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.     

Viruses,cancer and non-self recognition

Virus–host interactions form an essential part of every aspect of life, and this review is aimed at looking at the balance between the host and persistent viruses with a focus on the immune system. The virus–host interaction is like a cat-and-mouse game and viruses have developed ingenious mechanisms to manipulate cellular pathways, most notably the major histocompatibility (MHC) class I pathway, to reside within infected cell while evading detection and destruction by the immune system. However, some of the signals sensing and responding to viral infection are derived from viruses and the fact that certain viruses can prevent the infection of others, highlights a more complex coexistence between the host and the viral microbiota. Viral immune evasion strategies also illustrate that processes whereby cells detect and present non-self genetic material to the immune system are interlinked with other cellular pathways. Immune evasion is a target also for cancer cells and a more detailed look at the interfaces between viral factors and components of the MHC class I peptide-loading complex indicates that these interfaces are also targets for cancer mutations. In terms of the immune checkpoint, however, viral and cancer strategies appear different.     

Vaccinia Virus Proteins A52 and B14 Share a Bcl-2–Like Fold but Have Evolved to Inhibit NF-κB rather than Apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways.     

Virus proteins similar to human proteins as possible disturbance on human pathways

Cancer is not rare anywhere in the world now, and the global burden of cancer continues to increase largely every year. Previous research on infections and cancers reported that, about 17.8 % of the cancers worldwide, which are over 1.9 million cases of cancer, are related to viral infections. At least six oncoviruses, cancer-causing viruses, have been known so far, which include hepatitis B virus, hepatitis C virus, Epstein–Barr virus (EBV or HHV-4), human papillomavirus, human T lymphotropic virus type 1, Kaposi’s sarcoma-associated herpesvirus (KSHV or HHV-8), but the pathogenic mechanism is far from being completely understood. In this study, assuming that finding human proteins significantly similar to viral oncoproteins leads to a categorization of the cancer-related pathways that are currently not clearly known, we analyzed different types of virus-caused cancers based on their similarity in order to clarify the unknown cancer mechanisms. As a result, we obtained several potential tumor pathways that may be significant and essential in oncogenic cancer process, which will be helpful for further study on cancer mechanisms and the development of new drug targets.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-014-9141-y) contains supplementary material, which is available to authorized users.     

Paramyxovirus assembly and budding: Building particles that transmit infections

The paramyxoviruses define a diverse group of enveloped RNA viruses that includes a number of important human and animal pathogens. Examples include human respiratory syncytial virus and the human parainfluenza viruses, which cause respiratory illnesses in young children and the elderly; measles and mumps viruses, which have caused recent resurgences of disease in developed countries; the zoonotic Hendra and Nipah viruses, which have caused several outbreaks of fatal disease in Australia and Asia; and Newcastle disease virus, which infects chickens and other avian species. Like other enveloped viruses, paramyxoviruses form particles that assemble and bud from cellular membranes, allowing the transmission of infections to new cells and hosts. Here, we review recent advances that have improved our understanding of events involved in paramyxovirus particle formation. Contributions of viral matrix proteins, glycoproteins, nucleocapsid proteins, and accessory proteins to particle formation are discussed, as well as the importance of host factor recruitment for efficient virus budding. Trafficking of viral structural components within infected cells is described, together with mechanisms that allow for the selection of specific sites on cellular membranes for the coalescence of viral proteins in preparation of bud formation and virion release.     

Mutation of a Single Residue Renders Human Tetherin Resistant to HIV-1 Vpu-Mediated Depletion

The recently identified restriction factor tetherin/BST-2/CD317 is an interferon-inducible trans-membrane protein that restricts HIV-1 particle release in the absence of the HIV-1 countermeasure viral protein U (Vpu). It is known that Tantalus monkey CV1 cells can be rendered non-permissive to HIV-1 release upon stimulation with type 1 interferon, despite the presence of Vpu, suggesting species-specific sensitivity of tetherin proteins to viral countermeasures such as Vpu. Here we demonstrate that Tantalus monkey tetherin restricts HIV-1 by nearly two orders of magnitude, but in contrast to human tetherin the Tantalus protein is insensitive to HIV-1 Vpu. We have investigated tetherin''s sensitivity to Vpu using positive selection analyses, seeking evidence for evolutionary conflict between tetherin and viral countermeasures. We provide evidence that tetherin has undergone positive selection during primate evolution. Mutation of a single amino acid (showing evidence of positive selection) in the trans-membrane cap of human tetherin to that in Tantalus monkey (T45I) substantially impacts on sensitivity to HIV-1 Vpu, but not on antiviral activity. Finally, we provide evidence that cellular steady state levels of tetherin are substantially reduced by Vpu, and that the T45I mutation abrogates this effect. This study provides evidence that tetherin is important in protecting mammals against viral infection, and that the HIV-1 Vpu–mediated countermeasure is specifically adapted to act against human tetherin. It also emphasizes the power of selection analyses to illuminate the molecular details of host–virus interactions. This work suggests that tetherin binding agents might protect it from viral encoded countermeasures and thus make powerful antivirals.     

TNF‐α/IFN‐γ synergy amplifies senescence‐associated inflammation and SARS‐CoV‐2 receptor expression via hyper‐activated JAK/STAT1

Older age and underlying conditions such as diabetes/obesity or immunosuppression are leading host risk factors for developing severe complications from COVID‐19 infection. The pathogenesis of COVID‐19‐related cytokine storm, tissue damage, and fibrosis may be interconnected with fundamental aging processes, including dysregulated immune responses and cellular senescence. Here, we examined effects of key cytokines linked to cellular senescence on expression of SARS‐CoV‐2 viral entry receptors. We found exposure of human umbilical vein endothelial cells (HUVECs) to the inflammatory cytokines, TNF‐α + IFN‐γ or a cocktail of TNF‐α + IFN‐γ + IL‐6, increased expression of ACE2/DPP4, accentuated the pro‐inflammatory senescence‐associated secretory phenotype (SASP), and decreased cellular proliferative capacity, consistent with progression towards a cellular senescence‐like state. IL‐6 by itself failed to induce substantial effects on viral entry receptors or SASP‐related genes, while synergy between TNF‐α and IFN‐γ initiated a positive feedback loop via hyper‐activation of the JAK/STAT1 pathway, causing SASP amplification. Breaking the interactive loop between senescence and cytokine secretion with JAK inhibitor ruxolitinib or antiviral drug remdesivir prevented hyper‐inflammation, normalized SARS‐CoV‐2 entry receptor expression, and restored HUVECs proliferative capacity. This loop appears to underlie cytokine‐mediated viral entry receptor activation and links with senescence and hyper‐inflammation.     

Adenosine triphosphate energy‐independently controls protein homeostasis with unique structure and diverse mechanisms

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid–liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2–12 mM, much higher than required for its previously‐known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic‐acid‐binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding‐induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π‐π, π‐cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy‐independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein‐aggregation diseases.     

Regulation and roles of RNA modifications in aging‐related diseases

With the aging of the global population, accumulating interest is focused on manipulating the fundamental aging‐related signaling pathways to delay the physiological aging process and eventually slow or prevent the appearance or severity of multiple aging‐related diseases. Recently, emerging evidence has shown that RNA modifications, which were historically considered infrastructural features of cellular RNAs, are dynamically regulated across most of the RNA species in cells and thereby critically involved in major biological processes, including cellular senescence and aging. In this review, we summarize the current knowledge about RNA modifications and provide a catalog of RNA modifications on different RNA species, including mRNAs, miRNAs, lncRNA, tRNAs, and rRNAs. Most importantly, we focus on the regulation and roles of these RNA modifications in aging‐related diseases, including neurodegenerative diseases, cardiovascular diseases, cataracts, osteoporosis, and fertility decline. This would be an important step toward a better understanding of fundamental aging mechanisms and thereby facilitating the development of novel diagnostics and therapeutics for aging‐related diseases.     

Interactions between DNA viruses, ND10 and the DNA damage response

ND10 are small nuclear substructures that are defined by the presence the promyelocytic leukaemia protein PML. Many other proteins have been detected within ND10, a complexity that is reflected in reports of their involvement in multiple cellular pathways that include the regulation of gene expression, chromatin dynamics, protein modification, apoptosis, p53 function, senescence, DNA repair, the interferon response and viral infection. This review summarizes recent evidence of similarities between the behaviour of ND10 components and DNA repair pathway proteins in response to viral infection and DNA damage. ND10 structures become associated with the parental genomes and early replication compartments of many DNA viruses, and DNA repair pathway proteins are also recruited to these sites. Similarly, PML and DNA repair proteins are recruited to sites of DNA damage. The mechanisms by which these events might occur, and the implications for ND10 function in DNA virus infection and chromatin metabolism, are discussed.     

A stress-induced cellular aging model with postnatal neural stem cells

Aging refers to the physical and functional decline of the tissues over time that often leads to age-related degenerative diseases. Accumulating evidence implicates that the senescence of neural stem cells (NSCs) is of paramount importance to the aging of central neural system (CNS). However, exploration of the underlying molecular mechanisms has been hindered by the lack of proper aging models to allow the mechanistic examination within a reasonable time window. In the present study, we have utilized a hydroxyurea (HU) treatment protocol and effectively induced postnatal subventricle NSCs to undergo cellular senescence as determined by augmented senescence-associated-β-galactosidase (SA-β-gal) staining, decreased proliferation and differentiation capacity, increased G0/G1 cell cycle arrest, elevated reactive oxygen species (ROS) level and diminished apoptosis. These phenotypic changes were accompanied by a significant increase in p16, p21 and p53 expression, as well as a decreased expression of key proteins in various DNA repair pathways such as xrcc2, xrcc3 and ku70. Further proteomic analysis suggests that multiple pathways are involved in the HU-induced NSC senescence, including genes related to DNA damage and repair, mitochondrial dysfunction and the increase of ROS level. Intriguingly, compensatory mechanisms may have also been initiated to interfere with apoptotic signaling pathways and to minimize the cell death by downregulating Bcl2-associated X protein (BAX) expression. Taken together, we have successfully established a cellular model that will be of broad utilities to the molecular exploration of NSC senescence and aging.     

Genome 3'-end repair in dengue virus type 2

Genomes of RNA viruses encounter a continual threat from host cellular ribonucleases. Therefore, viruses have evolved mechanisms to protect the integrity of their genomes. To study the mechanism of 3′-end repair in dengue virus-2 in mammalian cells, a series of 3′-end deletions in the genome were evaluated for virus replication by detection of viral antigen NS1 and by sequence analysis. Limited deletions did not cause any delay in the detection of NS1 within 5 d. However, deletions of 7–10 nucleotides caused a delay of 9 d in the detection of NS1. Sequence analysis of RNAs from recovered viruses showed that at early times, virus progenies evolved through RNA molecules of heterogeneous lengths and nucleotide sequences at the 3′ end, suggesting a possible role for terminal nucleotidyl transferase activity of the viral polymerase (NS5). However, this diversity gradually diminished and consensus sequences emerged. Template activities of 3′-end mutants in the synthesis of negative-strand RNA in vitro by purified NS5 correlate well with the abilities of mutant RNAs to repair and produce virus progenies. Using the Mfold program for RNA structure prediction, we show that if the 3′ stem–loop (3′ SL) structure was abrogated by mutations, viruses eventually restored the 3′ SL structure. Taken together, these results favor a two-step repair process: non-template-based nucleotide addition followed by evolutionary selection of 3′-end sequences based on the best-fit RNA structure that can support viral replication.     

Adaptive Gene Amplification As an Intermediate Step in the Expansion of Virus Host Range

The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.     

Replication of Epstein–Barr Viral DNA

Epstein–Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV’s host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV’s lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding.     

PLC-γ1 Signaling Plays a Subtype-Specific Role in Postbinding Cell Entry of Influenza A Virus

Host signaling pathways and cellular proteins play important roles in the influenza viral life cycle and can serve as antiviral targets. In this study, we report the engagement of host phosphoinositide-specific phospholipase γ1 (PLC-γ1) in mediating cell entry of influenza virus H1N1 but not H3N2 subtype. Both PLC-γ1-specific inhibitor and short hairpin RNA (shRNA) strongly suppress the replication of H1N1 but not H3N2 viruses in cell culture, suggesting that PLC-γ1 plays an important subtype-specific role in the influenza viral life cycle. Further analyses demonstrate that PLC-γ1 activation is required for viral postbinding cell entry. In addition, H1N1, but not H3N2, infection leads to the phosphorylation of PLC-γ1 at Ser 1248 immediately after infection and independent of viral replication. We have further shown that H1N1-induced PLC-γ1 activation is downstream of epidermal growth factor receptor (EGFR) signaling. Interestingly, both H1N1 and H3N2 infections activate EGFR, but only H1N1 infection leads to PLC-γ1 activation. Taking our findings together, we have identified for the first time the subtype-specific interplay of host PLC-γ1 signaling and H1N1 virus that is critical for viral uptake early in the infection. Our study provides novel insights into how virus interacts with the cellular signaling network by demonstrating that viral determinants can regulate how the host signaling pathways function in virally infected cells.     

Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus–host protein–protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification–mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus–host protein–protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus–host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.     

Computational drug repositioning using similarity constrained weight regularization matrix factorization: A case of COVID‐19

Amid the COVID‐19 crisis, we put sizeable efforts to collect a high number of experimentally validated drug–virus association entries from literature by text mining and built a human drug–virus association database. To the best of our knowledge, it is the largest publicly available drug–virus database so far. Next, we develop a novel weight regularization matrix factorization approach, termed WRMF, for in silico drug repurposing by integrating three networks: the known drug–virus association network, the drug–drug chemical structure similarity network, and the virus–virus genomic sequencing similarity network. Specifically, WRMF adds a weight to each training sample for reducing the influence of negative samples (i.e. the drug–virus association is unassociated). A comparison on the curated drug–virus database shows that WRMF performs better than a few state‐of‐the‐art methods. In addition, we selected the other two different public datasets (i.e. Cdataset and HMDD V2.0) to assess WRMF''s performance. The case study also demonstrated the accuracy and reliability of WRMF to infer potential drugs for the novel virus. In summary, we offer a useful tool including a novel drug–virus association database and a powerful method WRMF to repurpose potential drugs for new viruses.     

Epigenetic mechanisms in virus-induced tumorigenesis

About 15-20% of human cancers worldwide have viral etiology. Emerging data clearly indicate that several human DNA and RNA viruses, such as human papillomavirus, Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, hepatitis B virus, hepatitis C virus, and human T-cell lymphotropic virus, contribute to cancer development. Human tumor-associated viruses have evolved multiple molecular mechanisms to disrupt specific cellular pathways to facilitate aberrant replication. Although oncogenic viruses belong to different families, their strategies in human cancer development show many similarities and involve viral-encoded oncoproteins targeting the key cellular proteins that regulate cell growth. Recent studies show that virus and host interactions also occur at the epigenetic level. In this review, we summarize the published information related to the interactions between viral proteins and epigenetic machinery which lead to alterations in the epigenetic landscape of the cell contributing to carcinogenesis.