SARS-CoV-2-Mediated Lung Edema and Replication Are Diminished by Cystic Fibrosis Transmembrane Conductance Regulator Modulators.

Coronaviruses (CoVs) of genera α, ß, γ, and δ encode proteins that have a PDZ-binding motif (PBM) consisting of the last four residues of the envelope (E) protein (PBM core). PBMs may bind over 400 cellular proteins containing PDZ domains (an acronym formed by the combination of the first letter of the names of the three first proteins where this domain was identified), making them relevant for the control of cell function. Three highly pathogenic human CoVs have been identified to date: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. The PBMs of the three CoVs were virulence factors. SARS-CoV mutants in which the E protein PBM core was replaced by the E protein PBM core from virulent or attenuated CoVs were constructed. These mutants showed a gradient of virulence, depending on whether the alternative PBM core introduced was derived from a virulent or an attenuated CoV. Gene expression patterns in the lungs of mice infected with SARS-CoVs encoding each of the different PBMs were analyzed by RNA sequencing of infected lung tissues. E protein PBM of SARS-CoV and SARS-CoV-2 dysregulated gene expression related to ion transport and cell homeostasis. Decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA, essential for alveolar edema resolution, was shown. Reduced CFTR mRNA levels were associated with edema accumulation in the alveoli of mice infected with SARS-CoV and SARS-CoV-2. Compounds that increased CFTR expression and activity, significantly reduced SARS-CoV-2 growth in cultured cells and protected against mouse infection, suggesting that E protein virulence is mediated by a decreased CFTR expression. IMPORTANCE Three highly pathogenic human CoVs have been identified: SARS-CoV, MERS-CoV, and SARS-CoV-2. The E protein PBMs of these three CoVs were virulence factors. Gene expression patterns associated with the different PBM motifs in the lungs of infected mice were analyzed by deep sequencing. E protein PBM motif of SARS-CoV and SARS-CoV-2 dysregulated the expression of genes related to ion transport and cell homeostasis. A decrease in the mRNA expression of the cystic fibrosis transmembrane conductance regulator (CFTR), which is essential for edema resolution, was observed. The reduction of CFTR mRNA levels was associated with edema accumulation in the lungs of mice infected with SARS-CoV-2. Compounds that increased the expression and activity of CFTR drastically reduced the production of SARS-CoV-2 and protected against its infection in a mice model. These results allowed the identification of cellular targets for the selection of antivirals.

Viral PDZ Binding Motifs Influence Cell Behavior Through the Interaction with Cellular Proteins Containing PDZ Domains.

Viruses have evolved to interact with their hosts. Some viruses such as human papilloma virus, dengue virus, SARS-CoV, or influenza virus encode proteins including a PBM that interact with cellular proteins containing PDZ domains. There are more than 400 cellular protein isoforms with these domains in the human genome, indicating that viral PBMs have a high potential to influence the behavior of the cell. In this review we analyze the most relevant cellular processes known to be affected by viral PBM-cellular PDZ interactions including the establishment of cell-cell interactions and cell polarity, the regulation of cell survival and apoptosis and the activation of the immune system. Special attention has been provided to coronavirus PBM conservation throughout evolution and to the role of the PBMs of human coronaviruses SARS-CoV and MERS-CoV in pathogenesis.

Genetically Engineered Live-Attenuated Middle East Respiratory Syndrome Coronavirus Viruses Confer Full Protection against Lethal Infection.

There are no approved vaccines against the life-threatening Middle East respiratory syndrome coronavirus (MERS-CoV). Attenuated vaccines have proven their potential to induce strong and long-lasting immune responses. We have previously described that severe acute respiratory syndrome coronavirus (SARS-CoV) envelope (E) protein is a virulence factor. Based on this knowledge, a collection of mutants carrying partial deletions spanning the C-terminal domain of the E protein (rMERS-CoV-E*) has been generated using a reverse genetics system. One of these mutants, MERS-CoV-E*Δ2in, was attenuated and provided full protection in a challenge with virulent MERS-CoV after a single immunization dose. The MERS-CoV-E*Δ2in mutant was stable as it maintained its attenuation after 16 passages in cell cultures and has been selected as a promising vaccine candidate.IMPORTANCE The emergence of the new highly pathogenic human coronavirus SARS-CoV-2 that has already infected more than 80 million persons, killing nearly two million of them, clearly indicates the need to design efficient and safe vaccines protecting from these coronaviruses. Modern vaccines can be derived from virus-host interaction research directed to the identification of signaling pathways essential for virus replication and for virus-induced pathogenesis, in order to learn how to attenuate these viruses and design vaccines. Using a reverse genetics system developed in our laboratory, an infectious cDNA clone of MERS-CoV was engineered. Using this cDNA, we sequentially deleted several predicted and conserved motifs within the envelope (E) protein of MERS-CoV, previously associated with the presence of virulence factors. The in vitro and in vivo evaluation of these deletion mutants highlighted the relevance of predicted linear motifs in viral pathogenesis. Two of them, an Atg8 protein binding motif (Atg8-BM), and a forkhead-associated binding motif (FHA-BM), when deleted, rendered an attenuated virus that was evaluated as a vaccine candidate, leading to full protection against challenge with a lethal dose of MERS-CoV. This approach can be extended to the engineering of vaccines protecting against the new pandemic SARS-CoV-2.

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies.

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross-species specificity. Here we isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of betacoronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Our work highlights an immunogenic and vulnerable site on the betacoronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.

Middle East Respiratory Syndrome Coronavirus Gene 5 Modulates Pathogenesis in Mice.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a highly lethal pneumonia that emerged in 2012. There is limited information on MERS-CoV pathogenesis, as data from patients are scarce and the generation of animal models reproducing MERS clinical manifestations has been challenging. Human dipeptidyl peptidase 4 knock-in (hDPP4-KI) mice and a mouse-adapted MERS-CoV strain (MERSMA-6-1-2) were recently described. hDPP4-KI mice infected with MERSMA-6-1-2 show pathological signs of respiratory disease, high viral titers in the lung, and death. In this work, a mouse-adapted MERS-CoV infectious cDNA was engineered by introducing nonsynonymous mutations contained in the MERSMA-6-1-2 genome into a MERS-CoV infectious cDNA, leading to a recombinant mouse-adapted virus (rMERS-MA) that was virulent in hDDP4-KI mice. MERS-CoV adaptation to cell culture or mouse lungs led to mutations and deletions in genus-specific gene 5 that prevented full-length protein expression. In contrast, analysis of 476 MERS-CoV field isolates showed that gene 5 is highly stable in vivo in both humans and camels. To study the role of protein 5, two additional viruses were engineered expressing a full-length gene 5 (rMERS-MA-5FL) or containing a complete gene 5 deletion (rMERS-MA-Δ5). rMERS-MA-5FL virus was unstable, as deletions appeared during passage in different tissue culture cells, highlighting MERS-CoV instability. The virulence of rMERS-MA-Δ5 was analyzed in a sublethal hDPP4-KI mouse model. Unexpectedly, all mice died after infection with rMERS-MA-Δ5, in contrast to those infected with the parental virus, which contains a 17-nucleotide (nt) deletion and a stop codon in protein 5 at position 108. Expression of interferon and proinflammatory cytokines was delayed and dysregulated in the lungs of rMERS-MA-Δ5-infected mice. Overall, these data indicated that the rMERS-MA-Δ5 virus was more virulent than the parental one and suggest that the residual gene 5 sequence present in the mouse-adapted parental virus had a function in ameliorating severe MERS-CoV pathogenesis.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus causing human infections with high mortality rate (∼35%). Animal models together with reverse-genetics systems are essential to understand MERS-CoV pathogenesis. We developed a reverse-genetics system for a mouse-adapted MERS-CoV that reproduces the virus behavior observed in humans. This system is highly useful to investigate the role of specific viral genes in pathogenesis. In addition, we described a virus lacking gene 5 expression that is more virulent than the parental one. The data provide novel functions in IFN modulation for gene 5 in the context of viral infection and will help to develop novel antiviral strategies.

Towards a solution to MERS: protective human monoclonal antibodies targeting different domains and functions of the MERS-coronavirus spike glycoprotein.

The Middle-East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus that causes severe and often fatal respiratory disease in humans. Efforts to develop antibody-based therapies have focused on neutralizing antibodies that target the receptor binding domain of the viral spike protein thereby blocking receptor binding. Here, we developed a set of human monoclonal antibodies that target functionally distinct domains of the MERS-CoV spike protein. These antibodies belong to six distinct epitope groups and interfere with the three critical entry functions of the MERS-CoV spike protein: sialic acid binding, receptor binding and membrane fusion. Passive immunization with potently as well as with poorly neutralizing antibodies protected mice from lethal MERS-CoV challenge. Collectively, these antibodies offer new ways to gain humoral protection in humans against the emerging MERS-CoV by targeting different spike protein epitopes and functions.

Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection.

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV-exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries-apart from conventional antibodies-have relatively unique, heavy chain-only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV-infected dromedaries, we identified several MERS-CoV-specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs-composed of the camel VHH linked to a human Fc domain lacking the CH1 exon-had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.

Role of Severe Acute Respiratory Syndrome Coronavirus Viroporins E, 3a, and 8a in Replication and Pathogenesis.

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.