Heng Zhang

Eukaryotic circular Rep encoding single-stranded DNA (CRESS DNA) viruses exhibit high genomic diversity. Four species of circoviruses within the genus Circovirus have been identified in pigs, including the non-pathogenic porcine circovirus type 1 (PCV1), and the pathogenic PCV2, PCV3, and PCV4. In this study, a novel single circular DNA virus was identified in pigs suffering from respiratory, diarrheal, and reproductive failure diseases. The viral Rep and Cap showed low amino acid sequence identities to the four PCV species, with the highest identity of 26.2% to PCV2 Rep and 20.8% to PCV4 Cap. The classification of CRESS DNA viruses primarily relies on Rep, and biochemical analyses of the viral Rep confirm its classification as a CRESS DNA virus. Thus, it was tentatively named porcine circovirus type 5 (PCV5). This tentative designation reflects its host and genomic organization; however, phylogenetic analysis indicates that it clusters outside the family Circoviridae and is most closely related to the fur seal faces-associated circular DNA virus (FSfaCV). The positive detection rate for PCV5 in tested samples was 54.4% (294 out of 540), with 24.3% (17 out of 70) of pig farms in China testing positive for the virus. A close correlation between the copy number of PCV5 and the severity of infectious diseases was observed. Moreover, a PCV5 virus-like particles (VLPs)-based enzyme-linked immunosorbent assay, which was developed, also elucidated that PCV5 has been circulating. We subsequently established an in vitro culture system for PCV5, successfully purifying the virus. Morphological identification revealed that the purified PCV5 has a diameter of approximately 20 nm, and the recombinant PCV5 Cap could assemble into VLPs similar to purified PCV5. These results indicate that PCV5, associated with porcine circovirus-associated disease, commonly circulates within Chinese swine. IMPORTANCE: The single-stranded circular DNA virus, tentatively named porcine circovirus type 5 (PCV5), belongs to the circular Rep encoding single-stranded DNA (CRESS DNA) viruses. Notably, PCV5 is not classified within the same family as the previously reported PCV1–PCV4. PCV5, which was associated with porcine circovirus associated-disease, is widely prevalent in Southern China. PCV5 is characterized by a larger genome (2,904 nt), along with longer Cap (1,182 nt) and Rep (1,056 nt) regions, as well as an extended stem-loop structure (stem: 9 nt and loop: 17 nt). Importantly, we subsequently established an in vitro culture system for PCV5, successfully purifying the virus. Morphological identification revealed that the purified PCV5 has a diameter of approximately 20 nm. Additionally, a variant of the PCV5 Cap, lacking the N-terminal 63 residues, was expressed in Escherichia coli , and purified Cap could assemble into virus-like particles similar to purified PCV5. These results hold significant implications for the study of CRESS DNA viruses. The emergence of PCV5 warrants further study.

African swine fever virus (ASFV) pB318L is an important protein for viral replication that acts as a membrane-bound trans-geranylgeranyl-diphosphate synthase (GGPPS) catalyzing the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates. Recently we solved the crystal structure pB318L lacking N-terminal transmembrane region and performed a preliminary structural analysis. In this study, structure-based mutagenesis study and geranylgeranyl pyrophosphate (GGPP) production assay further revealed the key residues for the GGPPS activity. Structural comparison showed pB318L displays a strong similarity to typical GGPPSs instead of protein prenyltransferases. The phylogenetic analysis indicated pB318L may share a common ancestor with the GGPPSs from Brassicaceae plants rather than from its natural host. The subcellular localization analysis showed pB318L is localized in both nucleus and cytoplasm (including the endoplasmic reticulum membrane and mitochondria outer membrane). A unique N-terminal nuclear localization signal (NLS) following the transmembrane region was discovered in pB318L and the NLS was confirmed to be required for the nuclear import. We further revealed the NLS plays an essential role in the interaction with nuclear transporter karyopherin subunit alpha 1 (KPNA1). Their interaction may suppress signal transducers and activators of transcription 1 (STAT1) translocation and subsequently competitively inhibit nuclear import of IFN-stimulated gene factor 3 (ISGF3) complex. Our biochemical, structural and cellular analyses provide novel insights to pB318L that acts as an essential GGPPS that promotes viral replication and as a nuclear import protein that may be involved in immune evasion of ASFV.

Cyanobacterial cytochrome c6 (Cyt c6) is crucial for electron transfer between the cytochrome b6f complex and photosystem I (PSI), playing a key role in photosynthesis and enhancing adaptation to extreme environments. This study investigates the high-resolution crystal structures of Cyt c6 from Synechococcus elongatus PCC 7942 and Synechocystis PCC 6803, focusing on its dimerization mechanisms and functional implications for photosynthesis. Cyt c6 was expressed in Escherichia coli using a dual-plasmid co-expression system and characterized in both oxidized and reduced states. X-ray crystallography revealed three distinct crystal forms, with asymmetric units containing 2, 4, or 12 molecules, all of which consist of repeating dimeric structures. Structural comparisons across species indicated that dimerization predominantly occurs through hydrophobic interactions within a conserved motif around the heme crevice, despite notable variations in dimer positioning. We propose that the dimerization of Cyt c6 enhances structural stability, optimizes electron transfer kinetics, and protects the protein from oxidative damage. Furthermore, we used AlphaFold3 to predict the structure of the PSI-Cyt c6 complex, revealing specific interactions that may facilitate efficient electron transfer. These findings provide new insights into the functional role of Cyt c6 dimerization and its contribution to improving cyanobacterial photosynthetic electron transport.

2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure–function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions. IMPORTANCE: Encephalomyocarditis virus (Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis, and reproductive disorders in various mammals. 2C protein is functionally indispensable and a promising target for drug development involving broad-spectrum picornaviral inhibitors. Here, an endogenous ATP molecule with a unique conformation was discovered by a combination of protein crystallography and high-performance liquid chromatography in the encephalomyocarditis virus (EMCV) 2C structure. Biochemical and structural characterization analysis of EMCV 2C revealed the critical role of conserved Arg311 in ATPase activity and self-oligomerization of EMCV 2C. The viral replication kinetics and infectivity study suggested that the residue negatively regulated the infectivity titer and virus encapsulation efficiency of EMCV and is, therefore, crucial for 2C protein to promote viral replication. Our systemic structure–function analysis provides unique insights into the function and regulation mechanism of cardioviral 2C protein.

African swine fever virus (ASFV) is a complex nucleocytoplasmic, large DNA virus that infects both domestic pigs and wild boar, but little is known about the process of genomic replication. The E301R protein (pE301R) from ASFV was previously predicted as a proliferating cell nuclear antigen (PCNA)-like protein through clamping DNA polymerase to the DNA duplex, but its exact structure and functions remain uncharacterized. Here, the crystal structure of pE301R revealed that it is composed of structurally similar head and tail domains and shares significant structural similarities to the DNA polymerase processivity factors, including sliding clamp and eukaryotic PCNA. More specifically, we demonstrated that pE301R exhibited multiple oligomeric states (with dimers and tetramers dominant), and the tetramers are consistent with the ring-shaped homotetramers in a head-to-tail manner generated by crystal packing. We also showed that pE301R interacted with the ASFV genome and viral DNA polymerase O174L. Furthermore, knockdown of E301R by specific small interfering RNAs (siRNAs) significantly decreased the virus genomic replication. Interestingly, the downregulation of PCNA by siRNAs significantly decreased the cell viability, whereas the inhibitory effect was reversed by pE301R overexpression. Notably, we demonstrated that overexpression of PCNA partially restored ASFV replication upon transfection of siRNAs targeting E301R . More importantly, T2 amino alcohol, a PCNA-specific inhibitor, markedly inhibited ASFV replication at the stage of viral genome replication. Taken together, we revealed that pE301R functions as a sliding clamp in ASFV genomic replication and can be used as a potential antiviral target. IMPORTANCE: Sliding clamp is a highly conserved protein in the evolution of prokaryotic and eukaryotic cells. The sliding clamp is required for genomic replication as a critical co-factor of DNA polymerases. However, the sliding clamp analogs in viruses remain largely unknown. We found that the ASFV E301R protein (pE301R) exhibited a sliding clamp-like structure and similar functions during ASFV replication. Interestingly, pE301R is assembled into a unique ring-shaped homotetramer distinct from sliding clamps or proliferating cell nuclear antigens (PCNAs) from other species. Notably, the E301R gene is required for viral life cycle, but the pE301R function can be partially restored by the porcine PCNA. This study not only highlights the functional role of the ASFV pE301R as a viral sliding clamp analog, but also facilitates the dissection of the complex replication mechanism of ASFV, which provides novel clues for developing antivirals against ASF.

Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3C^pro) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3C^pro with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3C^pro. Our lipid-binding assays showed that SVA 3C^pro displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3C^pro was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3C^pro-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3C^pro structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3C^pro, indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3C^pro, suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme’s proteolytic activity during infection.

Recent technological breakthroughs in machine-learning-based AlphaFold2 (AF2) are pushing the prediction accuracy of protein structures to an unprecedented level that is on par with experimental structural quality. Despite its outstanding structural modeling capability, further experimental validations and performance assessments of AF2 predictions are still required, thus necessitating the development of integrative structural biology in synergy with both computational and experimental methods. Focusing on the B318L protein that plays an essential role in the African swine fever virus (ASFV) for viral replication, we experimentally demonstrate the high quality of the AF2 predicted model and its practical utility in crystal structural determination. Structural alignment implies that the AF2 model shares nearly the same atomic arrangement as the B318L crystal structure except for some flexible and disordered regions. More importantly, side-chain-based analysis at the individual residue level reveals that AF2′s performance is likely dependent on the specific amino acid type and that hydrophobic residues tend to be more accurately predicted by AF2 than hydrophilic residues. Quantitative per-residue RMSD comparisons and further molecular replacement trials suggest that AF2 has a large potential to outperform other computational modeling methods in terms of structural determination. Additionally, it is numerically confirmed that the AF2 model is accurate enough so that it may well potentially withstand experimental data quality to a large extent for structural determination. Finally, an overall structural analysis and molecular docking simulation of the B318L protein are performed. Taken together, our study not only provides new insights into AF2′s performance in predicting side-chain conformations but also sheds light upon the significance of AF2 in promoting crystal structural determination, especially when the experimental data quality of the protein crystal is poor.

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB–BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Å from the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure–function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

ParE_SO-CopA_SO is a new type II toxin–antitoxin (TA) system in prophage CP4So that plays an essential role in circular CP4So maintenance after the excision in Shewanella oneidensis. The toxin ParE_SO severely inhibits cell growth, while CopA_SO functions as an antitoxin to neutralize ParE_SO toxicity through direct interactions. However, the molecular mechanism of the neutralization and autoregulation of the TA operon transcription remains elusive. In this study, we determined the crystal structure of a ParE_SO-CopA_SO complex that adopted an open V-shaped heterotetramer with the organization of ParE_SO-(CopA_SO)₂-ParE_SO. The structure showed that upon ParE_SO binding, the intrinsically disordered C-terminal domain of CopA_SO was induced to fold into a partially ordered conformation that bound into a positively charged and hydrophobic groove of ParE_SO. Thermodynamics analysis showed the DNA-binding affinity of CopA_SO was remarkably higher than that of the purified TA complex, accompanied by the enthalpy change reversion from an exothermic reaction to an endothermic reaction. These results suggested ParE_SO acts as a de-repressor of the TA operon transcription at the toxin:antitoxin level of 1:1. Site-directed mutagenesis of ParE_SO identified His91 as the essential residue for its toxicity by cell toxicity assays. Our structure-function studies therefore elucidated the transcriptional regulation mechanism of the ParE_SO-CopA_SO pair, and may help to understand the regulation of CP4So maintenance in S. oneidensis.

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease and it is urgently needed to develop effective anti-ASFV vaccines and drugs. The process of mRNA 5′-end capping is a common characteristic in eukaryotes and many viruses, and the cap structure is required for mRNA stability and efficient translation. The ASFV protein pNP868R was found to have guanylyltransferase (GTase) activity involved in mRNA capping. Here we report the crystal structure of pNP868R methyltransferase (MTase) domain (referred as pNP868R^MT) in complex with S-adenosyl-L-methionine (AdoMet). The structure shows the characteristic core fold of the class I MTase family and the AdoMet is bound in a negative-deep groove. Remarkably, the N-terminal extension of pNP868R^MT is ordered and keeps away from the AdoMet-binding site, distinct from the close conformation over the active site of poxvirus RNA capping D1 subunit or the largely disordered conformation in most cellular RNA capping MTases. Structure-based mutagenesis studies based on the pNP868R^MT-cap analog complex model revealed essential residues involved in substrate recognition and binding. Functional studies suggest the N-terminal extension may play an essential role in substrate recognition instead of AdoMet-binding. A positively charged path stretching from the N-terminal extension to the region around the active site was suggested to provide a favorable electrostatic environment for the binding and approaching of substrate RNA into the active site. Our structure and biochemical studies provide novel insights into the methyltransfer process of mRNA cap catalyzed by pNP868R. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic viral disease in pigs that is caused by African swine fever virus (ASFV). There are no effective drugs or vaccines for protection against ASFV infection till now. The protein pNP868R was predicted to be responsible for process of mRNA 5′-end capping in ASFV, which is essential for mRNA stability and efficient translation. Here, we solved the high-resolution crystal structure of the methyltransferase (MTase) domain of pNP868R. The MTase domain structure shows a canonical class I MTase family fold and the AdoMet binds into a negative pocket. Structure-based mutagenesis studies revealed critical and conserved residues involved in AdoMet-binding and substrate RNA-binding. Notably, both the conformation and the role in MTase activities of the N-terminal extension are distinct from those of previously characterized poxvirus MTase domain. Our structure-function studies provide the basis for potential anti-ASFV inhibitor design targeting the critical enzyme.