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COVID-19 vaccine design and vaccination rollout need to take into account a detailed understanding of antibody durability and cross-neutralizing potential against SARS-CoV-2 and emerging variants of concern (VOCs). Analyses of convalescent sera provide unique insights into antibody longevity and cross-neutralizing activity induced by variant spike proteins, which are putative vaccine candidates. Using sera from 38 individuals infected in wave 1, we show that cross-neutralizing activity can be detected up to 305 days pos onset of symptoms, although sera were less potent against B.1.1.7 (Alpha) and B1.351 (Beta). Over time, despite a reduction in overall neutralization activity, differences in sera neutralization potency against SARS-CoV-2 and the Alpha and Beta variants decreased, which suggests that continued antibody maturation improves tolerance to spike mutations. We also compared the cross-neutralizing activity of wave 1 sera with sera from individuals infected with the Alpha, the Beta or the B.1.617.2 (Delta) variants up to 79 days post onset of symptoms. While these sera neutralize the infecting VOC and parental virus to similar levels, cross-neutralization of different SARS-CoV-2 VOC lineages is reduced. These findings will inform the optimization of vaccines to protect against SARS-CoV-2 variants., The authors assess the durability and long-term cross-reactivity of neutralizing antibodies raised in response to infections with SARS-CoV-2 or variants of concern in humans.

The emergence of SARS-CoV-2 in late 2019 led to the COVID-19 pandemic all over the world. When the virus was first isolated and its genome was sequenced in the early months of 2020, the efforts to develop a vaccine began. Based on prior well-known knowledge about coronavirus, the SARS-CoV-2 spike (S) protein was selected as the main target. Currently, more than one hundred vaccines are being investigated and several of them are already authorized by medical agencies. This review summarizes and compares the current knowledge about main approaches for vaccine development, focusing on those authorized and specifically their immunogenicity, efficacy preventing severe disease, adverse side effects, protection, and ability to cope with emergent SARS-CoV-2 variants.

The COVID-19 pandemic has accelerated the need to identify new antiviral therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir, the first antiviral against SARS-CoV-2 approved for human use, using a quantum-inspired device. We modelled Remdesivir and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of lead compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. We also demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Lastly, we employed an in vitro polymerization assay to demonstrate that these compounds directly inhibit the RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. Together, our data reveal that our QUBO model performs accurate comparisons (BMS-986094) that differed from those predicted by Tanimoto (different forms of vitamin B12); all compounds inhibited replication of SARS-CoV-2 via direct action on RdRP, with both models being useful. While Tanimoto may be employed when performing relatively small comparisons, QUBO is also accurate and may be well suited for very complex problems where computational resources may limit the number and/or complexity of possible combinations to evaluate. Our quantum-inspired screening method can therefore be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

As SARS-CoV-2 variants continue to emerge globally, a major challenge for COVID-19 vaccination is the generation of a durable antibody response with cross-neutralizing activity against both current and newly emerging viral variants. Cross-neutralizing activity against major variants of concern (B.1.1.7, P.1 and B.1.351) has been observed following vaccination, albeit at a reduced potency, but whether vaccines based on the Spike glycoprotein of these viral variants will produce a superior cross-neutralizing antibody response has not been fully investigated. Here, we used sera from individuals infected in wave 1 in the UK to study the long-term cross-neutralization up to 10 months post onset of symptoms (POS), as well as sera from individuals infected with the B.1.1.7 variant to compare cross-neutralizing activity profiles. We show that neutralizing antibodies with cross-neutralizing activity can be detected from wave 1 up to 10 months POS. Although neutralization of B.1.1.7 and B.1.351 is lower, the difference in neutralization potency decreases at later timepoints suggesting continued antibody maturation and improved tolerance to Spike mutations. Interestingly, we found that B.1.1.7 infection also generates a cross-neutralizing antibody response, which, although still less potent against B.1.351, can neutralize parental wave 1 virus to a similar degree as B.1.1.7. These findings have implications for the optimization of vaccines that protect against newly emerging viral variants.

The COVID-19 pandemic has accelerated the need to identify new antiviral therapeutics at pace, including through drug repurposing. We employed a Quadratic Unbounded Binary Optimization (QUBO) model, to search for compounds similar to Remdesivir, the first antiviral against SARS-CoV-2 approved for human use, using a quantum-inspired device. We modelled Remdesivir and compounds present in the DrugBank database as graphs, established the optimal parameters in our algorithm and resolved the Maximum Weighted Independent Set problem within the conflict graph generated. We also employed a traditional Tanimoto fingerprint model. The two methods yielded different lists of lead compounds, with some overlap. While GS-6620 was the top compound predicted by both models, the QUBO model predicted BMS-986094 as second best. The Tanimoto model predicted different forms of cobalamin, also known as vitamin B12. We then determined the half maximal inhibitory concentration (IC50) values in cell culture models of SARS-CoV-2 infection and assessed cytotoxicity. We also demonstrated efficacy against several variants including SARS-CoV-2 Strain England 2 (England 02/2020/407073), B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta). Lastly, we employed an in vitro polymerization assay to demonstrate that these compounds directly inhibit the RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. Together, our data reveal that our QUBO model performs accurate comparisons (BMS-986094) that differed from those predicted by Tanimoto (different forms of vitamin B12); all compounds inhibited replication of SARS-CoV-2 via direct action on RdRP, with both models being useful. While Tanimoto may be employed when performing relatively small comparisons, QUBO is also accurate and may be well suited for very complex problems where computational resources may limit the number and/or complexity of possible combinations to evaluate. Our quantum-inspired screening method can therefore be employed in future searches for novel pharmacologic inhibitors, thus providing an approach for accelerating drug deployment.

Interferon (IFN) mobilizes a cellular anti-viral state by inducing the expression of IFN-stimulated genes (ISGs). Myxovirus resistance 2 (MX2/MxB) is an ISG that inhibits HIV-1 infection by suppressing viral import into the nucleus. The amino-terminal domain (NTD) of MX2 plays an essential role in viral inhibition and, here, we exploit proteomic screening to identify the myosin light chain phosphatase (MLCP) components MYPT1 and PPP1CB as key NTD binding partners. Experimental depletion of either protein, or pharmacologic inhibition of MLCP, reduces MX2 anti-viral activity. Analysis of post-translational modifications defined sites of phosphorylation within the NTD, and replacement of the serines at positions 14, 17 and 18 with the phosphomimetic aspartic acid phenocopies MLCP silencing by abrogating anti-viral function. Phosphorylation of this triple-serine motif impedes MX2’s interaction with its HIV-1 protein target, Capsid, reduces MX2 accumulation at the nuclear envelope, and weakens MX2’s ability to inhibit the nuclear import of non-viral karyophilic cargo. Finally, IFN itself acts to reduce levels of phosphorylation at these key regulatory serines. We therefore propose that homeostatic repression of MX2 through NTD phosphorylation, together with its reversal by MLCP and IFN, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy. Lungs from patients who died from COVID-19 show atypical fused cells, the formation of which is mediated by the SARS-CoV-2 spike protein, and drugs that inhibit TMEM16F can prevent spike-induced syncytia formation.

Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10–15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.

There is a worldwide shortage of reagents to perform detection of SARS-2. Many clinical diagnostic laboratories rely on commercial platforms that provide integrated end-to-end solutions. While this provides established robust pipelines, there is a clear bottleneck in the supply of reagents given the current situation of extraordinary high demand. Some laboratories resort to implementing kit-free handling procedures, but many other small laboratories will not have the capacity to develop those and/or will perform manual handling of their samples. In order to provide multiple workflows for SARS-CoV-2 nucleic acid detection we compared several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), the recently developed RNAdvance Blood (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared different 1-step RT-qPCR Master Mix brands: TaqMan™ Fast Virus 1-Step Master Mix (ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (NEB). We used the Centre for Disease Control (CDC) recommended primers that detect two regions of the viral N gene as well as those that detect the RdRP gene region as per Public Health England (PHE) guidelines (Charite/WHO/PHE). Our data show that the RNA extraction methods provide similar results. Amongst the qPCR reagents tested, TaqMan™ Fast Virus 1-Step Master Mix and Luna® Universal Probe One-Step RT-qPCR Kit proved most sensitive. The N1 and N2 primer-probes provide a more reliable detection than the RdRP-SARSr primer-probe set, particularly in samples with low viral titres. Importantly, we have implemented a protocol using heat inactivation and demonstrate that it has minimal impact on the sensitivity of the qPCR in clinical samples - potentially making SARS-CoV-2 testing portable to settings that do not have CL-3 facilities.

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spike’s polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection., Author summary IFNs play a critical role in regulating host immune responses to virus infections. In cultured cell systems, SARS-CoV-2 can trigger robust IFN and innate immune responses through activation of cytoplasmic RNA sensors, and is also highly sensitive to inhibition by IFN treatment. Consistent with this, ISGs are induced in patients with COVID-19, and inborn errors in the IFN system are associated with severe COVID-19. Our understanding of the repertoire and mechanisms of action of the ISGs that impact SARS-CoV-2 is currently incomplete. In this study, we demonstrate that the IFN-inducible short isoform of NCOA7 is a potent inhibitor of SARS-CoV-2 infection. Our data support the presence of two distinct pathways of SARS-CoV-2 entry and reveal that only the pH-dependent endo-lysosomal pathway is efficiently inhibited by NCOA7. Unravelling the biological significance of the polybasic furin cleavage site in the viral Spike protein, and the concomitant use of TMPRSS2 for cell surface fusion is an area of intense research. Our findings suggest that the acquisition of the polybasic furin cleavage site in Spike may have driven the co-option of the cell surface TMPRSS2 protease to evade the antiviral effects of NCOA7-mediated perturbation of the endo-lysosomal system.

Interaction of the SARS-CoV-2 Spike receptor binding domain (RBD) with the receptor ACE2 on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies, and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS-CoV-2 variants has revealed mutations arising in the RBD, N-terminal domain (NTD) and S2 subunits of Spike. To understand how these mutations affect Spike antigenicity, we isolated and characterized >100 monoclonal antibodies targeting epitopes on RBD, NTD, and S2 from SARS-CoV-2-infected individuals. Approximately 45% showed neutralizing activity, of which ∼20% were NTD specific. NTD-specific antibodies formed two distinct groups: the first was highly potent against infectious virus, whereas the second was less potent and displayed glycan-dependant neutralization activity. Mutations present in B.1.1.7 Spike frequently conferred neutralization resistance to NTD-specific antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes should be considered when investigating antigenic drift in emerging variants., Graphical abstract, The impact of mutations arising in SARS-CoV-2 Spike on antigenicity is still not known. Graham et al. isolate potent neutralizing monoclonal antibodies from individuals experiencing a range of COVID-19 disease severity that target RBD, NTD, and non-S1 epitopes. The B.1.1.7 variant of concern was most resistant to NTD-specific nAbs whereas RBD-specific nAbs retained potent neutralization.

Summary Myxovirus resistance 2 (MX2/MXB) is an interferon (IFN)-induced HIV-1 restriction factor that inhibits viral nuclear DNA accumulation. The amino-terminal domain of MX2 binds the viral capsid and is essential for inhibition. Using in vitro assembled Capsid-Nucleocapsid (CANC) complexes as a surrogate for the HIV-1 capsid lattice, we reveal that the GTPase (G) domain of MX2 contains a second, independent capsid-binding site. The importance of this interaction was addressed in competition assays using the naturally occurring non-antiviral short isoform of MX2 that lacks the amino-terminal 25 amino acids. Specifically, these experiments show that the G domain enhances MX2 function, and the foreshortened isoform acts as a functional suppressor of the full-length protein in a G-domain-dependent manner. The interaction of MX2 with its HIV-1 capsid substrate is therefore multi-faceted: there are dual points of contact that, together with protein oligomerization, contribute to the complexity of MX2 regulation., Graphical Abstract, Highlights • MX2 interacts with the HIV-1 capsid via N-terminal and GTPase (G) domains • The G-domain interaction enhances MX2 binding to the viral capsid • The MX2 short isoform is not antiviral and binds the capsid through its G domain • The MX2 short isoform suppresses the antiviral activity of the long isoform, Myxovirus resistance 2 (MX2) inhibits HIV-1 infection by interacting with the viral capsid. Betancor et al. show that in addition to its N-terminal domain, MX2 binds to the capsid through its GTPase domain, therefore strengthening the interaction. Consequently, the non-antiviral short isoform competes with the long isoform for capsid binding and downmodulates viral inhibition.

Myxovirus resistance 2 (MX2/MXB) is an interferon (IFN)-induced HIV-1 restriction factor that inhibits viral nuclear DNA accumulation. The amino-terminal domain of MX2 binds the viral capsid and is essential for inhibition. Using in vitro assembled Capsid-Nucleocapsid (CANC) complexes as a surrogate for the HIV-1 capsid lattice, we reveal that the GTPase (G) domain of MX2 contains a second, independent capsid binding site. The importance of this interaction was addressed in a series of competition assays using the naturally occurring non-antiviral short isoform of MX2 that lacks the amino-terminal 25 amino acids. Specifically, we show that the G domain enhances MX2 function, and that the foreshortened isoform acts as a functional suppressor of the full-length protein in a G domain-dependent manner. The interaction of MX2 with its HIV-1 capsid substrate is therefore multi-faceted: there are dual points of contact which, together with protein oligomerization, contribute to the complexity of MX2 regulation.

Type 1 interferon suppresses viral replication by upregulating the expression of interferon-stimulated genes with diverse antiviral properties1. The replication of human immunodeficiency virus type 1 (HIV-1) is naturally inhibited by interferon, with the steps between viral entry and chromosomal integration of viral DNA being notably susceptible2–5. The interferon-stimulated gene myxovirus resistance 2 has been defined as an effective postentry inhibitor of HIV-1, but is only partially responsible for interferon’s suppressive effect6–8. Using small interfering RNA-based library screening in interferon-α-treated cells, we sought to characterize further interferon-stimulated genes that target the pre-integration phases of HIV-1 infection, and identified human tripartite-containing motif 5α (TRIM5α) as a potent anti-HIV-1 restriction factor. Human TRIM5α, in contrast with many nonhuman orthologues, has not generally been ascribed substantial HIV-1 inhibitory function, a finding attributed to ineffective recognition of cytoplasmic viral capsids by TRIM5α2,9,10. Here, we demonstrate that interferon-α-mediated stimulation of the immunoproteasome, a proteasome isoform mainly present in immune cells and distinguished from the constitutive proteasome by virtue of its different catalytic β-subunits, as well as the proteasome activator 28 regulatory complex11–13, and the associated accelerated turnover of TRIM5α underpin the reprogramming of human TRIM5α for effective capsid-dependent inhibition of HIV-1 DNA synthesis and infection. These observations identify a mechanism for regulating human TRIM5α antiviral function in human cells and rationalize how TRIM5α participates in the immune control of HIV-1 infection. Using a small interfering RNA-based library screening in interferon-treated cells, the authors identified human tripartite-containing motif 5α as potent restriction factor that inhibits HIV-1 DNA synthesis and infection in a capsid- and immunoproteasome-dependent manner.

Viroporins are viral proteins with ion channel (IC) activity that play an important role in several processes, including virus replication and pathogenesis. While many coronaviruses (CoVs) encode two viroporins, severe acute respiratory syndrome CoV (SARS-CoV) encodes three: proteins 3a, E, and 8a. Additionally, proteins 3a and E have a PDZ-binding motif (PBM), which can potentially bind over 400 cellular proteins which contain a PDZ domain, making them potentially important for the control of cell function. In the present work, a comparative study of the functional motifs included within the SARS-CoV viroporins was performed, mostly focusing on the roles of the IC and PBM of E and 3a proteins. Our results showed that the full-length E and 3a proteins were required for maximal SARS-CoV replication and virulence, whereas viroporin 8a had only a minor impact on these activities. A virus missing both the E and 3a proteins was not viable, whereas the presence of either protein with a functional PBM restored virus viability. E protein IC activity and the presence of its PBM were necessary for virulence in mice. In contrast, the presence or absence of the homologous motifs in protein 3a did not influence virus pathogenicity. Therefore, dominance of the IC and PBM of protein E over those of protein 3a was demonstrated in the induction of pathogenesis in mice., IMPORTANCE Collectively, these results demonstrate key roles for the ion channel and PBM domains in optimal virus replication and pathogenesis and suggest that the viral viroporins and PBMs are suitable targets for antiviral therapy and for mutation in attenuated SARS-CoV vaccines.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a respiratory disease that has a 10% mortality rate. We previously showed that SARS-CoV lacking the E gene (SARS-CoV-¿E) is attenuated in several animal model systems. Here, we show that absence of the E protein resulted in reduced expression of proinflammatory cytokines, decreased numbers of neutrophils in lung infiltrates, diminished lung pathology, and increased mouse survival, suggesting that lung inflammation contributed to SARS-CoV virulence. Further, infection with SARS-CoV-¿E resulted in decreased activation of NF-¿B compared to levels for the wild-type virus. Most important, treatment with drugs that inhibited NF-¿B activation led to a reduction in inflammation and lung pathology in both SARS-CoV-infected cultured cells and mice and significantly increased mouse survival after SARS-CoV infection. These data indicated that activation of the NF-¿B signaling pathway represents a major contribution to the inflammation induced after SARS-CoV infection and that NF-¿B inhibitors are promising antivirals in infections caused by SARS-CoV and potentially other pathogenic human coronaviruses., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the Seventh Framework Programme (FP7/2007-2013) of the European Commission (EC) under the project EMPERIE (EC grant agreement number 223498), and the U.S. National Institutes of Health (2P01AI060699-06A1 and CRIP-HHSN266200700010C). M.L.D. received a contract from the project EMPERIE (EC grant agreement number 223498), and J.A.R.-N. and C.C.-R. received a contract from Fundación La Caixa.

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-beta, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis, This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2010-16705), the European Community's Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699-06A1, W000306844). JAR received a fellowship from the Fundacion La Caixa

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones, polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein, respectively, were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence, immunoelectron microscopy and cell surface protein labeling. In addition, measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed. © 2011 Elsevier Inc., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2007-60978 and BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/2007-2013) under the project “EMPERIE” EC Grant Agreement number 223498, and U.S. National Institutes of Health (ARRA-W000151845). JLN received a fellowship from the Consejo Superior de Investigaciones Cientificas (CSIC) of Spain

A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV., Author Summary Zoonotic coronaviruses, including SARS-CoV, Middle East respiratory syndrome (MERS-CoV), porcine epidemic diarrhea virus (PEDV) and swine delta coronavirus (SDCoV) have recently emerged causing high morbidity and mortality in human or piglets. No fully protective therapy is still available for these CoVs. Therefore, the development of efficient vaccines is a high priority. Live attenuated vaccines are considered most effective compared to other types of vaccines, as they induce a long-lived, balanced immune response. However, safety is the main concern of this type of vaccines because attenuated viruses can eventually revert to a virulent phenotype. Therefore, an essential feature of any live attenuated vaccine candidate is its stability. In addition, introduction of several safety guards is advisable to increase vaccine safety. In this manuscript, we analyzed the mechanisms by which an attenuated SARS-CoV reverted to a virulent phenotype and describe the introduction of attenuating deletions that maintained virus stability. The virus, engineered with two safety guards, provided full protection against challenge with a lethal SARS-CoV. Understanding the molecular mechanisms leading to pathogenicity and the in vivo evaluation of vaccine genetic stability contributed to a rational design of a promising SARS-CoV vaccine.

Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a respiratory disease with a mortality rate of 10%. A mouseadapted SARS-CoV (SARS-CoV-MA15) lacking the envelope (E) protein (rSARS-CoV-MA15-ΔE) is attenuated in vivo. To identify E protein regions and host responses that contribute to rSARS-CoV-MA15-ΔE attenuation, several mutants (rSARS-CoVMA15- E*) containing point mutations or deletions in the amino-terminal or the carboxy-terminal regions of the E protein were generated. Amino acid substitutions in the amino terminus, or deletion of regions in the internal carboxy-terminal region of E protein, led to virus attenuation. Attenuated viruses induced minimal lung injury, diminished limited neutrophil influx, and increased CD4+ and CD8+ T cell counts in the lungs of BALB/c mice, compared to mice infected with the wild-type virus. To analyze the host responses leading to rSARS-CoV-MA15-E* attenuation, differences in gene expression elicited by the native and mutant viruses in the lungs of infected mice were determined. Expression levels of a large number of proinflammatory cytokines associated with lung injury were reduced in the lungs of rSARS-CoV-MA15-E*-infected mice, whereas the levels of anti-inflammatory cytokines were increased, both at the mRNA and protein levels. These results suggested that the reduction in lung inflammation together with a more robust antiviral T cell response contributed to rSARS-CoV-MA15-E* attenuation. The attenuated viruses completely protected mice against challenge with the lethal parental virus, indicating that these viruses are promising vaccine candidates., This work was supported by grants from the Ministry of Science and Innovation of Spain (MICINN BIO2010-16705 and MINECO BIO2013- 42869-R), the European Community’s Seventh Framework Programme (FP7/2007–2013) under the project “EMPERIE” EC grant agreement number 223498, and a U.S. National Institutes of Health (NIH) project (2P01AI060699). J.A.R.-N. and C.C.-R. received a fellowship from the Fundacion La Caixa.

A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation., This work was supported by grants from the government of Spain (BIO2010-16705), the European Community’s Seventh Framework Programme (FP7/ 2007-2013) under the project ‘‘EMPERIE’’ EC Gran Agreement number 223498, and U.S. National Institutes of Health (NIH) (2P01AI060699, 0258-3413/ HHSN266200700010C). JMJG received a JAE fellowship from the CSIC-JAE Program co-funded by the European Social Fund. JARN and CCR received a contract from Fundación La Caixa.

The severe acute respiratory syndrome (SARS) coronavirus (CoV) was identified as the etiological agent of an acute respiratory disease causing atypical pneumonia and diarrhea with high mortality. Different types of SARS-CoV vaccines, including nonreplicative and vectored vaccines, have been developed. Administration of these vaccines to animal model systems has shown promise for the generation of efficacious and safe vaccines. Nevertheless, the identification of side effects, preferentially in the elderly animal models, indicates the need to develop novel vaccines that should be tested in improved animal model systems. Live attenuated viruses have generally proven to be the most effective vaccines against viral infections. A limited number of SARS-CoV attenuating modifications have been described, including mutations, and partial or complete gene deletions affecting the replicase, like the nonstructural proteins (nsp1 or nsp2), or the structural genes, and drastic changes in the sequences that regulate the expression of viral subgenomic mRNAs. A promising vaccine candidate developed in our laboratory was based on deletion of the envelope E gene alone, or in combination with the removal of six additional genes nonessential for virus replication. Viruses lacking E protein were attenuated, grew in the lung, and provided homologous and heterologous protection. Improvements of this vaccine candidate have been directed toward increasing virus titers using the power of viruses with mutator phenotypes, while maintaining the attenuated phenotype. The safety of the live SARS-CoV vaccines is being increased by the insertion of complementary modifications in genes nsp1, nsp2, and 3a, by gene scrambling to prevent the rescue of a virulent phenotype by recombination or remodeling of vaccine genomes based on codon deoptimization using synthetic biology. The newly generated vaccinecandidates are very promising, but need to be evaluated in animal model systems that include young and aged animals., This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT) Bio2007 – 60978, the Consejería de Educación y Cultura de la Comunidad de Madrid S-SAL-0185/06, Ministerio de Ciencia e Innovación (MICINN) Project PROFIT, CIT-010000-2007-8, Fort Dodge Veterinaria, and the European Communities (Frame VII, EMPERIE project HEALTH-F3-2009-223498, and PLAPROVA project KBBE-2008-227056). MLD, JLN, and JMJ received fellowships from the Department of Education and Science of Spain. The work is also supported by a grant from the National Institutes of Health (US) RO1 AI079424-01A1, W000151845 (LE).

To analyze the proteins interacting with the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein, a SARS-CoV was engineered including two tags associated to the E protein. Using this virus, complexes of SARS-CoV E and other proteins were purified using a tandem affinity purification system. Several viral and cell proteins including spike, membrane, non-structural protein 3 (nsp3), dynein heavy chain, fatty acid synthase and transmembrane protein 43 bound E protein. In the present work, we focused on the binding of E protein to nsp3 in infected cells and cell-free systems. This interaction was mediated by the N-terminal acidic domain of nsp3. Moreover, nsp3 and E protein colocalized during the infection. It was shown that E protein was ubiquitinated in vitro and in cell culture, suggesting that the interaction between nsp3 and E protein may play a role in the E protein ubiquitination status and therefore on its turnover. © 2010 Elsevier Inc., This work was supported by grants from the Ministry of Science and Innovation of Spain (BIO2007-60978), the European Union (Frame VII, RiViGene, SSPE-CT-2005-022639 and EMPIRE, 223498), and U.S. National Institutes of Health (ARRA-W000151845). Enrique Álvarez received a contract from the National Institute of Health (ISCIII) of Spain.

Coronaviruses (CoV) are vertebrate pathogens that cause common colds, bronchiolitis and acute respiratory distress syndrome. In fact, their relevance increased when the causative agent of the severe acute respiratory syndrome (SARS) was identified as a CoV. CoV E protein is a small transmembrane protein of between 76-109 amino acids in length that modulates coronavirus morphogenesis, tropism and virulence [1].We sought to elucidate the role of E protein IC activity in virus pathogenesis by combining our knowledge of residues essential for E protein ion conductivity with the manipulation of SARS-CoV genome. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived.We have shown that SARS-CoV E protein IC activity is a virulence determinant.[1] DeDiego, M.L., et al 2008. Virology 376, 379-389.[2] Verdia-Baguena C., et al. 2012. Virology. 432: 485-494.

Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence., Author Summary Several highly pathogenic viruses encode small transmembrane proteins with ion-conduction properties named viroporins. Viroporins are generally involved in virus production and maturation processes, which many times are achieved by altering the ion homeostasis of cell organelles. Cells have evolved mechanisms to sense these imbalances in ion concentrations as a danger signal, and consequently trigger the innate immune system. Recently, it has been demonstrated that viroporins are inducers of cytosolic macromolecular complexes named inflammasomes that trigger the activation of key inflammatory cytokines such as IL-1β. The repercussions of this system in viral pathogenesis or disease outcome are currently being explored. SARS-CoV infection induces an uncontrolled inflammatory response leading to pulmonary damage, edema accumulation, severe hypoxemia and eventually death. In this study, we report that SARS-CoV E protein ion channel activity is a determinant of virulence, as the elimination of this function attenuated the virus, reducing the harmful inflammatory cytokine burst produced after infection, in which inflammasome activation plays a critical role. This led to less pulmonary damage and to disease resolution. These novel findings may be of relevance for other viral infections and can possibly be translated in order to find therapies for their associated diseases.

Severe acute respiratory syndrome virus (SARS-CoV) that lacks the envelope (E) gene (rSARS-CoV-ΔE) is attenuated in vivo. To identify factors that contribute to rSARS-CoV-ΔE attenuation, gene expression in cells infected by SARS-CoV with or without E gene was compared. Twenty-five stress response genes were preferentially upregulated during infection in the absence of the E gene. In addition, genes involved in signal transduction, transcription, cell metabolism, immunoregulation, inflammation, apoptosis and cell cycle and differentiation were differentially regulated in cells infected with rSARS-CoV with or without the E gene. Administration of E protein in trans reduced the stress response in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, or treated with drugs, such as tunicamycin and thapsigargin that elicit cell stress by different mechanisms. In addition, SARS-CoV E protein down-regulated the signaling pathway inositol-requiring enzyme 1 (IRE-1) of the unfolded protein response, but not the PKR-like ER kinase (PERK) or activating transcription factor 6 (ATF-6) pathways, and reduced cell apoptosis. Overall, the activation of the IRE-1 pathway was not able to restore cell homeostasis, and apoptosis was induced probably as a measure to protect the host by limiting virus production and dissemination. The expression of proinflammatory cytokines was reduced in rSARS-CoV-ΔE-infected cells compared to rSARS-CoV-infected cells, suggesting that the increase in stress responses and the reduction of inflammation in the absence of the E gene contributed to the attenuation of rSARS-CoV-ΔE., Author Summary To identify potential mechanisms mediating the in vivo attenuation of SARS-CoV lacking the E gene (rSARS-CoV-ΔE), the effect of the presence of the E gene on host gene expression was studied. In rSARS-CoV-ΔE-infected cells, the expression of at least 25 stress response genes was preferentially upregulated, compared to cells infected with rSARS-CoV. E protein supplied in trans reversed the increase in stress response genes observed in cells infected with rSARS-CoV-ΔE or with respiratory syncytial virus, and by treatment with drugs causing stress by different mechanisms. Furthermore, in the presence of the E protein a subset (IRE-1 pathway), but not two others (PERK and ATF-6), of the unfolded protein response was also reduced. Nevertheless, the activation of the unfolded protein response to control cell homeostasis was not sufficient to alleviate cell stress, and an increase in cell apoptosis in cells infected with the virus lacking E protein was observed. This apoptotic response was probably induced to protect the host by limiting virus production and dissemination. In cells infected with rSARS-CoV-ΔE, genes associated with the proinflammatory pathway were down-regulated compared to cells infected with virus expressing E protein, supporting the idea that a reduction in inflammation was also relevant in the attenuation of the virus deletion mutant.

Severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a viroporin involved in virulence. E protein ion channel (IC) activity is specifically correlated with enhanced pulmonary damage, edema accumulation and death. IL-1β driven proinflammation is associated with those pathological signatures, however its link to IC activity remains unknown. In this report, we demonstrate that SARS-CoV E protein forms protein-lipid channels in ERGIC/Golgi membranes that are permeable to calcium ions, a highly relevant feature never reported before. Calcium ions together with pH modulated E protein pore charge and selectivity. Interestingly, E protein IC activity boosted the activation of the NLRP3 inflammasome, leading to IL-1β overproduction. Calcium transport through the E protein IC was the main trigger of this process. These findings strikingly link SARS-CoV E protein IC induced ionic disturbances at the cell level to immunopathological consequences and disease worsening in the infected organism., The work done by the authors was supported by Grants from the Government of Spain (BIO2013-42869-R, FIS2013-40473-P), Generalitat Valenciana (Prometeo 2012/069), Fundació Caixa Castelló-Bancaixa (P1-1B2012-03) and a U.S. National Institutes of Health (NIH) project (5P01 AI060699). JLN, JMJ and JAR received contracts from NIH. CCR received a contract from Fundacion La Caixa.

The antiviral cytokine interferon activates expression of interferon-stimulated genes to establish an antiviral state. Myxovirus resistance 2 (MX2, also known as MxB) is an interferon-stimulated gene that inhibits the nuclear import of HIV-1 and interacts with the viral capsid and cellular nuclear transport machinery. Here, we identified the myosin light chain phosphatase (MLCP) subunits myosin phosphatase target subunit 1 (MYPT1) and protein phosphatase 1 catalytic subunit-β (PPP1CB) as positively-acting regulators of MX2, interacting with its amino-terminal domain. We demonstrated that serine phosphorylation of the N-terminal domain at positions 14, 17 and 18 suppresses MX2 antiviral function, prevents interactions with the HIV-1 capsid and nuclear transport factors, and is reversed by MLCP. Notably, serine phosphorylation of the N-terminal domain also impedes MX2-mediated inhibition of nuclear import of cellular karyophilic cargo. We also found that interferon treatment reduces levels of phosphorylation at these serine residues and outline a homeostatic regulatory mechanism in which repression of MX2 by phosphorylation, together with MLCP-mediated dephosphorylation, balances the deleterious effects of MX2 on normal cell function with innate immunity against HIV-1.