Structure-Function Investigation of SARS-CoV-2 Viral Proteins

March 2022 marks two years since the Covid-19 Pandemic began. Two years on, battle with the virus still continues. Officially the number of lives lost to the virus is reported to be around 6 Million but a recent study highlights that this number could be a huge undercount and the true estimate may account to three times that of the reported value. With the virus still circulating worldwide, it holds the opportunity to continue evolving and emerge as a variant capable of evading the human immune response. Therefore new drugs will be needed apart from the approved ones to counter the looming threat of resistance. For doing so, deeper understanding of the mechanistic basis of how the different viral proteins function is needed. In this study, we unravel structural basis of NSP 7-8 complex formation and investigate its dynamic assembly to form an active NSP 7-8-12 core replication complex. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces of NSP 7-8, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. In another project, we investigated the pathogen-host interaction based on SARS-CoV-2 Nucleocapsid protein and human G3BP1 protein. This aspect of our study reveals the biochemical and structural basis of the SARS-CoV-2 N1-25-G3BP1NTF2 interaction, revealing a ‘ϕ-x-F’ motif to be the primary and indispensable determinant of the interaction and flanking residues underpins diverse secondary interactions. Mutation of the key interaction residues of the SARS-CoV-2 N1–25 -G3BP1NTF2 complex leads to disruption of the SARS-CoV-2 N- G3BP1 interaction in vitro. In all, our results provide a molecular basis of the strain- specific interaction between SARSCoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection

SARS-CoV-2 nucleocapsid protein targets a conserved surface groove of the NTF2-like domain of G3BP1.

Stress granule (SG) formation mediated by Ras GTPase-activating protein-binding protein 1 (G3BP1) constitutes a key obstacle for viral replication, which makes G3BP1 a frequent target for viruses. For instance, the SARS-CoV-2 nucleocapsid (N) protein interacts with G3BP1 directly to suppress SG assembly and promote viral production. However, the molecular basis for the SARS-CoV-2 N-G3BP1 interaction remains elusive. Here we report biochemical and structural analyses of the SARS-CoV-2 N-G3BP1 interaction, revealing differential contributions of various regions of SARS-CoV-2 N to G3BP1 binding. The crystal structure of the NTF2-like domain of G3BP1 (G3BP1NTF2) in complex with a peptide derived from SARS-CoV-2 N (residues 1- 25, N1-25) reveals that SARS-CoV-2 N1-25 occupies a conserved surface groove of G3BP1NTF2 via surface complementarity. We show that a φ-x-F (φ, hydrophobic residue) motif constitutes the primary determinant for G3BP1NTF2-targeting proteins, while the flanking sequence underpins diverse secondary interactions. We demonstrate that mutation of key interaction residues of the SARS-CoV-2 N1-25-G3BP1NTF2 complex leads to disruption of the SARS-CoV-2 N-G3BP1 interaction in vitro. Together, these results provide a molecular basis of the strain-specific interaction between SARS2-CoV-2 N and G3BP1, which has important implications for the development of novel therapeutic strategies against SARS-CoV-2 infection

Devil's tools: SARS-CoV-2 antagonists against innate immunity

The unprecedented Coronavirus pandemic of 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Like other coronaviruses, to establish its infection, SARS-CoV-2 is required to overcome the innate interferon (IFN) response, which is the first line of host defense. SARS-CoV-2 has also developed complex antagonism approaches involving almost all its encoding viral proteins. Here, we summarize our current understanding of these different viral factors and their roles in suppressing IFN responses. Some of them are conserved IFN evasion strategies used by SARS-CoV; others are novel countermeasures only employed by SARS-CoV-2. The filling of gaps in understanding these underlying mechanisms will provide rationale guidance for applying IFN treatment against SARS-CoV-2 infection

Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase.

DNA methyltransferases interact with their CpG target sites in the context of variable flanking sequences. We investigated DNA methylation by the human DNMT3B catalytic domain using substrate pools containing CpX target sites in randomized flanking context and identified combined effects of CpG recognition and flanking sequence interaction together with complex contact networks involved in balancing the interaction with different flanking sites. DNA methylation rates were more affected by flanking sequences at non-CpG than at CpG sites. We show that T775 has an essential dynamic role in the catalytic mechanism of DNMT3B. Moreover, we identify six amino acid residues in the DNA-binding interface of DNMT3B (N652, N656, N658, K777, N779, and R823), which are involved in the equalization of methylation rates of CpG sites in favored and disfavored sequence contexts by forming compensatory interactions to the flanking residues including a CpG specific contact to an A at the +1 flanking site. Non-CpG flanking preferences of DNMT3B are highly correlated with non-CpG methylation patterns in human cells. Comparison of the flanking sequence preferences of human and mouse DNMT3B revealed subtle differences suggesting a co-evolution of flanking sequence preferences and cellular DNMT targets

Mechanistic basis for maintenance of CHG DNA methylation in plants.

DNA methylation is an evolutionarily conserved epigenetic mechanism essential for transposon silencing and heterochromatin assembly. In plants, DNA methylation widely occurs in the CG, CHG, and CHH (H = A, C, or T) contexts, with the maintenance of CHG methylation mediated by CMT3 chromomethylase. However, how CMT3 interacts with the chromatin environment for faithful maintenance of CHG methylation is unclear. Here we report structure-function characterization of the H3K9me2-directed maintenance of CHG methylation by CMT3 and its Zea mays ortholog ZMET2. Base-specific interactions and DNA deformation coordinately underpin the substrate specificity of CMT3 and ZMET2, while a bivalent readout of H3K9me2 and H3K18 allosterically stimulates substrate binding. Disruption of the interaction with DNA or H3K9me2/H3K18 led to loss of CMT3/ZMET2 activity in vitro and impairment of genome-wide CHG methylation in vivo. Together, our study uncovers how the intricate interplay of CMT3, repressive histone marks, and DNA sequence mediates heterochromatic CHG methylation

Two conserved oligomer interfaces of NSP7 and NSP8 underpin the dynamic assembly of SARS-CoV-2 RdRP.

Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development

Two conserved oligomer interfaces of NSP7 and NSP8 underpin the dynamic assembly of SARS-CoV-2 RdRP.

Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development

Structural basis to characterise transactivation domain of BRCA1

<p>Familial inheritance of breast and ovarian cancer is attributed to mutations discovered in functional domains of BRCA1 gene. BRCA1 is a multifunctional protein responsible for maintaining the genomic integrity and has transcriptional regulatory function encoded in its C-terminal region. The different amino-terminal e extensions to BRCA1 BRCT domain are responsible for transcription activation. However, only BRCA1 BRCT (1649–1859) amino acids have been explored for its structural characteristics. Noting the importance of extended region to the N-terminus of BRCT different regions of BRCA1 which demonstrates maximum transactivation activity has been explored for their structure and functional activity. Secondary and tertiary structural analysis revealed a limited alpha-helical content with well-folded tertiary structure. <i>In silico</i> tools were used to corroborate the <i>in vitro</i> results. Amino acids composition and sequence analysis display a propensity for intrinsic disorder and coiled-coil formation in BRCA1 (1396–1863) (BRCA1-TAD). The results presented in this paper suggest the extreme flexibility in coiled-coil motif might be an important requirement in the establishment of protein–protein interaction networks for BRCA1.</p