RESUMO
Rift Valley Fever Virus (RVFV) is a negative sense segmented RNA virus that can cause severe hemorrhagic fever. The tri-segmented virus genome encodes for six (6) multifunctional proteins that engage host factors at a variety of different stages in the replication cycle. The S segment encodes nucleoprotein (N) and nonstructural protein S (NSs), the M segment encodes viral glycoproteins Gn and Gc as well as nonstructural protein M (NSm) and the L segment encodes the viral polymerase (L). Viral glycoproteins Gn and Gc are responsible for entry by binding to a number of host factors. Our recent studies identified a scavenger receptor, LDL receptor related protein 1 (Lrp1), as a potential pro-viral host factor for RVFV and related viruses, including Oropouche virus (OROV) infection. Coincidentally, several recent studies identified other LDL family proteins as viral entry factors and receptors for other viral families. Collectively, these observations suggest that highly conserved LDL family proteins may play a significant role in facilitating entry of viruses from several distinct families. Given the significant roles of viral and host factors during infection, characterization of these interactions is critical for therapeutic targeting with neutralizing antibodies and vaccines.
Assuntos
Vírus da Febre do Vale do Rift , Animais , Humanos , Vírus da Febre do Vale do Rift/genética , Anticorpos Neutralizantes/genética , Genoma Viral , GlicoproteínasRESUMO
Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor-related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.
Assuntos
Febre do Vale de Rift , Vírus da Febre do Vale do Rift , Animais , Camundongos , Febre do Vale de Rift/genética , Vírus da Febre do Vale do Rift/genética , África , Hepatócitos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genéticaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.
Assuntos
COVID-19 , Ubiquitina , Humanos , Ubiquitina/metabolismo , Peptídeo Hidrolases/metabolismo , SARS-CoV-2/metabolismo , Domínio Catalítico , Papaína/química , Papaína/metabolismo , Replicação ViralRESUMO
People infected with the mosquito-borne Rift Valley fever virus (RVFV) can suffer from eye-related problems resulting in ongoing vision issues or even permanent blindness. Despite ocular disease being the most frequently reported severe outcome, it is vastly understudied compared to other disease outcomes caused by RVFV. Ocular manifestations of RVFV include blurred vision, uveitis, and retinitis. When an infected individual develops macular or paramacular lesions, there is a 50% chance of permanent vision loss in one or both eyes. The cause of blinding ocular pathology remains unknown in part due to the lack of a tractable animal model. Using 3 relevant exposure routes, both subcutaneous (SC) and aerosol inoculation of Sprague Dawley rats led to RVFV infection of the eye. Surprisingly, direct inoculation of the conjunctiva did not result in successful ocular infection. The posterior segment of the eye, including the optic nerve, choroid, ciliary body, and retina, were all positive for RVFV antigen in SC-infected rats, and live virus was isolated from the eyes. Proinflammatory cytokines and increased leukocyte counts were also found in the eyes of infected rats. Additionally, human ocular cell lines were permissive for Lrp1-dependent RVFV infection. This study experimentally defines viral tropism of RVFV in the posterior segment of the rat eye and characterizes virally-mediated ocular inflammation, providing a foundation for evaluation of vaccines and therapeutics to protect against adverse ocular outcomes. IMPORTANCE Rift Valley fever virus (RVFV) infection leads to eye damage in humans in up to 10% of reported cases. Permanent blindness occurs in 50% of individuals with significant retinal scarring. Despite the prevalence and severity of this outcome, very little is known about the mechanisms of pathogenesis. We addressed this gap by developing a rodent model of ocular disease. Subcutaneous infection of Sprague Dawley rats resulted in infection of the uvea, retina, and optic nerve along with the induction of inflammation within the posterior eye. Infection of human ocular cells induced inflammatory responses and required host entry factors for RVFV infection similar to rodents. This work provides evidence of how RVFV infects the eye, and this information can be applied to help mitigate the devastating outcomes of RVF ocular disease through vaccines or treatments.
Assuntos
Oftalmopatias , Febre do Vale de Rift , Vírus da Febre do Vale do Rift , Ratos , Humanos , Animais , Vírus da Febre do Vale do Rift/fisiologia , Ratos Sprague-Dawley , Inflamação , Citocinas , Aerossóis , CegueiraRESUMO
Oropouche orthobunyavirus (OROV; Peribunyaviridae) is a mosquito-transmitted virus that causes widespread human febrile illness in South America, with occasional progression to neurologic effects. Host factors mediating the cellular entry of OROV are undefined. Here, we show that OROV uses the host protein low-density lipoprotein-related protein 1 (Lrp1) for efficient cellular infection. Cells from evolutionarily distinct species lacking Lrp1 were less permissive to OROV infection than cells with Lrp1. Treatment of cells with either the high-affinity Lrp1 ligand receptor-associated protein (RAP) or recombinant ectodomain truncations of Lrp1 significantly reduced OROV infection. In addition, chimeric vesicular stomatitis virus (VSV) expressing OROV glycoproteins (VSV-OROV) bound to the Lrp1 ectodomain in vitro. Furthermore, we demonstrate the biological relevance of the OROV-Lrp1 interaction in a proof-of-concept mouse study in which treatment of mice with RAP at the time of infection reduced tissue viral load and promoted survival from an otherwise lethal infection. These results with OROV, along with the recent finding of Lrp1 as an entry factor for Rift Valley fever virus, highlight the broader significance of Lrp1 in cellular infection by diverse bunyaviruses. Shared strategies for entry, such as the critical function of Lrp1 defined here, provide a foundation for the development of pan-bunyaviral therapeutics.
Assuntos
Infecções por Bunyaviridae , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Orthobunyavirus , Internalização do Vírus , Animais , Infecções por Bunyaviridae/metabolismo , Infecções por Bunyaviridae/virologia , Técnicas de Inativação de Genes , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Orthobunyavirus/fisiologia , América do SulRESUMO
Hantavirus nucleocapsid protein (NP) inhibits protein kinase R (PKR) dimerization by an unknown mechanism to counteract its antiviral responses during virus infection. Here we demonstrate that NP exploits an endogenous PKR inhibitor P58IPK to inhibit PKR. The activity of P58IPK is normally restricted in cells by the formation of an inactive complex with its negative regulator Hsp40. On the other hand, PKR remains associated with the 40S ribosomal subunit, a unique strategic location that facilitates its free access to the downstream target eIF2α. Although both NP and Hsp40 bind to P58IPK, the binding affinity of NP is much stronger compared to Hsp40. P58IPK harbors an NP binding site, spanning to N-terminal TPR subdomains I and II. The Hsp40 binding site on P58IPK was mapped to the TPR subdomain II. The high affinity binding of NP to P58IPK and the overlap between NP and Hsp40 binding sites releases the P58IPK from its negative regulator by competitive inhibition. The NP-P58IPK complex is selectively recruited to the 40S ribosomal subunit by direct interaction between NP and the ribosomal protein S19 (RPS19), a structural component of the 40S ribosomal subunit. NP has distinct binding sites for P58IPK and RPS19, enabling it to serve as bridge between P58IPK and the 40S ribosomal subunit. NP mutants deficient in binding to either P58IPK or RPS19 fail to inhibit PKR, demonstrating that selective engagement of P58IPK to the 40S ribosomal subunit is required for PKR inhibition. Cells deficient in P58IPK mount a rapid PKR antiviral response and establish an antiviral state, observed by global translational shutdown and rapid decline in viral load. These studies reveal a novel viral strategy in which NP releases P58IPK from its negative regulator and selectively engages it on the 40S ribosomal subunit to promptly combat the PKR antiviral responses.
Assuntos
Infecções por Hantavirus/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , eIF-2 Quinase/metabolismo , Células HEK293 , Orthohantavírus , Células HeLa , HumanosRESUMO
Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.
Assuntos
Interações Hospedeiro-Patógeno , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Vírus da Febre do Vale do Rift/fisiologia , Internalização do Vírus , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Encéfalo/patologia , Encéfalo/virologia , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Células Cultivadas , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Glicosilação , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Glicoproteínas de Membrana/metabolismo , Camundongos , Ligação Proteica , Desnaturação Proteica , Febre do Vale de Rift/patologia , Febre do Vale de Rift/prevenção & controle , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/imunologiaRESUMO
Bourbon virus (BRBV) was first isolated from a patient hospitalized at the University of Kansas Hospital in 2014. Since then, several deaths have been reported to be caused by BRBV infection in the Midwest and Southern United States. BRBV is a tick-borne virus that is widely carried by lone star ticks. It belongs to genus Thogotovirus of the Orthomyxoviridae family. Currently, there are no treatments or vaccines available for BRBV or thogotovirus infection caused diseases. In this study, we reconstituted a replicon reporter system, composed of plasmids expressing the RNA-dependent RNA polymerase (RdRP) complex (PA, PB1, and PB2), nucleocapsid (NP) protein, and a reporter gene flanked by the 3' and 5' untranslated region (UTR) of the envelope glycoprotein (GP) genome segment. By using the luciferase reporter, we screened a few small molecule compounds of anti-endonuclease that inhibited the nicking activity by parvovirus B19 (B19V) NS1, as well as FDA-approved drugs targeting the RdRP of influenza virus. Our results demonstrated that myricetin, an anti-B19V NS1 nicking inhibitor, efficiently inhibited the RdRP activity of BRBV and virus replication. The IC50 and EC50 of myricetin are 2.22 and 4.6 µM, respectively, in cells. Myricetin had minimal cytotoxicity in cells, and therefore the therapeutic index of the compound is high. In conclusion, the BRBV replicon system is a useful tool to study viral RNA replication and to develop antivirals, and myricetin may hold promise in treatment of BRBV infected patients.
RESUMO
During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD ] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.
Assuntos
Regulação Viral da Expressão Gênica , Íntrons , Proteínas do Tecido Nervoso/metabolismo , Parvovirus B19 Humano/genética , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/genética , Células Cultivadas , Células Precursoras Eritroides/virologia , Genoma Viral , Células-Tronco Hematopoéticas , Humanos , Proteínas do Tecido Nervoso/genética , Parvovirus B19 Humano/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication.IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.
Assuntos
Proteínas do Capsídeo/biossíntese , Núcleo Celular , Regulação Viral da Expressão Gênica , Bocavirus Humano/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro , RNA Viral , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas do Capsídeo/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células HEK293 , Bocavirus Humano/genética , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Human parvovirus B19 (B19V), a member of the genus Erythroparvovirus of the family Parvoviridae, is a small nonenveloped virus that has a single-stranded DNA (ssDNA) genome of 5.6 kb with two inverted terminal repeats (ITRs). B19V infection often results in severe hematological disorders and fetal death in humans. B19V replication follows a model of rolling hairpin-dependent DNA replication, in which the large nonstructural protein NS1 introduces a site-specific single-strand nick in the viral DNA replication origins, which locate at the ITRs. NS1 executes endonuclease activity through the N-terminal origin-binding domain. Nicking of the viral replication origin is a pivotal step in rolling hairpin-dependent viral DNA replication. Here, we developed a fluorophore-based in vitro nicking assay of the replication origin using the origin-binding domain of NS1 and compared it with the radioactive in vitro nicking assay. We used both assays to screen a set of small-molecule compounds (n = 96) that have potential antinuclease activity. We found that the fluorophore-based in vitro nicking assay demonstrates sensitivity and specificity values as high as those of the radioactive assay. Among the 96 compounds, we identified 8 which have an inhibition of >80% at 10 µM in both the fluorophore-based and radioactive in vitro nicking assays. We further tested 3 compounds that have a flavonoid-like structure and an in vitro 50% inhibitory concentration that fell in the range of 1 to 3 µM. Importantly, they also exhibited inhibition of B19V DNA replication in UT7/Epo-S1 cells and ex vivo-expanded human erythroid progenitor cells.
Assuntos
Antivirais/farmacologia , Replicação do DNA/efeitos dos fármacos , Infecções por Parvoviridae/tratamento farmacológico , Parvovirus B19 Humano/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Linhagem Celular , DNA Viral/genética , Desenvolvimento de Medicamentos , Células Precursoras Eritroides , Humanos , Infecções por Parvoviridae/virologia , Replicação Viral/genéticaRESUMO
Lytic infection of human parvovirus B19 (B19V) takes place exclusively in human erythroid progenitor cells of bone marrow and fetal liver, which disrupts erythropoiesis. During infection, B19V expresses three nonstructural proteins (NS1, 11-kDa, and 7.5-kDa) and two structural proteins (VP1 and VP2). While NS1 is essential for B19V DNA replication, 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of 11-kDa in viral DNA replication and elucidated the underlying mechanism. We found that 11-kDa specially interacts with cellular growth factor receptor-bound protein 2 (Grb2) during virus infection and in vitro We determined a high affinity interaction between 11-kDa and Grb2 that has an equilibrium dissociation constant (KD ) value of 18.13 nM. In vitro, one proline-rich motif was sufficient for 11-kDa to sustain a strong interaction with Grb2. In consistence, in vivo during infection, one proline-rich motif was enough for 11-kDa to significantly reduce phosphorylation of extracellular signal-regulated kinase (ERK). Mutations of all three proline-rich motifs of 11-kDa abolished its capability to reduce ERK activity and, accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding a short hairpin RNA (shRNA) targeting Grb2 decreased the expression of Grb2 as well as the level of ERK phosphorylation, which resulted in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa protein interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of nonimmunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins, VP1 and VP2, and three nonstructural proteins, NS1, 11-kDa, and 7.5-kDa. While NS1 is essential, 11-kDa plays an enhancing role in viral DNA replication. Here, we elucidated a mechanism underlying 11-kDa protein-regulated B19V DNA replication. 11-kDa is tightly associated with cellular growth factor receptor-bound protein 2 (Grb2) during infection. In vitro, 11-kDa interacts with Grb2 with high affinity through three proline-rich motifs, of which at least one is indispensable for the regulation of viral DNA replication. 11-kDa and Grb2 interaction disrupts extracellular signal-regulated kinase (ERK) signaling, which mediates upregulation of B19V replication. Thus, our study reveals a novel mechanism of how a parvoviral small nonstructural protein regulates viral DNA replication by interacting with a host protein that is predominately expressed in the cytoplasm.
Assuntos
Proteína Adaptadora GRB2/metabolismo , Infecções por Parvoviridae/metabolismo , Parvovirus B19 Humano/fisiologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Replicação do DNA , Humanos , Peso Molecular , Mutação , Parvovirus B19 Humano/metabolismo , Fosforilação , Prolina/metabolismo , Ligação Proteica , Replicação ViralRESUMO
Parvovirus B19 (B19V) is pathogenic to humans and causes bone marrow failure diseases and various other inflammatory disorders. B19V infection exhibits high tropism for human erythroid progenitor cells (EPCs) in the bone marrow and fetal liver. The exclusive restriction of B19V replication to erythroid lineage cells is partly due to the expression of receptor and co-receptor(s) on the cell surface of human EPCs and partly depends on the intracellular factors essential for virus replication. We first summarize the latest developments in the viral entry process and the host cellular factors or pathways critical for B19V replication. We discuss the role of hypoxia, erythropoietin signaling and STAT5 activation in the virus replication. The B19V infection-induced DNA damage response (DDR) and cell cycle arrest at late S-phase are two key events that promote B19V replication. Lately, the virus infection causes G2 arrest, followed by the extensive cell death of EPCs that leads to anemia. We provide the current understanding of how B19V exploits the cellular resources and manipulate pathways for efficient virus replication. B19V encodes a single precursor mRNA (pre-mRNA), which undergoes alternate splicing and alternative polyadenylation to generate at least 12 different species of mRNA transcripts. The post-transcriptional processing of B19V pre-mRNA is tightly regulated through cis-acting elements and trans-acting factors flanking the splice donor or acceptor sites. Overall, in this review, we focus on the recent advances in the molecular virology and pathogenesis of B19V infection.
Assuntos
Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/fisiologia , Parvovirus B19 Humano/patogenicidade , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Pontos de Checagem do Ciclo Celular , Dano ao DNA , Células Precursoras Eritroides/virologia , Regulação Viral da Expressão Gênica , Humanos , Parvovirus B19 Humano/genética , Fator de Transcrição STAT5/metabolismo , Tropismo Viral , VirulênciaRESUMO
Human parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5' donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified that cis-acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5'-UGUGUG-3'. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 element in vitro The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein.IMPORTANCE B19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral receptors and coreceptors on the cell surface but also on the intracellular host factors that support B19V replication. Our present study shows that B19V uses a host factor, RNA binding motif protein 38 (RBM38), for the processing of its pre-mRNA during virus replication. Specifically, RBM38 interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression, thereby regulating the 11-kDa protein-mediated augmentation of B19V replication. The identification of this novel host-pathogen interaction will provide mechanistic insights into B19V replication and aid in finding new targets for anti-B19V therapeutics.
Assuntos
Replicação do DNA/fisiologia , DNA Viral/metabolismo , Regulação para Baixo/fisiologia , Eritema Infeccioso/metabolismo , Regulação Viral da Expressão Gênica/fisiologia , Parvovirus B19 Humano/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas não Estruturais Virais/biossíntese , Replicação Viral/fisiologia , DNA Viral/genética , Eritema Infeccioso/genética , Humanos , Proteínas de Ligação a RNA/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.
Assuntos
Divisão Celular , Replicação do DNA , Interações Hospedeiro-Patógeno , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano/genética , Parvovirus B19 Humano/metabolismo , Replicação Viral , Bromodesoxiuridina/metabolismo , Antígenos CD36/análise , Antígenos CD36/metabolismo , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Dano ao DNA , DNA Polimerase III , DNA Polimerase beta , Reparo do DNA , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/virologia , Morte Fetal , Regulação Viral da Expressão Gênica/fisiologia , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Parvovirus B19 Humano/patogenicidade , Fosforilação , Mapas de Interação de Proteínas , Aplasia Pura de Série Vermelha/virologia , Proteína de Replicação A/genética , Fase S , Transcriptoma , Viremia/virologiaRESUMO
Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor.IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.
RESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus.IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Proteínas do Nucleocapsídeo/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Humanos , Ligação ProteicaRESUMO
Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.
Assuntos
Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Parvovirus B19 Humano/genética , Fator de Transcrição STAT5/metabolismo , Replicação Viral , Células Precursoras Eritroides/virologia , Eritropoetina/genética , Eritropoetina/metabolismo , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Parvovirus B19 Humano/fisiologia , Fosforilação , Fator de Transcrição STAT5/genética , Transdução de SinaisRESUMO
Human parvovirus B19 (B19V) infection of primary human erythroid progenitor cells (EPCs) arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR) that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2) within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related) activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.