Involvement of SARS‐CoV‐2 accessory proteins in immunopathogenesis
Hayato ItoTomokazu TamuraLei WangKento MoriMasumi TsudaRigel SuzukiSaori SuzukiKumiko YoshimatsuShinya TanakaTakasuke Fukuhara
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Abstract Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the largest single‐stranded RNA virus known to date. Its genome contains multiple accessory protein genes that act against host immune responses but are not required for progeny virus production. The functions of the accessory proteins in the viral life cycle have been examined, but their involvement in viral pathogenicity remains unclear. Here, we investigated the roles of the accessory proteins in viral immunopathogenicity. To this end, recombinant SARS‐CoV‐2 possessing nonsense mutations in the seven accessory protein open reading frames (ORFs) (ORF3a, ORF3b, ORF6, ORF7a, ORF8, ORF9b, and ORF10) was de novo generated using an early pandemic SARS‐CoV‐2 strain as a backbone. We confirmed that the resultant virus (termed ORF3–10 KO) did not express accessory proteins in infected cells and retained the desired mutations in the viral genome. In cell culture, the ORF3–10 KO virus exhibited similar virus growth kinetics as the parental virus. In hamsters, ORF3–10 KO virus infection resulted in mild weight loss and reduced viral replication in the oral cavity and lung tissue. ORF3–10 KO virus infection led to mild inflammation, indicating that an inability to evade innate immune sensing because of a lack of accessory proteins impairs virus growth in vivo and results in quick elimination from the body. Overall, we showed that SARS‐CoV‐2 accessory proteins are involved in immunopathogenicity.Keywords:
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Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.
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In humans, acute and chronic respiratory infections caused by viruses are associated with considerable morbidity and mortality. Respiratory viruses infect airway epithelial cells and induce oxidative stress, yet the exact pathogenesis remains unclear. Oxidative stress activates the transcription factor NRF2, which plays a key role in alleviating redox-induced cellular injury. The transcriptional activation of NRF2 has been reported to affect both viral replication and associated inflammation pathways. There is complex bidirectional crosstalk between virus replication and the NRF2 pathway because virus replication directly or indirectly regulates NRF2 expression, and NRF2 activation can reversely hamper viral replication and viral spread across cells and tissues. In this review, we discuss the complex role of the NRF2 pathway in the regulation of the pathogenesis of the main respiratory viruses, including coronaviruses, influenza viruses, respiratory syncytial virus (RSV), and rhinoviruses. We also summarize the scientific evidence regarding the effects of the known NRF2 agonists that can be utilized to alter the NRF2 pathway.
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Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.
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Introduction In addition to the structural proteins required for viral replication, HIV expresses several additional proteins. Although these proteins are not required for viral replication in tissue culture systems, they are important factors for the development of pathogenesis. These factors, known as accessory proteins, include the HIV proteins Nef, Vpr, Vpu, and Vif. The accessory proteins are a defining feature of lentiviruses, and are necessary for HIV to cause AIDS. Because the accessory proteins play an important role in causing the clinical manifestations of HIV disease, they have become potential targets for antiviral therapies, and many basic research studies have been designed to gain an understanding of their modes of action. The following discussion highlights a number of important insights that have emerged over the past year into how this important class of HIV protein functions. Nef Nef is a 27 kDa myristolated protein that is the first viral protein to accumulate to detectable levels in a cell following HIV infection [1]. It is clear that Nef is necessary for the maintenance of high viral loads and viral pathogenesis in HIV-infected individuals. This finding was originally demonstrated in experiments using the SIV model for HIV infection, whereby macaques infected with Nef mutant virus did not develop normal disease [2]. Instead, only low-level viral replication was detected in these animals, and the normal course of disease progression was severely delayed. However, if viral revertants emerged that restored normal Nef function, viral loads increased and the test animals progressed to AIDS in a normal fashion. These observations correlated with the findings of a study focused on an Australian cohort of patients infected with a Nef-defective derivative of HIV [3]. As observed in the SIV system, individuals exhibited only low-level HIV replication and significantly delayed progression to AIDS, thus confirming the role of Nef in HIV pathogenesis and the relevance of the SIV model to the study of HIV infection. Nef is clearly the most versatile of the accessory proteins, as it has been shown to have multiple activities that may influence HIV replication after infection. These activities can be divided into three separate classes that appear to be genetically distinct because they can be divided by their sensitivity to certain mutations in the Nef protein. For the purposes of this discussion, these three classes will be defined as Nef-mediated cell activation, altered intracellular trafficking of normal cell surface proteins, and the stimulation of the infectivity of virions produced after infection. Several lines of research suggest that Nef contributes to viral-mediated cellular activation by altering T-cell signal transduction and activation. Previous studies utilizing Nef transgenic mice revealed that Nef expression led to elevated T-cell signaling [4]. Additionally, recent reports have shown that Nef interacts with lipid rafts, which play an important role in T-cell signaling, as interaction with the rafts is required for Nef to prime T cells for activation [5]. Nef-mediated activation is thought to occur through the mimicking of signaling through the T-cell receptor. This concept was best demonstrated in a study by McMichael et al. utilizing a Jurkat cell line in which Nef expression could be induced by the addition of tetracycline [6]. Through the use of gene chip analysis, these studies revealed that gene expression stimulated by Nef expression was 97% identical to that observed after stimulation of the T-cell receptor. Use of mutant cell lines revealed that the tyrosine kinase Zap70 and the zeta chain of the T-cell receptor are required for Nef-mediated stimulation of the entire set of the normally induced genes. These studies support the model that Nef expression activates the infected T cell to provide an optimal environment for viral replication. One of the genes stimulated by Nef is FasL, the ligand for the death receptor Fas [7]. It is believed that the presence of FasL may lead to the death of reactive CD8 cells in vivo. One argument against this model is that the expression of FasL should lead to the death of cells expressing Nef through intercellular binding to Fas. However, recent results by Greene et al. have found that the virus can evade this potential dilemma through Nef interaction with the apoptosis stimulating kinase [8]. This interaction blocks apoptosis stimulating kinase function, leading to a block in the induction of apoptosis by signaling through Fas. Nef is believed to activate T cells by direct association with a factor designated the Nef-associated kinase (NAK) [9]. Two recent publications substantiate the model that NAK is the p21-associated kinase 2 (PAK2) [10,11]. These studies have found that, although only a fraction of the PAK2 present in cells is associated with Nef, most of the PAK2 kinase activity is associated with Nef. This finding suggests that binding of NAK to the SH3 domain of Nef leads to activation of its kinase activity, which could potentially activate a program of gene expression similar to that observed after T-cell activation [12]. Nef may also contribute to HIV pathogenicity by altering intracellular trafficking of normal cell surface proteins. One of the earliest observations related to the action of Nef is the downregulation of cell surface expression of CD4, the receptor for HIV [13]. Subsequent studies revealed that Nef acts post-translationally by increasing the rate of CD4 endocytosis and lysomal degradation [14]. The cytoplasmic tail of CD4, particularly a di-leucine repeat sequence contained in its membrane proximal region, is key for the effective CD4 response to Nef [14]. CD4 downregulation appears to be advantageous to viral production because an excess of CD4 on the cell surface has been found to inhibit env incorporation and virion budding [15,16]. Demonstration of direct binding of Nef to the cytoplasmic tail of CD4 has been difficult, suggesting that the interaction is relatively weak. Using nuclear magnetic resonance methods, studies of the interaction between CD4 and Nef suggest that the interaction is indeed weak, with a dissociation constant of slightly less than 1 μM. Subsequently, it was shown that Nef can also downregulate class I MHC for the purpose of immune evasion [17,18]. Studies in the past year add the co-stimulatory protein CD28 to the list of proteins whose cell surface expression are downregulated by the expression of Nef [5]. Interestingly, the downregulation of all three proteins can be genetically separated. This observation suggests that the ability of Nef to downregulate these proteins was independently selected. The mechanism of downregulation of MHC class I is distinct from that of CD4 and CD28 [5,19,20]. While CD4 and CD28 are downregulated through clatherin-mediated endocytosis, MHC class I downregulation is mediated by a distinct mechanism. Consistent with this model, Garcia et al. found that ikarugamycin, a naturally occurring anti-protozoan inhibitor of clatherin-mediated endocytosis, blocks Nef-mediated downregulation of CD4 cell surface expression [21]. After endocytosis, Nef redirects CD4 to late endosomes, and subsequently lysosomes, where it is degraded. Normally, CD4 is recycled to the cell surface. This alteration of the normal trafficking of CD4 is believed to be mediated by a di-acidic motif present in Nef. One group believes that this domain specifically interacts with the adaptor protein B-cop, which mediates delivery to late endosomes [22]. In contrast, another group finds that the di-acidic domain is not important for B-cop binding or CD4 downregulation [23]. Recent studies by Carl et al. have revealed that Nef-mediated downregulation of CD4 is important for normal viral replication and pathogenesis in the SIV model [24]. Macaques were infected with a virus expressing three point mutations in Nef, which had previously been shown to be involved in CD4 downregulation. Initially, only low-level viral replication was detected. Subsequent increases in viral replication coincided with reversion of the mutations or the appearance of second-site mutations. The mutated derivative of SIV Nef, which contained two point mutations, led to defects in both CD4 downregulation and the enhancement of infectivity. The accessory protein Nef can also work to stimulate the infectivity of HIV virions [25]. Although part of this effect is a consequence of CD4 downregulation, even in the absence of CD4, HIV-1 particles produced in the presence of Nef are significantly more infectious than virions produced in the absence of Nef. Nef is packaged into virions, where it can be cleaved by the viral protease during virion maturation [26]. Nef is also present in isolated virion cores [27]. Virions produced in the absence of Nef are less efficient for proviral DNA synthesis, although Nef does not appear to influence directly the process of reverse transcription [28]. This finding has led to a model suggesting that Nef plays a role in the uncoating of the viral core after fusion. An alternative model, suggesting that Nef is important for efficient viral entry, has been gaining support. This new model is supported by the observation that the importance of Nef for infectivity is dependent on the mechanism of virion entry. When entry is mediated by the vesticular stomatitis virus, which enters via receptor-mediated endocytosis, the presence of Nef does not significantly stimulate infectivity [29]. Consistent with the path of entry being important, it was recently shown that virions pseudotyped with the ebola virus envelope, which also enters via receptor-mediated endocytosis and acidification, are also not sensitive to the presence of Nef [30]. These studies suggest that Nef may only be important for the HIV entry pathway. This possibility is supported by a report that finds virion fusion is more efficient in the presence of Nef [31]. Finally, Zhou and Aiken have found new evidence that Nef is influencing virion entry. Their group has developed a system in which only virions that fuse together are infectious [32]. With this system, they have demonstrated that Nef can influence infectivity after introduction in trans, being introduced by intravirion fusion. Interestingly, Nef only complements when produced in the envelope protein containing component. This suggests that Nef has a role at the virion envelope. Although these new results argue that Nef plays a role in stimulating the efficiency of viral entry, more studies are required to determine the exact role that Nef plays in stimulating the infectivity of HIV. Vpr The Vpr protein, like the Nef protein, is present in viral particles. Although it has been suggested that Vpr can be incorporated into assembling virions in amounts equimolar with Gag (1500-2000 copies) [33], experimental studies suggest that much smaller amounts are present in particles, in the range of 100-200 copies [34,35]. A minor portion of the virion-associated Vpr is phosphorylated. Incorporation of Vpr into virions is mediated through specific interactions with the carboxyl terminal region of p55 Gag [36], which corresponds to p6 in the proteolytically processed protein. Interestingly, after maturation, Vpr is associated with the virion core, while the p6 protein is not [37]. Vpr also appears to be an HIV protein that provides multiple functions for HIV replication. These multiple activities include induction of cell-cycle arrest, transactivation of viral and cellular gene expression, participation in the nuclear import of the HIV genome, modulation of viral replication kinetics, and induction of apoptosis [38]. Although it was initially believed that many of these functions were the consequence of a single activity of Vpr, it has recently been shown that the activities leading to cell-cycle arrest, induction of apoptosis, and the activation of at least some genes can be separated through the use of certain mutations and cellular contexts [39]. For instance, the ability of Vpr to act as a glucocorticoid receptor co-activator and the ability to mediate cell-cycle arrest can be separated by mutagenesis [40]. Recently, a new function for Vpr has been proposed. Studies of Jurkat cells constitutively expressing Vpr revealed that these cells could not be efficiently infected. Subsequent characterization found that expression of Vpr lead to a decrease in the cell-surface expression of CD4 [41]. Understanding the mechanism of this new action of Vpr requires further study. Vpr has been demonstrated to play a role in the ability of HIV to infect non-dividing cells by facilitating the nuclear localization of the pre-integration complex [42]. Consistent with this model, Vpr is present in the reverse transcription complex and the pre-integration complex [43]. This potential activity has lead to extensive study of the nuclear localization activity contained in the Vpr protein. Various studies, including two published in the past year, have suggested that Vpr contains two sequences that can mediate nuclear localization [44,45]. A lack of sequence similarity and functional differences in the action of the two putative nuclear localization signals (NLS) suggests that they function through distinct pathways or provide distinct functions. For instance, one of the sequences may act like a nucleocytoplasmic transport factor by directly tethering the viral genome to the nuclear pore rather than a traditional NLS. Consistent with this model, Vpr is found to be associated with the nuclear pore when expressed in cells and can be biochemically demonstrated to bind to components of the nuclear pore complex [46]. The complexity of the NLS in Vpr has made the understanding of their function a contentious issue. One explanation for the complexity was recently revealed by the identification of a nuclear export signal (NES) in Vpr [45,47]. A NES functions to export proteins from the nucleus. When a protein contains both a NLS and a NES, as in the case of Vpr, it continually shuttles between the nuclear and cytoplasmic compartment. The reason for Vpr shuttling has yet to be defined. An additional activity of Vpr is demonstrated by its ability to block cell division [48]. Vpr expression causes cells to accumulate in the G2 phase of the cell cycle [49]. The expression of Vpr has been shown to prevent the activation of the cyclin B/p34cdc2 complex, which is a regulator of the cell cycle important for entry into mitosis [50,51]. Consistent with this model, it was recently shown that several yeast cell-cycle proteins are required for Vpr-mediated cell-cycle arrest in fission yeast [52]. Vpu The Vpu polypeptide is an integral membrane phosphoprotein that is primarily localized in the internal membranes of the cell [53]. The two functions of Vpu, the downmodulation of CD4 expression and the enhancement of virion release, can be genetically separated [54]. In HIV-infected cells, the simultaneous expression of both the viral receptor, CD4, and the viral envelope protein in the endoplasmic reticulum leads to the formation of complexes that trap both proteins within this compartment. The formation of intracellular Env-CD4 complexes decreases the amount of envelope protein, interfering with virion assembly. Vpu liberates the viral envelope by facilitating the ubiquitin-mediated degradation of CD4 molecules complexed with Env [55]. Proteosome-mediated degradation of CD4 complexed with Vpu is mediated by interaction between Vpu and βTrCP, a component of the SCF ubiquitin ligase complex [56]. Interestingly, Vpu itself is not degraded. Recent studies find that Vpu can act as a general inhibitor of βTrCP function [57]. This is best demonstrated by the observation that Vpu blocks the normal degradation of IκBa, which is typically degraded by interaction with βTrCP, after stimuli that activate the transcription factor nuclear factor-κB (NF-κB). Interestingly, NF-κB is a key player in the activation of the HIV LTR promoter. Therefore, Vpu may be a modulator of NF-κB-regulated viral and cellular gene expression. Vpu also stimulates HIV replication by facilitating the release of HIV from the surface of an infected cell. In the absence of Vpu, large numbers of virions can be seen attached to the surface of infected cells [58]. The cell-bound virus is infectious, as recently demonstrated by Bour et al.[57]. Using a cell culture system that selects for cell-cell transmission, these experiments led to the selection of a spontaneous Vpu mutation. Vpu is believed to form a channel-like structure in the plasma membrane that plays a role in virion release. Recent structural studies support the idea that Vpu forms ion channels in lipid bilayers [59]. It was found that pores were formed in a manner consistent with the self-assembly of oligomers and the structural findings. A study by Strebel et al. found that cell surface CD4 can inhibit virion release by disrupting Vpu function [60]. It is believed that Vpu-CD4 interactions block formation of the oligomers necessary to form the pores required for Vpu to facilitate virion release. Together, these results obtained in the past year support a model where Vpu facilitates virion release through the formation of ion-conducting channels in the plasma membrane. Vif Vif is a 23 kD a polypeptide that is essential for the replication of HIV in peripheral blood lymphocytes, macrophages, and certain cell lines [61]. In most cell lines, Vif is not required. These cell lines are called permissive for Vif mutants of HIV. Virions generated in permissive cells can infect non-permissive cells but the virus subsequently produced is not infectious. Complementation studies reveal that it is possible to restore the infectivity of HIV Vif mutants by expression of Vif in producer cells but not in target cells [62]. These results show that Vif must be present during virion assembly. Vif can be found at low levels in virions of HIV [63]. However, the incorporation of Vif may be non-specific, as Vif is also incorporated into heterologous retroviruses such as murine leukemia viruses [64]. Vif co-localizes with Gag [65] and has recently been shown to be present at higher levels in virions that contain unprocessed Gag proteins or mutations in the capsid region of the Gag precursor [65,66]. These results suggest that Vif packaging is regulated and that virion-associated Vif might not have a specific function. There are two obvious models for the permissive phenotype of Vif: either the non-permissive cell line lacks a factor that has a Vif-like function, or non-permissive cells contain an antiviral factor that is blocked by Vif. These models were tested using heterokaryons generated by the fusion of permissive and non-permissive cells. Vif-negative virus produced from the heterokaryons was non-infectious, revealing that non-permissive cells contain a naturally occurring antiviral factor that is overcome by Vif [67,68]. Further substantiation for a model that Vif is counteracting an antiviral cellular factor came from the finding that Vif proteins from different lentiviruses are species specific [69]. For instance, HIV Vif can stimulate the infectivity of HIV-2 and SIV produced in human non-permissive cells, while SIV Vif protein does not function in human cells. Together, these observations suggest that cellular factors, rather than viral components, are the target of Vif action. Vif-defective HIV strains can enter cells but cannot efficiently synthesize the proviral DNA [62]. It is not clear whether Vif facilitates reverse transcription per se, viral uncoating, or the overall stability of the viral nucleoprotein complex. Studies by Madani and Kabat reveal that Vif works at a post-penetration stage of the viral life-cycle [70]. These investigators found that murine leukemia virus-pseudotyped HIV capsids required Vif for replication, while murine leukemia virus produced from the same cells was infectious. Other results recently obtained by Pomerantz et al. support a role for Vif in reverse transcription [71]. By exposing Vif-defective particles to high concentrations of nucleotides, it is possible to stimulate reverse transcription within virions and to partially restore infectivity. Vif mutant virions have improperly packed nucleoprotein cores, as revealed by electron microscopic analyses [72]. A recent study has also shown that Vif-defective virions have cores that are less stable than wild-type virions [73]. Together, these observations suggest a model where Vif facilitates a post-entry step that is essential for the completion of reverse transcription. A new model for Vif function has been suggested in three recent studies that found Vif is an RNA binding protein that binds to intracellular genomic HIV RNA [74-76]. A recent report found that FIV Vif localized primarily in the nucleus is in support of an association with virion RNA [77]. To make these observations compatible with the results suggesting a post-entry role, it has been proposed that Vif promotes efficient reverse transcription after virion entry by modulating nucleic acid components within the virion core. Conclusion The accessory proteins represent an unexploited target for therapies directed to treat HIV infection. Although they can be shown to be non-essential for viral replication in certain cell culture contexts, they are clearly required for the generation of high viral loads and pathogenesis. Future studies should be focused on further elucidation of their mechanisms of action and their association with elements contributing to HIV disease.
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Background: The recent coronavirus disease-19 (COVID-19) emergence worldwide associated with pneumonia-like symptoms poses a great threat to human health. This article highlights the genomic structure, viral replication, classification of structural and non-structural proteins according to their primary function, and viral pathogenesis. Search Methodology: Literature of this review was collected from different databases like PubMed, Embase, Scopus, Medline, and Web of Science by using following keywords COVID-19, Genomic structure, Non-structural proteins, Viral replication, Viral pathogenesis. Results: Severe acute respiratory syndrome-2 (SARS-CoV-2) is single-stranded RNA enveloped virus containing less conserved matrix (M), nucleocapsid (N), and spike (S) proteins involved in viral entry, virion assembly, nucleocapsid formation and defining coronavirus shape. S protein and Angiotensin-converting enzyme 2(ACE-2) receptor binding facilitates virus entry and membrane fusion. M and N proteins are expressed together to form mature virion assembly, transported in secretory vesicles for release towards the cell membrane. SARS-CoV-2 pathogenesis is complex involving pathological pathways such as cytokines dysregulation, cytopathic effects, innate immune response deficiencies, ACE-2 down regulation and direct immune cell evasion, causing severe injury to lung tissues. Conclusion: A broad understanding of replicase transcriptase complex (RTC) intricacies in viral replication and the role of different accessory and structural proteins in viral pathogenesis enhances the ability to develop therapeutic tools with reducing disease potential.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the current pandemic of coronavirus disease 2019 (COVID-19) that has killed nearly one million people so far. While this is a respiratory virus, surprisingly, it has been recognized that patients with cardiovascular disease are likely to be affected severely and die of COVID-19. This phenomenon cannot be explained by the generally accepted logic that the SARS-CoV-2 infection/replication is the sole determinant of the actions of the virus to define the fate of host cells. I herein propose the viral protein fragment theory of COVID-19 pathogenesis based on my observations in cultured human vascular cells that SARS-CoV-2 spike protein can activate cell signaling events without the rest of the viral components. It is generally thought that SARS-CoV-2 and other single-stranded RNA viruses attach to the host cells through the interactions between surface proteins of the viral capsid and the host cell receptors; the fusion and the entry of the viral components, resulting in the replication of the viruses; and the host cell responses are the consequence of these events. I hypothesize that, as humans are infected with SARS-CoV-2, the virus releases (a) fragment(s) of the spike protein that can target host cells for eliciting cell signaling without the rest of the viral components. Thus, COVID-19 patients are subjected to the intact virus infecting the host cells for the replication and amplification as well as the spike protein fragments that are capable of affecting the host cells. I propose that cell signaling elicited by the spike protein fragments that occur in cardiovascular cells would predispose infected individuals to develop complications that are seen in severe and fatal COVID-19 conditions. If this hypothesis is correct, then the strategies to treat COVID-19 should include, in addition to agents that inhibit the viral replication, therapeutics that inhibit the viral protein fragment-mediated cardiovascular cell signaling.
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Abstract Glioma tumor suppressor candidate region gene 2 protein (GLTSCR2) is a nucleolar protein. In the investigation of the role of GLTSCR2 that played in the cellular innate immune response to viral infection, we found GLTSCR2 supported viral replication of rhabdovirus, paramyxovirus, and coronavirus in cells. Viral infection induced translocation of GLTSCR2 from nucleus to cytoplasm that enabled GLTSCR2 to attenuate type I interferon IFN-β and support viral replication. Cytoplasmic GLTSCR2 was able to interact with retinoic acid-inducible gene I (RIG-I) and the ubiquitin-specific protease 15 (USP15), and the triple interaction induced USP15 activity to remove K63-linked ubiquitination of RIG-I, leading to attenuation of RIG-I and IFN-β. Blocking cytoplasmic translocation of GLTSCR2, by deletion of its nuclear export sequence (NES), abrogated its ability to attenuate IFN-β and support viral replication. GLTSCR2-mediated attenuation of RIG-I and IFN-β led to alleviation of host cell innate immune response to viral infection. Our findings suggested that GLTSCR2 contributed to efficient viral replication, and GLTSCR2 should be considered as a potential target for therapeutic control of viral infection.
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Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications. Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.
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