Complete genome sequence of a novel fusarivirus from the phytopathogenic fungus Corynespora cassiicola

Corynespora cassiicola is an important phytopathogenic fungus and it has severely impaired the production of crops. In this study, we report on the molecular characterization of a novel (+) ssRNA mycovirus, Corynespora cassiicola fusarivirus 1 (CcFV1) isolated from C. cassiicola strain 20200826-3-1. Excluding a poly (A) tail, the genome of the virus is 6491 nt containing three putative open reading frames. The large ORF1 encodes a polypeptide of 1524aa with a conserved RNA-dependent RNA polymerase (RdRp) domain, a helicase (Hel) domain, and a Phage-holin-3-6 (Phage-holin) domain. ORF2 encodes a polypeptide with a conserved Chromosome segregation ATPase (Smc) domain. The smallest ORF3 encodes a putative protein with an unknown function. Phylogenetic analysis based on the ORF1 and ORF2 of CcFV1 encoded polypeptide showed that CcFV1 is phylogenetically related to the newly proposed family Fusariviridae. Thus, we suggest that CcFV1 might be a novel member of the family Fusariviridae and is also the first discovered in C. cassiicola.

Molecular Identification of a Novel Iflavirus in Brown-Spotted Pitvipers (Protobothrops Mucrosquamatus)

Background: Iflaviridae is a family of small non-enveloped viruses with monopartite, positive-stranded RNA genomes, which are identified in arthropod hosts, primarily infecting insect species. Herein, we firstly identify the sequence of an iflavirus (YB-PMP20) found in brown-spotted pitvipers in China. Results: The sequence of YB-PMP20 showed high identity to the sequences of Hubei picorna-like virus (HUPV) (99.2% in nt), Vespa velutina-associated iflavirus like virus (VVAIV) (58.6% in nt) and Lygus lineolaris virus (LyIV-1) (46.6% in nt) in nucleotides encoding polyproteins. It contained a single large ORF (304–9291 nt) encoding 2996 amino acids. The deduced amino acid sequences were compared with those of iflavirus. Helicase, protease and the RdRp domain were found to be located at the 3´ end, and structural genes (VP1, VP2 and VP3) were found to be located at the 5´ end. Phylogenetic analysis indicated that YB-PMP20 belongs to the iflavirus cluster, and is similar to HUPV, LyIV-1 and VVAIV. Conclusion: The present study described the genetic characterization of a PmIFV strain in brown-spotted pitvipers. Our genomic data extend knowledge of the diversity of viruses in snakes.

A crystal structure of alphavirus nonstructural protein 4 (nsP4) reveals an intrinsically dynamic RNA-dependent RNA polymerase

Alphaviruses such as Ross River virus (RRV), chikungunya virus and Venezuelan equine encephalitis virus are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report a crystal structure of the RdRp domain of nsP4 from RRV determined at a resolution of 2.6 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses and picornaviruses. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods, we showed that in-solution nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were able to catalyze in vitro RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication.

Amino Acid Substitutions in NS5 Contribute Differentially to Tembusu Virus Attenuation in Ducklings and Cell Cultures

Tembusu virus (TMUV), a highly infectious pathogenic flavivirus, causes severe egg-drop and encephalitis in domestic waterfowl, while the determinants responsible for viral pathogenicity are largely unknown. In our previous studies, virulent strain JXSP2-4 had been completely attenuated by successive passages in BHK-21 cells and the avirulent strain was designated as JXSP-310. Based on the backbone of JXSP2-4, a series of chimeric viruses were generated according to the amino acid substitutions in NS5 and their infectivities were also analyzed in cell cultures and ducklings. The results showed that the viral titers of RNA-dependent RNA polymerase (RdRp) domain-swapped cheimeric mutant (JXSP-310RdRp) in cells and ducklings were both markedly decreased compared with JXSP2-4, indicating that mutations in the RdRp domain affected viral replication. There are R543K and V711A two amino acid substitutions in the RdRp domain. Further site-directed mutagenesis showed that single-point R543K mutant (JXSP-R543K) exhibited similar replication efficacy compared with JXSP2-4 in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP2-4 and higher than JXSP-310RdRp. Surprisingly, the single-point V711A mutation we introduced rapidly reverted. In addition, qRT-PCR and Western blot confirmed that the mutations in the RdRp domain significantly affected the replication of the virus. Taken together, these results show that R543K substitution in the RdRp domain impairs the in vivo growth of TMUV, but sustaining its attenuated infectivity requires the concurrent presence of the V711A mutation.

Complete Genome Sequence of a Novel Mitovirus From the Phytopathogenic Fungus Fusarium Oxysporum

Fusarium oxysporum is a cosmopolitan plant pathogen causing Fusarium wilt and Fusarium root rot in many economically important crops. There is still limited information about mycoviruses that infect F. oxysporum. Here, a novel mitovirus tentatively named Fusarium oxysporum mitovirus 1 (FoMV1) was identified from F. oxysporum strain B2-10. The genome of FoMV1 is 2,453 nt in length with a predicted AU content of 71.6%, and contains one large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF encodes RNA-dependent RNA polymerases (RdRp) of 723 amino acids with a molecular mass of 84.98 kDa. The RdRp domain of FoMV1 shares 29.01–68.43% sequence similarity to the members of the family Mitoviridae. Phylogenetic analysis further suggested that FoMV1 is a new member of a distinct species in the genus Mitovirus.

Molecular Characterization of a Novel Single-stranded RNA Virus, ChRV1, Isolated From the Plant Pathogenic Fungus Colletotrichum Higginsianum

In this study, a novel single-stranded RNA virus was isolated from the plant pathogenic fungus, Colletotrichum higginsianum strain HTC-5, named “Colletotrichum higginsianum ssRNA virus 1” (ChRV1). The complete genome of ChRV1 is 3850 bp in length with a GC content of 52 % and encodes two in-frame open reading frames (ORFs): ORF1 (smaller) and ORF2 (larger). ORF1 encodes a protein with highest similarity to proteins encoded by Phoma matteucciicola RNA virus 1 (PmRV1, 47.99% identity) and Periconia macrospinosa ambiguivirus 1 (PmAV1, 50.73% identity). ORF2 encodes a protein with a conserved RNA-dependent RNA polymerase (RdRp) domain with similarity to RdRps of PmRV1 (61.41% identity) and PmAV1 (60.61% identity), which are unclassified (+)ssRNA mycoviruses reported recently. Phylogenetic analysis of the RdRp domain suggested that ChRV1 grouped together with PmRV1, PmAV1 and other unclassified (+)ssRNA mycoviruses, and had a distant relationship to invertebrate viruses and plant viruses of the family Tombusviridae. This is the first report of a novel (+)ssRNA virus infecting the phytopathogenic fungus C. higginsianum.

Flavivirus RNA-Dependent RNA Polymerase Interacts with Genome UTRs and Viral Proteins to Facilitate Flavivirus RNA Replication

Flaviviruses, most of which are emerging and re-emerging human pathogens and significant public health concerns worldwide, are positive-sense RNA viruses. Flavivirus replication occurs on the ER and is regulated by many mechanisms and factors. NS5, which consists of a C-terminal RdRp domain and an N-terminal methyltransferase domain, plays a pivotal role in genome replication and capping. The C-terminal RdRp domain acts as the polymerase for RNA synthesis and cooperates with diverse viral proteins to facilitate productive RNA proliferation within the replication complex. Here, we provide an overview of the current knowledge of the functions and characteristics of the RdRp, including the subcellular localization of NS5, as well as the network of interactions formed between the RdRp and genome UTRs, NS3, and the methyltransferase domain. We posit that a detailed understanding of RdRp functions may provide a target for antiviral drug discovery and therapeutics.

Molecular Characterization and Geographic Distribution of a Mymonavirus in the Population of Botrytis cinerea

Here, we characterized a negative single-stranded (−ss)RNA mycovirus, Botrytis cinerea mymonavirus 1 (BcMyV1), isolated from the phytopathogenic fungus Botrytis cinerea. The genome of BcMyV1 is 7863 nt in length, possessing three open reading frames (ORF1–3). The ORF1 encodes a large polypeptide containing a conserved mononegaviral RNA-dependent RNA polymerase (RdRp) domain showing homology to the protein L of mymonaviruses, whereas the possible functions of the remaining two ORFs are still unknown. The internal cDNA sequence (10-7829) of BcMyV1 was 97.9% identical to the full-length cDNA sequence of Sclerotinia sclerotiorum negative stranded RNA virus 7 (SsNSRV7), a virus-like contig obtained from Sclerotinia sclerotiorum metatranscriptomes, indicating BcMyV1 should be a strain of SsNSRV7. Phylogenetic analysis based on RdRp domains showed that BcMyV1 was clustered with the viruses in the family Mymonaviridae, suggesting it is a member of Mymonaviridae. BcMyV1 may be widely distributed in regions where B. cinerea occurs in China and even over the world, although it infected only 0.8% of tested B. cinerea strains.

Proposing drug fragments for dengue virus NS5 protein

Dengue fever is a febrile illness caused by Dengue Virus, which belongs to the Flaviviridae family. Among its proteome, the nonstructural protein 5 (NS5) is the biggest and most conserved. It has a primer-independent RNA-dependent RNA polymerase (RdRp) domain at its C-Terminus. Zou et al. studied the biological relevance of the two conserved cavities (named A and B) within the NS5 proteins of dengue virus (DENV) and West Nile Virus (WNV) using mutagenesis and revertant analysis and found four mutations located at cavity B having effects on viral replication. They recommended Cavity B, but not Cavity A as a potential target for drugs against flavivirus RdRp. In this study, we virtually screened the MayBridge drug fragments dataset for potential small molecule binders of cavity B using both AutoDock Vina, the standard docking tool, and QuickVina 2, our previously developed tool. We selected 16 fragments that appeared in the top 100 docking results of each of the representative structures of NS5. Visual inspection suggests that they have reasonable binding poses. The 16 predicted fragments are plausible drug candidates and should be considered for further validation, optimization, and linking to come up with a suitable inhibitor of dengue virus.

Structural insight and flexible features of NS5 proteins from all four serotypes ofDengue virusin solution

Infection by the four serotypes ofDengue virus(DENV-1 to DENV-4) causes an important arthropod-borne viral disease in humans. The multifunctional DENV nonstructural protein 5 (NS5) is essential for capping and replication of the viral RNA and harbours a methyltransferase (MTase) domain and an RNA-dependent RNA polymerase (RdRp) domain. In this study, insights into the overall structure and flexibility of the entire NS5 of all fourDengue virusserotypes in solution are presented for the first time. The solution models derived revealed an arrangement of the full-length NS5 (NS5FL) proteins with the MTase domain positioned at the top of the RdRP domain. The DENV-1 to DENV-4 NS5 forms are elongated and flexible in solution, with DENV-4 NS5 being more compact relative to NS5 from DENV-1, DENV-2 and DENV-3. Solution studies of the individual MTase and RdRp domains show the compactness of the RdRp domain as well as the contribution of the MTase domain and the ten-residue linker region to the flexibility of the entire NS5. Swapping the ten-residue linker between DENV-4 NS5FL and DENV-3 NS5FL demonstrated its importance in MTase–RdRp communication and in concerted interaction with viral and host proteins, as probed by amide hydrogen/deuterium mass spectrometry. Conformational alterations owing to RNA binding are presented.