N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Role of the membrane-associated accessory protein (MAAP) in adeno-associated virus (AAV) infection.

Adeno-associated viruses (AAVs) package a single-stranded (ss) DNA genome of 4.7 kb in their capsid of ~20 nm in diameter. AAV replication requires co-infection of a helper virus, such as adenovirus. During the optimization of recombinant AAV production, a small viral nonstructural protein, membrane-associated accessory protein (MAAP), was identified. However, the function of the MAAP in the context of AAV infection remains unknown. Here, we investigated the expression strategy and function of the MAAP during infection of both AAV2 and AAV5 in human embryonic kidney (HEK)293 cells. We found that AAV2 MAAP2 and AAV5 MAAP5 are expressed from the capsid gene (cap)-transcribing mRNA spliced from the donor to the second splice site that encodes VP2 and VP3. Thus, this AAV cap gene transcribes a multicistronic mRNA that can be translated to four viral proteins, MAAP, VP2, AAP, and VP3 in order. In AAV2 infection, MAAP2 predominantly localized in the cytoplasm, alongside the capsid, near the nuclear and plasma membranes, but a fraction of MAAP2 exhibited nuclear localization. In AAV5 infection, MAAP5 revealed a distinct pattern, predominantly localizing within the nucleus. In the cells infected with an MAAP knockout mutant of AAV2 or AAV5, both viral DNA replication and virus replication increased, whereas virus egress decreased, and the decrease in virus egress can be restored by providing MAAP in trans. In summary, MAAP, a novel AAV nonstructural protein translated from a multicistronic viral cap mRNA, not only facilitates cellular egress of AAV but also likely negatively affects viral DNA replication during infection. IMPORTANCE: Recombinant adeno-associated virus (rAAV) has been used as a gene delivery vector in clinical gene therapy. In current gene therapies employing rAAV, a high dose of the vector is required. Consequently, there is a high demand for efficient and high-purity vector production systems. In this study, we demonstrated that membrane-associated accessory protein (MAAP), a small viral nonstructural protein, is translated from the same viral mRNA transcript encoding VP2 and VP3. In AAV-infected cells, apart from its prevalent expression in the cytoplasm with localization near the plasma and nuclear membranes, the MAAP also exhibits notable localization within the nucleus. During AAV infection, MAAP expression increases the cellular egress of progeny virions and decreases viral DNA replication and progeny virion production. Thus, the choice of MAAP expression has pros and cons during AAV infection, which could provide a guide to rAAV production.

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium.

IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

Inhibition of DNA-dependent protein kinase catalytic subunit boosts rAAV transduction of polarized human airway epithelium.

Adeno-associated virus 2.5T (AAV2.5T) was selected from the directed evolution of AAV capsid library in human airway epithelia. This study found that recombinant AAV2.5T (rAAV2.5T) transduction of well-differentiated primary human airway epithelia induced a DNA damage response (DDR) characterized by the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all three phosphatidylinositol 3-kinase-related kinases: ataxia telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). While suppressing the expression of ATR by a specific pharmacological inhibitor or targeted gene silencing inhibited rAAV2.5T transduction, DNA-PKcs inhibition or targeted gene silencing significantly increased rAAV2.5T transgene expression. Notably, DNA-PKcs inhibitors worked as a "booster" to further increase rAAV2.5T transgene expression after treatment with doxorubicin and did not compromise epithelial integrity. Thus, our study provides evidence that DDR is associated with rAAV transduction in well-differentiated human airway epithelia, and DNA-PKcs inhibition has the potential to boost rAAV transduction. These findings highlight that the application of DDR inhibition-associated pharmacological interventions has the potential to increase rAAV transduction and thus to reduce the required vector dose.

Identification of Host Restriction Factors Critical for Recombinant AAV Transduction of Polarized Human Airway Epithelium.

Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L , TM9SF2 , VPS51 , and VPS54 , as well as a novel gene WDR63 . We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T. Significance: The essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63 , whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

SARS-CoV-2 infection of polarized human airway epithelium induces necroptosis that causes airway epithelial barrier dysfunction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause the ongoing pandemic of coronavirus disease 2019 (COVID19). One key feature associated with COVID-19 is excessive pro-inflammatory cytokine production that leads to severe acute respiratory distress syndrome. Although the cytokine storm induces inflammatory cell death in the host, which type of programmed cell death mechanism that occurs in various organs and cells remains elusive. Using an in vitro culture model of polarized human airway epithelium (HAE), we observed that necroptosis, but not apoptosis or pyroptosis, plays an essential role in the damage of the epithelial barrier of polarized HAE infected with SARS-CoV-2. Pharmacological inhibitors of necroptosis, necrostatin-2 and necrosulfonamide, efficiently prevented cell death and epithelial barrier dysfunction caused by SARS-CoV-2 infection. Moreover, the silencing of genes that are involved in necroptosis, RIPK1, RIPK3, and MLKL, ameliorated airway epithelial damage of the polarized HAE infected with SARS-CoV-2. This study, for the first time, confirms that SARS-CoV-2 infection triggers necroptosis that disrupts the barrier function of human airway epithelia in vitro.

Repurposing Niclosamide as a Novel Anti-SARS-CoV-2 Drug by Restricting Entry Protein CD147.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the global coronavirus disease 2019 (COVID-19) pandemic, and the search for effective treatments has been limited. Furthermore, the rapid mutations of SARS-CoV-2 have posed challenges to existing vaccines and neutralizing antibodies, as they struggle to keep up with the increased viral transmissibility and immune evasion. However, there is hope in targeting the CD147-spike protein, which serves as an alternative point for the entry of SARS-CoV-2 into host cells. This protein has emerged as a promising therapeutic target for the development of drugs against COVID-19. Here, we demonstrate that the RNA-binding protein Human-antigen R (HuR) plays a crucial role in the post-transcriptional regulation of CD147 by directly binding to its 3'-untranslated region (UTR). We observed a decrease in CD147 levels across multiple cell lines upon HuR depletion. Furthermore, we identified that niclosamide can reduce CD147 by lowering the cytoplasmic translocation of HuR and reducing CD147 glycosylation. Moreover, our investigation revealed that SARS-CoV-2 infection induces an upregulation of CD147 in ACE2-expressing A549 cells, which can be effectively neutralized by niclosamide in a dose-dependent manner. Overall, our study unveils a novel regulatory mechanism of regulating CD147 through HuR and suggests niclosamide as a promising therapeutic option against COVID-19.

Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5' untranslated region.

One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as C30 and C34, which bind to a four-way RNA helix called SL5 in the 5' UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2'-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of C30 in the SARS-CoV-2 5' UTR, which was validated through mutagenesis and in vitro binding experiments. C30 was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5' UTR expressing cells. We further explored another RLR conjugation site on the E ring of C30/C34 and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells.

Repurposing niclosamide as a novel anti-SARS-Cov-2 drug by restricting entry protein CD147.

Background The burst of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global COVID-19 pandemic. But until today only limited numbers of drugs are discovered to treat COVID-19 patients. Even worse, the rapid mutations of SARS-CoV-2 compromise the effectiveness of existing vaccines and neutralizing antibodies due to the increased viral transmissibility and immune escape. CD147-spike protein, one of the entries of SRAR-CoV-2 into host cells, has been reported as a promising therapeutic target for developing drugs against COVID-19. Methods CRISPR-Cas9 induced gene knockout, western blotting, tet-off protein overexpression, ribonucleoprotein IP and RNA-IP were used to confirm the regulation of HuR on mRNA of CD147. Regulation of niclosamide on HuR nucleo-translocation was assessed by immunofluorescence staining of cell lines, IHC staining of tissue of mouse model and western blotting. Finally, the suppression of niclosamide on SARS-CoV-2 infection induced CD147 was evaluated by ACE2-expressing A549 cells and western blotting. Results We first discovered a novel regulation mechanism of CD147 via the RNA-binding protein HuR. We found that HuR regulates CD147 post-transcription by directly bound to its 3'-UTR. The loss of HuR reduced CD147 in multiple cell lines. Niclosamide inhibited CD147 function by blocking HuR cytoplasmic translocation and diminishing CD147 glycosylation. SARS-CoV-2 infection induced CD147 in ACE2-expressing A549 cells, which could be neutralized by niclosamide in a dose-dependent manner. Conclusion Together, our study reveals a novel regulation mechanism of CD147 and niclosamide can be repurposed as an effective COVID-19 drug by targeting the virus entry, CD147-spike protein.

Identification of AXL as a co-receptor for human parvovirus B19 infection of human erythroid progenitors.

Parvovirus B19 (B19V) infects human erythroid progenitor cells (EPCs) and causes several hematological disorders and fetal hydrops. Amino acid (aa) 5-68 of minor capsid protein VP1 (VP1u5-68aa) is the minimal receptor binding domain for B19V to enter EPCs. Here, we carried out a genome-wide CRISPR-Cas9 guide RNA screen and identified tyrosine protein kinase receptor UFO (AXL) as a proteinaceous receptor for B19V infection of EPCs. AXL gene silencing in ex vivo expanded EPCs remarkably decreased B19V internalization and replication. Additions of the recombinant AXL extracellular domain or a polyclonal antibody against it upon infection efficiently inhibited B19V infection of ex vivo expanded EPCs. Moreover, B19V VP1u interacted with the recombinant AXL extracellular domain in vitro at a relatively high affinity (KD = 103 nM). Collectively, we provide evidence that AXL is a co-receptor for B19V infection of EPCs.

Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication.

Adeno-associated virus (AAV) belongs to the Dependoparvovirus genus of the Parvoviridae family. AAV replication relies on a helper virus, such as adenovirus (Ad). Co-infection of AAV and Ad induces a DNA damage response (DDR), although its function in AAV DNA replication remains unknown. In this study, monoinfection of AAV2 in HEK293T cells expressing a minimal set of Ad helper genes was used to investigate the role of the DDR solely induced by AAV. We found that AAV2 DNA replication, but not single stranded (ss)DNA genome accumulation and Rep expression only, induced a robust DDR in HEK293T cells. The induced DDR featured the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all 3 phosphatidylinositol 3-kinase-related kinases (PIKKs). We also found that the kinase ataxia telangiectasia and Rad3-related protein (ATR) plays a major role in AAV2 DNA replication and that Y family DNA repair DNA polymerases η (Pol η) and Pol κ contribute to AAV2 DNA replication both in vitro and in HEK293T cells. Knockout of Pol η and Pol κ in HEK293T cells significantly decreased wild-type AAV2 replication and recombinant AAV2 production. Thus, our study has proven that AAV2 DNA replication induces a DDR, which in turn initiates a DNA repairing process that partially contributes to the viral genome amplification in HEK293T cells. IMPORTANCE Recombinant AAV (rAAV) has emerged as one of the preferred delivery vectors for clinical gene therapy. rAAV production in HEK293 cells by transfection of a rAAV transgene plasmid, an AAV Rep and Cap expression packaging plasmid, and an Ad helper plasmid remains the popular method. Here, we demonstrated that the high fidelity Y family DNA repair DNA polymerase, Pol η, and Pol κ, plays a significant role in AAV DNA replication and rAAV production in HEK293T cells. Understanding the AAV DNA replication mechanism in HEK293T cells could provide clues to increase rAAV vector yield produced from the transfection method. We also provide evidence that the ATR-mediated DNA repair process through Pol η and Pol κ is one of the mechanisms to amplify AAV genome, which could explain AAV replication and rAAV ssDNA genome conversion in mitotic quiescent cells.

Autologous Bioactive Compound Concentrated Growth Factor Ameliorates Fistula Healing of Anal Fistula in a Pig Model and Promotes Proliferation and Migration of Human Skin Fibroblasts via Regulating the MEK/ERK Pathway.

Recent evidence suggested that autologous concentrated growth factor (CGF), a new bioactive compound from autologous blood is used widely as an ingenious biomaterial in tissue regeneration with anti-inflammatory properties. This study investigated whether CGF could be involved in the treatment of fistula healing in the anal fistula. For this purpose, the porcine anal fistula model was conducted using the rubber band ligation method and collected pig autogenic CGF to treat the fistulas. CGF treatment promoted fistula healing, which was reflected in the downregulation of inflammatory factors, upregulation of growth factors, and promoted epithelial-mesenchymal transition with increased collagen synthesis. Besides, 16S rRNA gene sequencing analysis of fistula tissues between the control and CGF groups showed that the microbial populations exhibiting significant differences were VadinCA02, Blastomonas, Deinococcus, Devosia, Sphingomonas, Rubrobacteria, and GW_34. CGF of volunteers were collected to process small interfering RNA- (siRNA-) ERK or siRNA-negative control transfected human skin fibroblasts (HSF). The results showed that CGF also promoted the proliferation and extracellular matrix-related functions in HSF, as well as activated the MEK/ERK pathway in vitro and in vivo. Finally, knockdown ERK reversed the effects of CGF in promoting wound healing in HSF. Collectively, our results suggest that the CGF as the bioactive compound from autologous blood exhibited great potential for repairing fistulas as well as promoting the proliferation and migration of human skin fibroblasts by triggering MEK/ERK signaling. These findings provided a fresh perspective for understanding the role of CGF in the management of fistulas.

Capsid Structure of Aleutian Mink Disease Virus and Human Parvovirus 4: New Faces in the Parvovirus Family Portrait.

Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10-30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.

5-methylcytosine (m5C) RNA modification controls the innate immune response to virus infection by regulating type I interferons.

5-methylcytosine (m5C) is one of the most prevalent modifications of RNA, playing important roles in RNA metabolism, nuclear export, and translation. However, the potential role of RNA m5C methylation in innate immunity remains elusive. Here, we show that depletion of NSUN2, an m5C methyltransferase, significantly inhibits the replication and gene expression of a wide range of RNA and DNA viruses. Notably, we found that this antiviral effect is largely driven by an enhanced type I interferon (IFN) response. The antiviral signaling pathway is dependent on the cytosolic RNA sensor RIG-I but not MDA5. Transcriptome-wide mapping of m5C following NSUN2 depletion in human A549 cells revealed a marked reduction in the m5C methylation of several abundant noncoding RNAs (ncRNAs). However, m5C methylation of viral RNA was not noticeably altered by NSUN2 depletion. In NSUN2-depleted cells, the host RNA polymerase (Pol) III transcribed ncRNAs, in particular RPPH1 and 7SL RNAs, were substantially up-regulated, leading to an increase of unshielded 7SL RNA in cytoplasm, which served as a direct ligand for the RIG-I-mediated IFN response. In NSUN2-depleted cells, inhibition of Pol III transcription or silencing of RPPH1 and 7SL RNA dampened IFN signaling, partially rescuing viral replication and gene expression. Finally, depletion of NSUN2 in an ex vivo human lung model and a mouse model inhibits viral replication and reduces pathogenesis, which is accompanied by enhanced type I IFN responses. Collectively, our data demonstrate that RNA m5C methylation controls antiviral innate immunity through modulating the m5C methylome of ncRNAs and their expression.

The small nonstructural protein NP1 of human bocavirus 1 directly interacts with Ku70 and RPA70 and facilitates viral DNA replication.

Human bocavirus 1 (HBoV1), a member of the genus Bocaparvovirus of the family Parvoviridae, causes acute respiratory tract infections in young children. Well-differentiated pseudostratified human airway epithelium cultured at an air-liquid interface (HAE-ALI) is an ideal in vitro culture model to study HBoV1 infection. Unique to other parvoviruses, bocaparvoviruses express a small nonstructured protein NP1 of ~25 kDa from an open reading frame (ORF) in the center of the viral genome. NP1 plays an important role in viral DNA replication and pre-mRNA processing. In this study, we performed an affinity purification assay to identify HBoV1 NP1-inteacting proteins. We identified that Ku70 and RPA70 directly interact with the NP1 at a high binding affinity, characterized with an equilibrium dissociation constant (KD) of 95 nM and 122 nM, respectively. Furthermore, we mapped the key NP1-interacting domains of Ku70 at aa266-439 and of RPA70 at aa181-422. Following a dominant negative strategy, we revealed that the interactions of Ku70 and RPA70 with NP1 play a significant role in HBoV1 DNA replication not only in an in vitro viral DNA replication assay but also in HBoV1-infected HAE-ALI cultures. Collectively, our study revealed a novel mechanism by which HBoV1 NP1 enhances viral DNA replication through its direct interactions with Ku70 and RPA70.

Eight years' advances on Bourbon virus, a tick-born Thogotovirus of the Orthomyxovirus family.

Bourbon virus (BRBV) was first isolated from a blood sample collected from a male patient living in Bourbon county, Kansas, during the spring of 2014. The patient later died due to complications associated with multiorgan failure. Currently, several BRBV infection-caused deaths have been reported in the United States, and misdiagnosed cases are often undercounted. BRBV is a member of the genus Thogotovirus of the Orthomyxoviridae family, and is transmitted through the Lone Star tick, Amblyomma Americanum, in North America. Currently, there are no specific antivirals or vaccinations available to treat or prevent BRBV infection. Several small molecular compounds have been identified to effectively inhibit BRBV infection of in vitro cell cultures at a single- or sub-micromolar level. Favipiravir, an RNA-dependent RNA polymerase inhibitor, prevented the death of Type I interferon receptor knockout mice infected with BRBV infection.

High-Throughput Screening Identifies Inhibitors for Parvovirus B19 Infection of Human Erythroid Progenitors.

Parvovirus B19 (B19V) infection can cause hematological disorders and fetal hydrops during pregnancy. Currently, no antivirals or vaccines are available for the treatment or prevention of B19V infection. To identify novel small-molecule antivirals against B19V replication, we developed a high-throughput screening (HTS) assay, which is based on an in vitro nicking assay using recombinant N-terminal amino acids 1 to 176 of the viral large nonstructural protein (NS1N) and a fluorescently labeled DNA probe (OriQ) that spans the nicking site of the viral DNA replication origin. We collectively screened 17,040 compounds and identified 2,178 (12.78%) hits that possess >10% inhibition of the NS1 nicking activity, among which 84 hits were confirmed to inhibit nicking in a dose-dependent manner. Using ex vivo-expanded primary human erythroid progenitor cells (EPCs) infected by B19V, we validated 24 compounds that demonstrated >50% in vivo inhibition of B19V infection at 10 µM, which can be categorized into 7 structure scaffolds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, the top 4 compounds were chosen to examine their inhibitions of B19V infection in EPCs at two times of the 90% maximal effective concentration (EC90). A purine derivative (P7) demonstrated an antiviral effect (EC50 = 1.46 µM) without prominent cytotoxicity (CC50 = 71.8 µM) in EPCs and exhibited 92% inhibition of B19V infection in EPCs at 3.32 µM, which can be used as the lead compound in future studies for the treatment of B19V infection-caused hematological disorders. IMPORTANCE B19V encodes a large nonstructural protein, NS1. Its N-terminal domain (NS1N) consisting of amino acids 1 to 176 binds to viral DNA and serves as an endonuclease to nick the viral DNA replication origins, which is a pivotal step in rolling-hairpin-dependent B19V DNA replication. For high-throughput screening (HTS) of anti-B19V antivirals, we miniaturized a fluorescence-based in vitro nicking assay, which employs a fluorophore-labeled probe spanning the terminal resolution site (trs) and the NS1N protein, into a 384-well-plate format. The HTS assay showed high reliability and capability in screening 17,040 compounds. Based on the therapeutic index (half-maximal cytotoxic concentration [CC50]/half-maximal effective concentration [EC50] ratio) in EPCs, a purine derivative demonstrated an antiviral effect of 92% inhibition of B19V infection in EPCs at 3.32 µM (two times the EC90). Our study demonstrated a robust HTS assay for screening antivirals against B19V infection.

The Large Nonstructural Protein (NS1) of Human Bocavirus 1 Directly Interacts with Ku70, Which Plays an Important Role in Virus Replication in Human Airway Epithelia.

Human bocavirus 1 (HBoV1), an autonomous human parvovirus, causes acute respiratory tract infections in young children. HBoV1 infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI). HBoV1 expresses a large nonstructural protein, NS1, that is essential for viral DNA replication. HBoV1 infection of polarized human airway epithelial cells induces a DNA damage response (DDR) that is critical to viral DNA replication involving DNA repair with error-free Y-family DNA polymerases. HBoV1 NS1 or the isoform NS1-70 per se induces a DDR. In this study, using the second-generation proximity-dependent biotin identification (BioID2) approach, we identified that Ku70 is associated with the NS1-BioID2 pulldown complex through a direct interaction with NS1. Biolayer interferometry (BLI) assay determined a high binding affinity of NS1 with Ku70, which has an equilibrium dissociation constant (KD) value of 0.16 µM and processes the strongest interaction at the C-terminal domain. The association of Ku70 with NS1 was also revealed during HBoV1 infection of HAE-ALI. Knockdown of Ku70 and overexpression of the C-terminal domain of Ku70 significantly decreased HBoV1 replication in HAE-ALI. Thus, our study provides, for the first time, a direct interaction of parvovirus large nonstructural protein NS1 with Ku70. IMPORTANCE Parvovirus infection induces a DNA damage response (DDR) that plays a pivotal role in viral DNA replication. The DDR includes activation of ATM (ataxia telangiectasia mutated), ATR (ATM- and RAD3-related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). The large nonstructural protein (NS1) often plays a role in the induction of DDR; however, how the DDR is induced during parvovirus infection or simply by the NS1 is not well studied. Activation of DNA-PKcs has been shown as one of the key DDR pathways in DNA replication of HBoV1. We identified that HBoV1 NS1 directly interacts with Ku70, but not Ku80, of the Ku70/Ku80 heterodimer at high affinity. This interaction is also important for HBoV1 replication in HAE-ALI. We propose that the interaction of NS1 with Ku70 recruits the Ku70/Ku80 complex to the viral DNA replication center, which activates DNA-PKcs and facilitates viral DNA replication.