activation of interferon signalling. Conditional mouse models and ex vivo analysis showed that protection against IAV infection of lung was independent of phagocytes and other leucocytes. This establishes non‐canonical autophagy in airway epithelial cells as a novel innate defence that restricts IAV infection and lethal inflammation at respiratory surfaces.     

A plant RNA virus hijacks endocytic proteins to establish its infection in plants

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor‐mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin‐related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co‐opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV‐infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1‐dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2β) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2β impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2β are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.