Interactomes of SARS‐CoV‐2 and human coronaviruses reveal host factors potentially affecting pathogenesis

Host–virus protein–protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity‐labeling strategies and identified 437 human proteins as the high‐confidence interacting proteins. Further characterization of these interactions and comparison to other large‐scale study of cellular responses to SARS‐CoV‐2 infection elucidated how distinct SARS‐CoV‐2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID‐19. The interactomes of two key SARS‐CoV‐2‐encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein–protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS‐CoV‐2 provides valuable resources for the understanding and treating of this disease.     

The effect of host Chlorella NC64A carbon : phosphorus ratio on the production of Paramecium bursaria Chlorella Virus-1

1. We used the freshwater alga Chlorella NC64A (Division Chlorophyta) and its virus Paramecium bursaria Chlorella virus‐1 (PBCV‐1) as a model system to test for potential stoichiometric constraints on a virus–host interaction. 2. Media phosphorus concentrations were manipulated to create Chlorella NC64A host cells with low (91 ± 23) or high (453 ± 246) C : P ratio. In contrast, the C : P ratio of PBCV‐1, calculated from its biochemical composition, was 17 : 1. 3. Stoichiometric theory predicts that infection success and postinfection viral production should be depressed in high C : P cultures due to insufficient intracellular P for production of P‐rich viral particles. 4. Consistent with this hypothesis, viral production was strongly affected by host C : P ratio. While host C : P ratio did not affect viral attachment or the percentage of new viral particles that were infectious, in the low C : P Chlorella NC64A treatment, nine times more viruses were produced per infected cell than in the high C : P treatment (158 ± 138 versus 18 ± 18), indicating that the low C : P cells were higher quality for PBCV‐1 proliferation. 5. This result implies that the stoichiometric quality of algal cells can have a major effect on host–virus population dynamics.     

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.     

In Vivo Anti-HIV Activity of the Heparin-Activated Serine Protease Inhibitor Antithrombin III Encapsulated in Lymph-Targeting Immunoliposomes

Endogenous serine protease inhibitors (serpins) are anti-inflammatory mediators with multiple biologic functions. Several serpins have been reported to modulate HIV pathogenesis, or exhibit potent anti-HIV activity in vitro, but the efficacy of serpins as therapeutic agents for HIV in vivo has not yet been demonstrated. In the present study, we show that heparin-activated antithrombin III (hep-ATIII), a member of the serpin family, significantly inhibits lentiviral replication in a non-human primate model. We further demonstrate greater than one log₁₀ reduction in plasma viremia in the nonhuman primate system by loading of hep-ATIII into anti-HLA-DR immunoliposomes, which target tissue reservoirs of viral replication. We also demonstrate the utility of hep-ATIIII as a potential salvage agent for HIV strains resistant to standard anti-retroviral treatment. Finally, we applied gene-expression arrays to analyze hep-ATIII-induced host cell interactomes and found that downstream of hep-ATIII, two independent gene networks were modulated by host factors prostaglandin synthetase-2, ERK1/2 and NFκB. Ultimately, understanding how serpins, such as hep-ATIII, regulate host responses during HIV infection may reveal new avenues for therapeutic intervention.     

Proteomic analysis of host brain components that bind to infectious particles in Creutzfeldt‐Jakob disease

Transmissible encephalopathies (TSEs), such as Creutzfeldt‐Jakob disease (CJD) and scrapie, are caused by infectious agents that provoke strain‐specific patterns of disease. Misfolded host prion protein (PrP‐res amyloid) is believed to be the causal infectious agent. However, particles that are stripped of PrP retain both high infectivity and viral proteins not detectable in uninfected mouse controls. We here detail host proteins bound with FU‐CJD agent infectious brain particles by proteomic analysis. More than 98 proteins were differentially regulated, and 56 FU‐CJD exclusive proteins were revealed after PrP, GFAP, C1q, ApoE, and other late pathologic response proteins were removed. Stripped FU‐CJD particles revealed HSC70 (144× the uninfected control), cyclophilin B, an FU‐CJD exclusive protein required by many viruses, and early endosome‐membrane pathways known to facilitate viral processing, replication, and spread. Synaptosomal elements including synapsin‐2 (at 33×) and AP180 (a major FU‐CJD exclusive protein) paralleled the known ultrastructural location of 25 nm virus‐like TSE particles and infectivity in synapses. Proteins without apparent viral or neurodegenerative links (copine‐3), and others involved in viral‐induced protein misfolding and aggregation, were also identified. Human sCJD brain particles contained 146 exclusive proteins, and heat shock, synaptic, and viral pathways were again prominent, in addition to Alzheimer, Parkinson, and Huntington aggregation proteins. Host proteins that bind TSE infectious particles can prevent host immune recognition and contribute to prolonged cross‐species transmissions (the species barrier). Our infectious particle strategy, which reduces background sequences by >99%, emphasizes host targets for new therapeutic initiatives. Such therapies can simultaneously subvert common pathways of neurodegeneration.     

From deep sequencing to viral tagging: Recent advances in viral metagenomics

Culture‐independent high‐throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non‐viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high‐throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation‐independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts.     

Global Interactome Mapping of Mitochondrial Intermembrane Space Proteases Identifies a Novel Function for HTRA2

A number of unique proteases localize to specific sub‐compartments of the mitochondria, but the functions of these enzymes are poorly defined. Here, in vivo proximity‐dependent biotinylation (BioID) is used to map the interactomes of seven proteases localized to the mitochondrial intermembrane space (IMS). In total, 802 high confidence proximity interactions with 342 unique proteins are identified. While all seven proteases co‐localized with the IMS markers OPA1 and CLPB, 230 of the interacting partners are unique to just one or two protease bait proteins, highlighting the ability of BioID to differentiate unique interactomes within the confined space of the IMS. Notably, high‐temperature requirement peptidase 2 (HTRA2) interacts with eight of 13 components of the mitochondrial intermembrane space bridging (MIB) complex, a multiprotein assembly essential for the maintenance of mitochondrial cristae structure. Knockdown of HTRA2 disrupts cristae in HEK 293 and OCI‐AML2 cells, and leads to increased intracellular levels of the MIB subunit IMMT. Using a cell‐free assay it is demonstrated that HTRA2 can degrade recombinant IMMT but not two other core MIB complex subunits, SAMM50 and CHCHD3. The IMS protease interactome thus represents a rich dataset that can be mined to uncover novel IMS protease biology.     

Comparative interactomes of SIRT6 and SIRT7: Implication of functional links to aging

Sirtuins are NAD⁺‐dependent deacetylases that regulate a range of cellular processes. Although diverse functions of sirtuins have been proposed, those functions of SIRT6 and SIRT7 that are mediated by their interacting proteins remain elusive. In the present study, we identified SIRT6‐ and SIRT7‐interacting proteins, and compared their interactomes to investigate functional links. Our interactomes revealed 136 interacting proteins for SIRT6 and 233 for SIRT7 while confirming seven and 111 proteins identified previously for SIRT6 and SIRT7, respectively. Comparison of SIRT6 and SIRT7 interactomes under the same experimental conditions disclosed 111 shared proteins, implying related functional links. The interaction networks of interactomes indicated biological processes associated with DNA repair, chromatin assembly, and aging. Interactions of two highly acetylated proteins, nucleophosmin (NPM1) and nucleolin, with SIRT6 and SIRT7 were confirmed by co‐immunoprecipitation. NPM1 was found to be deacetylated by both SIRT6 and SIRT7. In senescent cells, the acetylation level of NPM1 was increased in conjunction with decreased levels of SIRT6 and SIRT7, suggesting that the acetylation of NPM1 could be regulated by SIRT6 and SIRT7 in the aging process. Our comparative interactomic study of SIRT6 and SIRT7 implies important functional links to aging by their associations with interacting proteins. All MS data have been deposited in the ProteomeXchange with identifiers PXD000159 and PXD000850 ( http://proteomecentral.proteomexchange.org/dataset/PXD000159 , http://proteomecentral.proteomexchange.org/dataset/PXD000850 ).     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.     

Real‐time monitoring of influenza virus production kinetics in HEK293 cell cultures

There is an increased interest from the vaccine industry to use mammalian cell cultures for influenza vaccine manufacturing. Therefore, it became important to study the influenza infection mechanism, the viral–host interaction, and the replication kinetics from a bioprocessing stand point to maximize the influenza viral production yield in cell culture. In the present work, influenza replication kinetics was studied in HEK293 cells. Two infection conditions were evaluated, a low (0.01) and a high multiplicity of infection (1.0). Critical time points of the viral production cycle (infection, protein synthesis, viral assembly and budding, viral release, and host‐cell death) were identified in small‐scale cell cultures. Additionally, cell growth, viability, and viral titers were monitored in the viral production process. The infection state of the cultivated cell population was assessed by influenza immunolabeling throughout the culture period. Influenza virus production kinetics were also on‐line monitored by dielectric spectroscopy and successfully correlated to real‐time capacitance measures. Overall, this work provided insights into the mechanisms associated with the infection of human HEK293 cell line by the influenza virus and demonstrated, once again, the usefulness of multifrequency scanning permittivity for in‐line monitoring and supervision of cell‐based viral production processes. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Biological and molecular events associated with simultaneous transmission of plant viruses by invertebrate and fungal vectors

Viruses are likely to be the most dangerous parasites of living organisms because of their widespread occurrence, possible deleterious effects on their hosts and high rates of evolution. Virus host‐to‐host transmission is a critical step in the virus life cycle, because it enables survival in a given environment and efficient dissemination. As hosts of plant viruses are not mobile, these pathogens have adopted diverse transmission strategies involving various vector organisms, mainly arthropods, nematodes, fungi and protists. In nature, plants are often infected with more than one virus at a time, thereby creating potential sources for vectors to acquire and transmit simultaneously two or more viruses. Simultaneous transmission can result in multiple infections of new host plants, which become subsequent potential sources of the viruses, thus enhancing the spread of the diseases caused by these pathogens. Moreover, it can contribute to the maintenance of viral genetic diversity in the host communities. However, despite its possible significance, the problem of the simultaneous transmission of plant viruses by vectors has not been investigated in detail. In this review, the current knowledge on multiple viral transmissions by aphids, whiteflies, leafhoppers, planthoppers, nematodes and fungi is outlined.     

Fundamentals of protein interaction network mapping

Studying protein interaction networks of all proteins in an organism (“interactomes”) remains one of the major challenges in modern biomedicine. Such information is crucial to understanding cellular pathways and developing effective therapies for the treatment of human diseases. Over the past two decades, diverse biochemical, genetic, and cell biological methods have been developed to map interactomes. In this review, we highlight basic principles of interactome mapping. Specifically, we discuss the strengths and weaknesses of individual assays, how to select a method appropriate for the problem being studied, and provide general guidelines for carrying out the necessary follow‐up analyses. In addition, we discuss computational methods to predict, map, and visualize interactomes, and provide a summary of some of the most important interactome resources. We hope that this review serves as both a useful overview of the field and a guide to help more scientists actively employ these powerful approaches in their research.     

Truncation of a P1 leader proteinase facilitates potyvirus replication in a non‐permissive host

The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self‐cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N‐terminal domain is relevant for host factor interaction and suggested that the C‐terminal domain is also involved. In the absence of plant factors, the N‐terminal end of Plum pox virus P1 antagonizes protease self‐processing. We performed extended deletion mutagenesis analysis to define the N‐terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain‐of‐function phenotype, strongly increasing local infection in a non‐permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids.     

Entry of the DNA virus, Ectocarpus fasciculatus virus type 1 (Phycodnaviridae), into host cell cytosol and nucleus

The process by which Ectocarpus fasciculatus virus type 1 (EfasV‐1) infects zoospores of its brown algal host was studied by electron microscopy. Upon virus attachment to the target cell, the integral membrane component of the viral capsid fuses with the host plasma membrane, and the 140‐nm viral DNA‐protein core enters the cytosol. Within 5 min after infection, particles resembling viral cores appeared in the nucleus. The entry mechanism of EfasV‐1 into the host nucleus remains enigmatic.     

Heat shock protein 70 is necessary for Rice stripe virus infection in plants

Heat shock proteins 70 (HSP70s) are a highly conserved family of genes in eukaryotes, and are involved in a remarkable variety of cellular processes. In many plant positive‐stranded RNA viruses, HSP70 participates in the construction of a viral replication complex and plays various roles during viral infection. Here, we found increased expression of HSP70 following infection by Rice stripe virus (RSV), a negative‐stranded RNA virus, in both rice (the natural host) and Nicotiana benthamiana (an experimental host). Heat treatment of N. benthamiana (Nb) plants enhanced viral infection, whereas RSV infection was retarded and viral RNAs accumulated at a low level when HSP70 was silenced. In both bimolecular fluorescence complement and in vitro pull‐down assays, the N‐terminus of RSV RNA‐dependent RNA polymerase (RdRp) interacted and co‐localized with the HSP70s of both plants (OsHSP70 and NbHSP70). The localization of the N‐terminus of RdRp when expressed alone was not obviously different from when it was co‐expressed with OsHSP or NbHSP, and vice versa. RSV infection also had no effect on the localization of host HSP70. These results demonstrate that host HSP70 is necessary for RSV infection and probably plays a role in viral replication by interacting with viral RdRp, which provides the first evidence of an interacting host protein related to RSV replication, which has been little studied to date.     

Bending of the BST‐2 coiled‐coil during viral budding

BST‐2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV‐1 and other viruses by inhibiting viral spreading. Structurally, BST‐2 is a homo‐dimeric coiled‐coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The C‐terminal membrane anchor of BST‐2 is inserted into the budding virus while the N‐terminal membrane anchor remains in the host cell membrane creating a viral tether. The structural mechanism of viral budding and tethering as mediated by BST‐2 is not clear. To more fully describe the mechanism of viral tethering, we created a model of BST‐2 embedded in a membrane and used steered molecular dynamics to simulate the transition from the host cell membrane associated form to the cell‐virus membrane bridging form. We observed that BST‐2 did not transition as a rigid structure, but instead bent at positions with a reduced interface between the helices of the coiled‐coil. The simulations for the human BST‐2 were then compared with simulations on the mouse homolog, which has no apparent weak spots. We observed that the mouse homolog spread the bending across the ectodomain, rather than breaking at discrete points as observed with the human homolog. These simulations support previous biochemical and cellular work suggesting some flexibility in the coiled‐coil is necessary for viral tethering, while also highlighting how subtle changes in protein sequence can influence the dynamics and stability of proteins with overall similar structure.