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1. Structural domains of SARS-CoV-2 nucleocapsid protein coordinate to compact long nucleic acid substrates

   https://doi.org/10.1093/nar/gkac1179  

   Morse, Michael ; Sefcikova, Jana ; Rouzina, Ioulia ; Beuning, Penny J. ; Williams, Mark C. ( December 2022 , Nucleic Acids Research)

   The SARS-CoV-2 nucleocapsid (N) protein performs several functions including binding, compacting, and packaging the ∼30 kb viral genome into the viral particle. N protein consists of two ordered domains, with the N terminal domain (NTD) primarily associated with RNA binding and the C terminal domain (CTD) primarily associated with dimerization/oligomerization, and three intrinsically disordered regions, an N-arm, a C-tail, and a linker that connects the NTD and CTD. We utilize an optical tweezers system to isolate a long single-stranded nucleic acid substrate to measure directly the binding and packaging function of N protein at a single molecule level in real time. We find that N protein binds the nucleic acid substrate with high affinity before oligomerizing and forming a highly compact structure. By comparing the activities of truncated protein variants missing the NTD, CTD, and/or linker, we attribute specific steps in this process to the structural domains of N protein, with the NTD driving initial binding to the substrate and ensuring high localized protein density that triggers interprotein interactions mediated by the CTD, which forms a compact and stable protein-nucleic acid complex suitable for packaging into the virion.
2. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

   https://doi.org/10.1038/s41467-021-21953-3  

   Cubuk, Jasmine ; Alston, Jhullian J. ; Incicco, J. Jeremías ; Singh, Sukrit ; Stuchell-Brereton, Melissa D. ; Ward, Michael D. ; Zimmerman, Maxwell I. ; Vithani, Neha ; Griffith, Daniel ; Wagoner, Jason A. ; et al ( March 2021 , Nature Communications)

   The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.
3. Conserved Genomic Terminals of SARS-CoV-2 as Coevolving Functional Elements and Potential Therapeutic Targets

   https://doi.org/10.1128/mSphere.00754-20  

   Chan, Agnes P. ; Choi, Yongwook ; Schork, Nicholas J. ( December 2020 , mSphere)

   Lee, Benhur (Ed.)

   ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 40 million people worldwide, with over 1 million deaths as of October 2020 and with multiple efforts in the development and testing of antiviral drugs and vaccines under way. In order to gain insights into SARS-CoV-2 evolution and drug targets, we investigated how and to what extent the SARS-CoV-2 genome sequence differs from those of other well-characterized human and animal coronavirus genomes, as well as how polymorphic SARS-CoV-2 genomes are generally. We ultimately sought to identify features in the SARS-CoV-2 genome that may contribute to its viral replication, host pathogenicity, and vulnerabilities. Our analyses suggest the presence of unique sequence signatures in the 3′ untranslated region (3′-UTR) of betacoronavirus lineage B, which phylogenetically encompasses SARS-CoV-2 and SARS-CoV as well as multiple groups of bat and animal coronaviruses. In addition, we identified genome-wide patterns of variation across different SARS-CoV-2 strains that likely reflect the effects of selection. Finally, we provide evidence for a possible host-microRNA-mediated interaction between the 3′-UTR and human microRNA hsa-miR-1307-3p based on the results of multiple computational target prediction analyses and an assessment of similar interactions involving the influenza A H1N1 virus. This interaction also suggests amore »possible survival mechanism, whereby a mutation in the SARS-CoV-2 3′-UTR leads to a weakened host immune response. The potential roles of host microRNAs in SARS-CoV-2 replication and infection and the exploitation of conserved features in the 3′-UTR as therapeutic targets warrant further investigation. IMPORTANCE The coronavirus disease 2019 (COVID-19) outbreak is having a dramatic global effect on public health and the economy. As of October 2020, SARS-CoV-2 has been detected in over 189 countries, has infected over 40 million people, and is responsible for more than 1 million deaths. The genome of SARS-CoV-2 is small but complex, and its functions and interactions with human host factors are being studied extensively. The significance of our study is that, using extensive SARS-CoV-2 genome analysis techniques, we identified potential interacting human host microRNA targets that share similarity with those of influenza A virus H1N1. Our study results will allow the development of virus-host interaction models that will enhance our understanding of SARS-CoV-2 pathogenesis and motivate the exploitation of both the interacting viral and host factors as therapeutic targets.« less
4. Highly conserved s2m element of SARS-CoV-2 dimerizes via a kissing complex and interacts with host miRNA-1307-3p

   https://doi.org/10.1093/nar/gkab1226  

   Imperatore, Joshua A. ; Cunningham, Caylee L. ; Pellegrene, Kendy A. ; Brinson, Robert G. ; Marino, John P. ; Evanseck, Jeffrey D. ; Mihailescu, Mihaela Rita ( December 2021 , Nucleic Acids Research)

   The ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the genome 3′ untranslated region is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the SARS-CoV-2 s2m element dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular microRNA (miRNA) 1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19more »and other coronavirus-related diseases.

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5. Computational Saturation Mutagenesis of SARS-CoV-1 Spike Glycoprotein: Stability, Binding Affinity, and Comparison With SARS-CoV-2

   https://doi.org/10.3389/fmolb.2021.784303  

   Sobitan, Adebiyi ; Mahase, Vidhyanand ; Rhoades, Raina ; Williams, Dejaun ; Liu, Dongxiao ; Xie, Yixin ; Li, Lin ; Tang, Qiyi ; Teng, Shaolei ( December 2021 , Frontiers in Molecular Biosciences)

   Severe Acute respiratory syndrome coronavirus (SARS-CoV-1) attaches to the host cell surface to initiate the interaction between the receptor-binding domain (RBD) of its spike glycoprotein (S) and the human Angiotensin-converting enzyme (hACE2) receptor. SARS-CoV-1 mutates frequently because of its RNA genome, which challenges the antiviral development. Here, we per-formed computational saturation mutagenesis of the S protein of SARS-CoV-1 to identify the residues crucial for its functions. We used the structure-based energy calculations to analyze the effects of the missense mutations on the SARS-CoV-1 S stability and the binding affinity with hACE2. The sequence and structure alignment showed similarities between the S proteins of SARS-CoV-1 and SARS-CoV-2. Interestingly, we found that target mutations of S protein amino acids generate similar effects on their stabilities between SARS-CoV-1 and SARS-CoV-2. For example, G839W of SARS-CoV-1 corresponds to G857W of SARS-CoV-2, which decrease the stability of their S glycoproteins. The viral mutation analysis of the two different SARS-CoV-1 isolates showed that mutations, T487S and L472P, weakened the S-hACE2 binding of the 2003–2004 SARS-CoV-1 isolate. In addition, the mutations of L472P and F360S destabilized the 2003–2004 viral isolate. We further predicted that many mutations on N-linked glycosylation sites would increase the stability of the Smore »glycoprotein. Our results can be of therapeutic importance in the design of antivirals or vaccines against SARS-CoV-1 and SARS-CoV-2.« less