TRIM21 restricts influenza A virus replication by ubiquitination-dependent degradation of M1.

Tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin ligase, plays a critical role in the host antiviral response. However, the mechanism and antiviral spectrum of TRIM21 in influenza A virus (IAV) remain unclear. Here, we report that TRIM21 inhibits the replication of various IAV subtypes by targeting matrix protein 1 (M1) from H3/H5/H9, but not H1 and H7 M1. Mechanistically, TRIM21 binds to the residue R95 of M1 and facilitates K48 ubiquitination of M1 K242 for proteasome-dependent degradation, leading to the inhibition of H3, H5, and H9 IAV replication. Interestingly, the recombinant viruses with M1 R95K or K242R mutations were resistance to TRIM21 and exhibited more robust replication and severe pathogenicity. Moreover, the amino acid sequence M1 proteins, mainly from avian influenza such as H5N1, H7N9, H9N2, ranging from 1918 to 2022, reveals a gradual dominant accumulation of the TRIM21-driven R95K mutation when the virus jumps into mammals. Thus, TRIM21 in mammals' functions as a host restriction factor and drives a host adaptive mutation of influenza A virus.

Protein kinase Cdc7 supports viral replication by phosphorylating Avibirnavirus VP3 protein.

IMPORTANCE: The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.

Nanobody peptide conjugate: a novel CD163 based broad neutralizing strategy against porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a prevalent swine pathogen, which has caused adverse impact on the global swine industry for almost 30 years. However, due to the immune suppression caused by the virus and the genetic diversity in PRRSV, no virus-targeting broad neutralizing strategy has been successfully developed yet. Antiviral peptide and nanobody have attracted extensive attention with the ease in production and the efficacy in practice. In this study, four new fusion proteins named nanobody peptide conjugates (NPCs) were developed by combining PRRSV specific non-neutralizing nanobodies with CD163-derived peptides targeting the receptor binding domain (RBD) of PRRSV proteins. RESULTS: Four NPCs were successfully constructed using two nanobodies against PRRSV N and nsp9 individually, recombining with two antiviral peptides 4H7 or 8H2 from porcine CD163 respectively. All four NPCs demonstrated specific capability of binding to PRRSV and broad inhibitory effect against various lineages of PRRSV in a dose-dependent manner. NPCs interfere with the binding of the RBD of PRRSV proteins to CD163 in the PRRSV pre-attachment stage by CD163 epitope peptides in the assistance of Nb components. NPCs also suppress viral replication during the stage of post-attachment, and the inhibitory effects depend on the antiviral functions of Nb parts in NPCs, including the interference in long viral RNA synthesis, NF-κB and IFN-ß activation. Moreover, an interaction was predicted between aa K31 and T32 sites of neutralizing domain 4H7 of NPC-N/nsp9-4H7 and the motif 171NLRLTG176 of PRRSV GP2a. The motif 28SSS30 of neutralizing domain 8H2 of NPC-N/nsp9-8H2 could also form hydrogens to bind with the motif 152NAFLP156 of PRRSV GP3. The study provides valuable insights into the structural characteristics and potential functional implications of the RBD of PRRSV proteins. Finally, as indicated in a mouse model, NPC intranasally inoculated in vivo for 12-24 h sustains the significant neutralizing activity against PRRSV. These findings inspire the potential of NPC as a preventive measure to reduce the transmission risk in the host population against respiratory infectious agents like PRRSV. CONCLUSION: The aim of the current study was to develop a peptide based bioactive compound to neutralize various PRRSV strains. The new antiviral NPC (nanobody peptide conjugate) consists of a specific nanobody targeting the viral protein and a neutralizing CD163 epitope peptide for virus blocking and provides significant antiviral activity. The study will greatly promote the antiviral drug R&D against PRRSV and enlighten a new strategy against other viral diseases.

Porcine Circovirus Type 2 Hijacks Host IPO5 to Sustain the Intracytoplasmic Stability of Its Capsid Protein.

Nuclear entrance and stability of porcine circovirus type 2 (PCV2), the smallest virus in mammals, are crucial for its infection and replication. However, the mechanisms are not fully understood. Here, we found that the PCV2 virion maintains self-stability via the host importin 5 (IPO5) during infection. Coimmunoprecipitation combined with mass spectrometry and glutathione S-transferase pulldown assays showed that the capsid protein (Cap) of PCV2 binds directly to IPO5. Fine identification demonstrated that the N-terminal residue arginine24 of Cap is the most critical to efficient binding to the proline709 residue of IPO5. Detection of replication ability further showed that IPO5 supports PCV2 replication by promoting the nuclear import of incoming PCV2 virions. Knockdown of IPO5 delayed the nuclear transport of incoming PCV2 virions and significantly decreased the intracellular levels of overexpressed PCV2 Cap, which was reversed by treatment with a proteasome inhibitor or by rescuing IPO5 expression. Cycloheximide treatment showed that IPO5 increases the stability of the PCV2 Cap protein. Taken together, our findings demonstrated that during infection, IPO5 facilitates PCV2 replication by directly binding to the nuclear localization signal of Cap to block proteasome degradation. IMPORTANCE Circovirus is the smallest virus to cause immune suppression in pigs. The capsid protein (Cap) is the only viral structural protein that is closely related to viral infection. The nuclear entry and stability of Cap are necessary for PCV2 replication. However, the molecular mechanism maintaining the stability of Cap during nuclear trafficking of PCV2 is unknown. Here, we report that IPO5 aggregates within the nuclear periphery and combines with incoming PCV2 capsids to promote their nuclear entry. Concurrently, IPO5 inhibits the degradation of newly synthesized Cap protein, which facilitates the synthesis of virus proteins and virus replication. These findings highlight a mechanism whereby IPO5 plays a dual role in PCV2 infection, which not only enriches our understanding of the virus replication cycle but also lays the foundation for the subsequent development of antiviral drugs.

A Case of Fetal Familial Hemophagocytic Lymphohistiocytosis Type 5 caused by STXBP2 Gene Mutation.

BACKGROUND: Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is a rare hyper-inflammatory syndrome caused by mutations in STXBP2. Most cases present at 2 - 6 months of age, and FHL-5 is extremely rare in neonates. METHODS: Appropriate laboratory tests, abdominal ultrasonography and whole exome sequencing were carried out. Respiratory support, antibiotics, and transfusion of blood products were done. RESULTS: Laboratory tests revealed metabolic acidosis, thrombocytopenia, mild anemia, and low fibrinogen level. Blood culture, metagenomics, and TORCH screening were negative. Liver and spleen enlargements were confirmed by abdominal ultrasonography. Whole exome sequencing identified a homozygous mutation in STXBP2 c. 1432del G (p. V478Sfs*5). The heterozygous STXBP2 mutation was identified in the paternal grandfather, maternal grandfather, and parents. CONCLUSIONS: Here we report a case with a novel homozygous deletion in exon 16 of STXBP2, which caused the earliest reported case of FHL-5 in a neonate. Our results identify a new pathogenic variant for the early identification and clinical consultation of FHL-5.

A New Segmented Virus Associated with Human Febrile Illness in China.

BACKGROUND: In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected. METHODS: We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients. RESULTS: We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness. CONCLUSIONS: A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.).

ATM-mediated DNA double-strand break response facilitated oncolytic Newcastle disease virus replication and promoted syncytium formation in tumor cells.

Deoxyribonucleic acid (DNA) damage response (DDR) is the fundamental cellular response for maintaining genomic integrity and suppressing tumorigenesis. The activation of ataxia telangiectasia-mutated (ATM) kinase is central to DNA double-strand break (DSB) for maintaining host-genome integrity in mammalian cells. Oncolytic Newcastle disease virus (NDV) can selectively replicate in tumor cells; however, its influence on the genome integrity of tumor cells is not well-elucidated. Here, we found that membrane fusion and NDV infection triggered DSBs in tumor cells. The late replication and membrane fusion of NDV mechanistically activated the ATM-mediated DSB pathway via the ATM-Chk2 axis, as evidenced by the hallmarks of DSBs, i.e., auto-phosphorylated ATM and phosphorylated H2AX and Chk2. Immunofluorescence data showed that multifaceted ATM-controlled phosphorylation markedly induced the formation of pan-nuclear punctum foci in response to NDV infection and F-HN co-expression. Specific drug-inhibitory experiments on ATM kinase activity further suggested that ATM-mediated DSBs facilitated NDV replication and membrane fusion. We confirmed that the Mre11-RAD50-NBS1 (MRN) complex sensed the DSB signal activation triggered by NDV infection and membrane fusion. The pharmacological inhibition of MRN activity also significantly inhibited intracellular and extracellular NDV replication and syncytia formation. Collectively, these data identified for the first time a direct link between the membrane fusion induced by virus infection and DDR pathways, thereby providing new insights into the efficient replication of oncolytic NDV in tumor cells.

Cytoplasmic Cargo Receptor p62 Inhibits Avibirnavirus Replication by Mediating Autophagic Degradation of Viral Protein VP2.

Selective autophagy regulates the degradation of cytoplasmic cargos, such as damaged organelles, invading pathogens, and aggregated proteins. Furthermore, autophagy is capable of degrading avibirnavirus, but the mechanism responsible for this process is unclear. Here, we show that autophagy cargo receptor p62 regulates the degradation of the avibirnavirus capsid protein VP2. Binding of p62 to VP2 enhances autophagic induction and promotes autophagic degradation of viral protein VP2. Further study showed that the interaction of p62 with viral protein VP2 is dependent on ubiquitination at the K411 site of VP2 and the ubiquitin-associated domain of p62. Mutation analysis showed that the K411R mutation of viral protein VP2 prohibits its p62-mediated degradation. Consistent with this finding, p62 lacking the ubiquitin-associated domain or the LC3-interacting region no longer promoted the degradation of VP2. Virus production revealed that the knockout of p62 but not the overexpression of p62 promotes the replication of avibirnavirus. Collectively, our findings suggest that p62 mediates selective autophagic degradation of avibirnavirus protein VP2 in a ubiquitin-dependent manner and is an inhibitor of avibirnavirus replication.IMPORTANCE Avibirnavirus causes severe immunosuppression and mortality in young chickens. VP2, the capsid protein of avibirnavirus, is responsible for virus assembly, maturation, and replication. Previous study showed that avibirnavirus particles could be engulfed into the autophagosome and degradation of virus particles took apart. Selective autophagy is a highly specific and regulated degradation pathway for the clearance of damaged or unwanted cytosolic components and superfluous organelles as well as invading microbes. However, whether and how selective autophagy removes avibirnavirus capsids is largely unknown. Here, we have shown that selective autophagy specifically clears ubiquitinated avibirnavirus protein VP2 by p62 recognition and that p62 is an inhibitor of avibirnavirus replication, highlighting the role of p62 as a potential drug target for mediating the removal of ubiquitinated virus components from cells.

Porcine Epidemic Diarrhea Virus Deficient in RNA Cap Guanine-N-7 Methylation Is Attenuated and Induces Higher Type I and III Interferon Responses.

The 5' cap methylation of viral RNA plays important roles in RNA stability, efficient translation, and immune evasion. Thus, RNA cap methylation is an attractive target for antiviral discovery and development of new live attenuated vaccines. For coronaviruses, RNA cap structure is first methylated at the guanine-N-7 (G-N-7) position by nonstructural protein 14 (nsp14), which facilitates and precedes the subsequent ribose 2'-O methylation by the nsp16-nsp10 complex. Using porcine epidemic diarrhea virus (PEDV), an Alphacoronavirus, as a model, we showed that G-N-7 methyltransferase (G-N-7 MTase) of PEDV nsp14 methylated RNA substrates in a sequence-unspecific manner. PEDV nsp14 can efficiently methylate RNA substrates with various lengths in both neutral and alkaline pH environments and can methylate cap analogs (GpppA and GpppG) and single-nucleotide GTP but not ATP, CTP, or UTP. Mutations to the S-adenosyl-l-methionine (SAM) binding motif in the nsp14 abolished the G-N-7 MTase activity and were lethal to PEDV. However, recombinant rPEDV-D350A with a single mutation (D350A) in nsp14, which retained 29.0% of G-N-7 MTase activity, was viable. Recombinant rPEDV-D350A formed a significantly smaller plaque and had significant defects in viral protein synthesis and viral replication in Vero CCL-81 cells and intestinal porcine epithelial cells (IPEC-DQ). Notably, rPEDV-D350A induced significantly higher expression of both type I and III interferons in IPEC-DQ cells than the parental rPEDV. Collectively, our results demonstrate that G-N-7 MTase activity of PEDV modulates viral replication, gene expression, and innate immune responses.IMPORTANCE Coronaviruses (CoVs) include a wide range of important human and animal pathogens. Examples of human CoVs include severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recently emerged SARS-CoV-2. Examples of pig CoVs include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and swine enteric alphacoronavirus (SeACoV). There are no vaccines or antiviral drugs for most of these viruses. All known CoVs encode a bifunctional nsp14 protein which possesses ExoN and guanine-N-7 methyltransferase (G-N-7 MTase) activities, responsible for replication fidelity and RNA cap G-N-7 methylation, respectively. Here, we biochemically characterized G-N-7 MTase of PEDV nsp14 and found that G-N-7 MTase-deficient PEDV was defective in replication and induced greater responses of type I and III interferons. These findings highlight that CoV G-N-7 MTase may be a novel target for rational design of live attenuated vaccines and antiviral drugs.

The serine-48 residue of nucleolar phosphoprotein nucleophosmin-1 plays critical role in subcellular localization and interaction with porcine circovirus type 3 capsid protein.

The transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.