Nucleocapsid Domain Research Articles

Highly sensitive and ultra-rapid antigen-based detection of SARS-CoV-2 using nanomechanical sensor platform

The rapid spread of COVID-19 including recent emergence of new variants with its extreme range of pathologies create an urgent need to develop a versatile sensor for a rapid, precise, and highly sensitive detection of SARS-CoV-2. Herein, we report a microcantilever-based optical detection of SARS-CoV-2 antigenic proteins in just few minutes with high specificity by employing fluidic-atomic force microscopy (f-AFM) mediated nanomechanical deflection method. The corresponding antibodies against the target antigens were first grafted on the gold-coated microcantilever surface pre-functionalized with EDC-NHS chemistry for a suitable antibody-antigen interaction. Rapid detection of SARS-CoV-2 nucleocapsid (N) and spike (S1) receptor binding domain (RBD) proteins was first demonstrated at a clinically relevant concentration down to 1 ng/mL (33 pM) by real-time monitoring of nanomechanical signal induced by antibody-antigen interaction. More importantly, we further show high specific detection of antigens with nasopharyngeal swab specimens from patients pre-determined with qRT-PCR. The results take less than 5 min (swab to signal ≤5 min) and exhibit high selectivity and analytical sensitivity (LoD: 100 copies/ ml; 0.71 ng/ml of N protein). These findings demonstrate potential for nanomechanical signal transduction towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.

Stability and conformation of the dimeric HIV-1 genomic RNA 5′UTR

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5′ untranslated region (5′UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5′UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5′UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5′UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5′UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5′UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.

Evaluation of a SARS-CoV-2 Capture IgM Antibody Assay in Convalescent Sera.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic with over 152 million cases and 3.19 million deaths reported by early May 2021. Understanding the serological response to SARS-CoV-2 is critical to determining the burden of infection and disease (coronavirus disease 2019 [COVID-19]) and transmission dynamics. We developed a capture IgM assay because it should have better sensitivity and specificity than the commonly used indirect assay. Here, we report the development and performance of a capture IgM enzyme-linked immunosorbent assay (ELISA) and a companion indirect IgG ELISA for the spike (S) and nucleocapsid (N) proteins and the receptor-binding domain (RBD) of S. We found that among the IgM ELISAs, the S ELISA was positive in 76% of 55 serum samples from SARS-CoV-2 PCR-positive patients, the RBD ELISA was positive in 55% of samples, and the N ELISA was positive in 15% of samples. The companion indirect IgG ELISAs were positive for S in 89% of the 55 serum samples, RBD in 78%, and N in 85%. While the specificities for IgM RBD, S, and N ELISAs and IgG S and RBD ELISAs were 97% to 100%, the specificity of the N IgG ELISA was lower (89%). RBD-specific IgM antibodies became undetectable by 3 to 6 months, and S IgM reached low levels at 6 months. The corresponding IgG S, RBD, and N antibodies persisted with some decreases in levels over this time period. These capture IgM ELISAs and the companion indirect IgG ELISAs should enhance serologic studies of SARS-CoV-2 infections.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has inflicted tremendous loss of lives, overwhelmed health care systems, and disrupted all aspects of life worldwide since its emergence in Wuhan, China, in December 2019. Detecting current and past infection by PCR or serology is important to understanding and controlling SARS-CoV-2. With increasing prevalence of past infection or vaccination, IgG antibodies are less helpful in diagnosing a current infection. IgM antibodies indicate a more recent infection and can supplement PCR diagnosis. We report an alternative method, capture IgM, to detect serum IgM antibodies, which should be more sensitive and specific than most currently used methods. We describe this capture IgM assay and a companion indirect IgG assay for the SARS-CoV-2 spike (S), nucleocapsid (N), and receptor-binding domain (RBD) proteins. These assays can add value to diagnostic and serologic studies of coronavirus disease 2019 (COVID-19).

5′-Cap sequestration is an essential determinant of HIV-1 genome packaging

HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. ΨCES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to ΨCES to enable cotranscriptional shedding of the 5' cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.

Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

Maturation of the matrix and viral membrane of HIV-1

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. We determined the structure and arrangement of MA within immature and mature HIV-1 through cryo-electron tomography. We found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Our data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

Structural changes in HIV maturation

Structural Biology Nascent HIV particles assemble at the plasma membrane of an infected cell and bud into a membrane-enveloped, immature virion. Assembly and budding are driven by a polyprotein called Gag, which consists of a matrix domain (MA) that is recruited to the plasma membrane, a capsid domain (CA) responsible for self-assembly, and a nucleocapsid domain (NC) that recruits the viral RNA genome. Gag cleavage results in a structural rearrangement that produces the mature virion. Qu et al. imaged mature and immature HIV particles by electron tomography and focused in on the MA domain (see the Perspective by Hikichi and Freed). They found that MA rearranges between two distinct hexameric lattices, and mature MA modulates the viral membrane by binding to a membrane lipid. This finding suggests that MA may play functional roles in the mature virion. Science , abe6821, this issue p. [700][1]; see also abj9075, p. [621][2] [1]: /lookup/doi/10.1126/science.abe6821 [2]: /lookup/doi/10.1126/science.abj9075

AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG.

BackgroundSerological tests detecting severe acute respiratory syndrome coronavirus−2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use.MethodsSARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD).ResultsIn-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen’s Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits.ConclusionThe assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.

Structural and conformational analysis of SARS CoV 2 N-CTD revealing monomeric and dimeric active sites during the RNA-binding and stabilization: Insights towards potential inhibitors for N-CTD

The advent of SARS-CoV-2 has become a universal health issue with no appropriate cure available to date. The coronavirus nucleocapsid (N) protein combines viral genomic RNA into a ribonucleoprotein and protects the viral genome from the host's nucleases. Structurally, the N protein comprises two independent domains: the N-terminal domain (NTD) for RNA-binding and C-terminal domain (CTD) involved in RNA-binding, protein dimerization, and nucleocapsid stabilization. The present study explains the structural aspects associated with the involvement of nucleocapsid C-terminal domain in the subunit assembly that helps the RNA binding and further stabilizing the virus assembly by protecting RNA from the hosts exonucleases degradation. The molecular dynamics (MD) simulations of the N-CTD and RNA complex suggests two active sites (site I: a monomer) and (site II: a dimer) with structural stability (RMSD: ~2 Å), Cα fluctuations (RMSF: ~3 Å) and strong protein-ligand interactions were estimated through the SiteMap module of Schrodinger. Virtual screening of 2456 FDA-approved drugs using structure-based docking identified top two leads distinctively against Site-I (monomer): Ceftaroline fosamil (MM-GBSA = −47.12 kcal/mol) and Cefoperazone (−45.84 kcal/mol); and against Site-II (dimer): Boceprevir, (an antiviral protease inhibitor, −106.78 kcal/mol) and Ceftaroline fosamil (−99.55 kcal/mol). The DCCM and PCA of drugs Ceftaroline fosamil (PC1+PC2 = 71.9%) and Boceprevir (PC1 +PC2 = 61.6%) show significant correlated residue motions which suggests highly induced conformational changes in the N-CTD dimer. Therefore, we propose N-CTD as a druggable target with two active binding sites (monomer and dimer) involved in specific RNA binding and stability. The RNA binding site with Ceftaroline fosamil binding can prevent viral assembly and can act as an antiviral for coronavirus.

CryoET structures of immature HIV Gag reveal six-helix bundle

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

SARS-CoV-2 recombinant proteins stimulate distinct cellular and humoral immune response profiles in samples from COVID-19 convalescent patients

OBJECTIVES:In this preliminary study we investigated cellular and humoral immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in blood samples from 14 recovered coronavirus disease 2019 (COVID-19) patients and compared them to those in samples from 12 uninfected/unvaccinated volunteers.METHODS:Cellular immunity was assessed by intracellular detection of IFN-γ in CD3+ T lymphocytes after stimulation with SARS-CoV-2 spike (S1), nucleocapsid (NC), or receptor-binding domain (RBD) recombinant proteins or overlapping peptide pools covering the sequence of SARS-CoV-2 spike, membrane and nucleocapsid regions. The humoral response was examined by ELISAs and/or chemiluminescence assays for the presence of serum IgG antibodies directed to SARS-CoV-2 proteins.RESULTS:We observed differences between humoral and cellular immune profiles in response to stimulation with the same proteins. Assays of IgG antibodies directed to SARS-CoV-2 NC, RBD and S1/S2 recombinant proteins were able to differentiate convalescent from uninfected/unvaccinated groups. Cellular immune responses to SARS-CoV-2 protein stimuli did not exhibit a specific response, as T cells from both individuals with no history of contact with SARS-CoV-2 and from recovered donors were able to produce IFN-γ.CONCLUSIONS:Determination of the cellular immune response to stimulation with a pool of SARS-CoV-2 peptides but not with SARS-CoV-2 proteins is able to distinguish convalescent individuals from unexposed individuals. Regarding the humoral immune response, the screening for serum IgG antibodies directed to SARS-CoV-2 proteins has been shown to be specific for the response of recovered individuals.

A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting.

Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay–based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0–1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.

High-resolution structure and biophysical characterization of the nucleocapsid phosphoprotein dimerization domain from the Covid-19 severe acute respiratory syndrome coronavirus 2

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N.

Overview of the Nucleic-Acid Binding Properties of the HIV-1 Nucleocapsid Protein in Its Different Maturation States.

HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with nucleic acids, and p1 and p6, two unstructured regions, p6 containing the motifs to bind ALIX, the cellular ESCRT factor TSG101 and the viral protein Vpr. The processing of Gag by the viral protease subsequently liberates NCp15 (NC-p1-p6), NCp9 (NC-p1) and NCp7, NCp7 displaying the optimal chaperone activity of nucleic acids. This review focuses on the nucleic acid binding properties of the NC domain in the different maturation states during the HIV-1 viral cycle.

Comparison of HIV-1 Gag and NCp7 in their selectivity for package signal, affinity for stem-loop 3, and Zn2+ content

The human immunodeficiency virus type 1 (HIV-1) Gag recognizes viral packaging signal (Psi) specifically via its nucleocapsid (NC) domain, resulting in the encapsidation of two copies of genomic RNA (gRNA) into the viral particle. The NCp7, which is cleaved from Gag during viral maturation, is a nucleic acid chaperone, coating and protecting the gRNA. In this study, an RT-qPCR-based approach was developed to quantitatively compare the Psi-selectivity of Gag and NCp7 in the presence of bacterial or 293T total RNAs. The binding affinity of Gag and NCp7 to the stem-loop (SL) 3 of Psi was also compared using surface plasmon resonance. We found that Gag selected more Psi-RNA than NCp7 from both E. coli BL21 (DE3) and in vitro binding reactions, and Gag bound to SL3-RNA with a higher affinity than NCp7. Moreover, Gag contained two Zn2+ whereas NCp7 contained one. The N-terminal zinc-finger motif of NCp7 lost most of its Zn2+-binding activity. Deletion of N-terminal amino acids 1–11 of NCp7 resulted in increased Psi-selectivity, SL3-affinity and Zn2+ content. These results indicated that Zn2+ coordination of Gag is critical for Psi-binding and selection. Removal of Zn2+ from the first zinc-finger motif during or after Gag cleavage to generate mature NCp7 might serve as a switch to regulate the functions of Gag NC domain and mature NCp7. Our study will be helpful to elucidate the important roles that Zn2+ plays in the viral life cycle, and may benefit further investigations of the function of HIV-1 Gag and NCp7.

The nucleic acid chaperone activity of the HIV-1 Gag polyprotein is boosted by its cellular partner RPL7: a kinetic study.

The HIV-1 Gag protein playing a key role in HIV-1 viral assembly has recently been shown to interact through its nucleocapsid domain with the ribosomal protein L7 (RPL7) that acts as a cellular co-factor promoting Gag's nucleic acid (NA) chaperone activity. To further understand how the two proteins act together, we examined their mechanism individually and in concert to promote the annealing between dTAR, the DNA version of the viral transactivation element and its complementary cTAR sequence, taken as model HIV-1 sequences. Gag alone or complexed with RPL7 was found to act as a NA chaperone that destabilizes cTAR stem-loop and promotes its annealing with dTAR through the stem ends via a two-step pathway. In contrast, RPL7 alone acts as a NA annealer that through its NA aggregating properties promotes cTAR/dTAR annealing via two parallel pathways. Remarkably, in contrast to the isolated proteins, their complex promoted efficiently the annealing of cTAR with highly stable dTAR mutants. This was confirmed by the RPL7-promoted boost of the physiologically relevant Gag-chaperoned annealing of (+)PBS RNA to the highly stable tRNALys3 primer, favoring the notion that Gag recruits RPL7 to overcome major roadblocks in viral assembly.

How HIV-1 Gag Manipulates Its Host Cell Proteins: A Focus on Interactors of the Nucleocapsid Domain.

The human immunodeficiency virus (HIV-1) polyprotein Gag (Group-specific antigen) plays a central role in controlling the late phase of the viral lifecycle. Considered to be only a scaffolding protein for a long time, the structural protein Gag plays determinate and specific roles in HIV-1 replication. Indeed, via its different domains, Gag orchestrates the specific encapsidation of the genomic RNA, drives the formation of the viral particle by its auto-assembly (multimerization), binds multiple viral proteins, and interacts with a large number of cellular proteins that are needed for its functions from its translation location to the plasma membrane, where newly formed virions are released. Here, we review the interactions between HIV-1 Gag and 66 cellular proteins. Notably, we describe the techniques used to evidence these interactions, the different domains of Gag involved, and the implications of these interactions in the HIV-1 replication cycle. In the final part, we focus on the interactions involving the highly conserved nucleocapsid (NC) domain of Gag and detail the functions of the NC interactants along the viral lifecycle.

Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles' heel to which HIV therapeutics may be targeted.

Zinc Fingers in HIV-1 Gag Precursor Are Not Equivalent for gRNA Recruitment at the Plasma Membrane

The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol.

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths. Currently, there is no specific viral protein-targeted therapeutics. Viral nucleocapsid protein is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. However, the structural information of SARS-CoV-2 nucleocapsid protein remains unclear. Herein, we have determined the 2.7 Å crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein. Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain, the surface electrostatic potential characteristics between them are distinct. Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside the β-sheet core. Complemented by in vitro binding studies, our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain, guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.