Essential Host Factors for Human Parvovirus B19 Replication

Parvovirus B19 (B19V) is a small, non-enveloped virus that contains a single-stranded DNA (ssDNA) genome of 5.6 kb in size. 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. On this rationale, we tried to investigate the essential host factors for efficient virus replication. Our results demonstrated that signal transducer and activator of transcription 5 (STAT5) and RNA-binding motif protein-38 (RBM38) are two of these important host factors that ensure virus DNA replication and pre-mRNA processing, respectively, during B19V infection. The stages of erythropoiesis during which STAT5 is activated and RBM38 is expressed, are highly susceptible to B19V infection, thus suggesting that these two factors are among the key determinants of the B19V restriction to human EPCs. B19V requires erythropoietin (EPO) signaling and hypoxia for its efficient replication. EPO to EPO receptor signaling activates JAK2-STAT5 pathway that phosphorylates STAT5. In our first study, we show that phosphorylated STAT5 is critical for B19V replication. Upon in-silico analysis, we identified a consensus STAT5 binding element adjacent to the NS1-binding elements within the minimal origin of viral DNA replication (Ori) in the B19V genome. The phosphorylated STAT5 specifically interacts with viral Ori both in vivo and in vitro, and is actively recruited within the viral DNA replication centers. Furthermore, our study shows a novel interaction between STAT5 and the minichromosome maintenance (MCM) complex. Our proposed model suggests that STAT5 directly facilitates viral DNA replication by recruiting MCM complex into viral DNA replication centers. Interestingly, we found that pimozide, a STAT5 phosphorylation inhibitor and an FDA-approved drug, inhibits B19V replication in ex vivo expanded EPCs, suggesting that pimozide could be a promising antiviral drug for the treatment of B19V-related pathologies. B19V expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternate polyadenylation to generate 12 viral mRNA transcripts. Splicing at the second 5’ donor site (D2) of the B19V pre-mRNA is essential for the expression of VP1, VP2 and 11-kDa. We have previously identified that a cis-acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates recognition of the D2 donor for its efficient splicing. In our second study, we described that ISE2 harbors a consensus RBM38 binding sequence–5’-UGUGUG-3’. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that the RBM38 binds specifically with the ISE2 element in vitro. Knockdown of RBM38 significantly decreases the level of the spliced mRNA at D2 that encodes 11-kDa protein and, thereafter, the expression of the 11-kDa protein. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, knockdown of RBM38 decreases virus replication via downregulating 11-kDa expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication, and that RBM38 is an essential host factor for the splicing of B19V pre-mRNA from D2 to A2-2 sites and for the expression of the 11-kDa protein. In conclusion, we identified two host factors, STAT5 and RBM38, which play important roles in B19V replication. We provide a mechanistic overview of how STAT5 facilitates virus DNA replication and RBM38 promotes the splicing of B19V pre-mRNA that ensures the expression of 11-kDa protein

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site

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

Oropouche orthobunyavirus infection is mediated by the cellular host factor Lrp1

Oropouche orthobunyavirus (OROV

Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice

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

Recent Advances in Replication and Infection of Human Parvovirus B19

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

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

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

RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers

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.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

pSTAT5, but not NS1, interacts with the MCM complex.

<p>(A) Immunoprecipitation (IP) assay. Cell lysates of NS1^Flag-expressing UT7/Epo-S1 cells were prepared for pull-down assays with either anti-Flag-conjugated beads or control beads. Immunoprecipitated proteins were examined for the presence of MCM2 by Western blotting. Blots were reprobed with rabbit anti-pSTAT5(Y694), anti-E2F5, and anti-Flag antibodies. Detection of E2F5 was used as a positive control for NS1 IP. (B) Co-IP assay. UT7/Epo-S1 cells were collected, washed, and lysed with RIPA buffer. After centrifugation, the supernatant was incubated with either rabbit anti-pSTAT5(Y694) or control IgG antibody. Immunoprecipitated proteins were blotted for the presence of the MCM complex with an anti-MCM5 antibody and for pSTAT5 with rabbit anti-pSTAT5(Y694). (C) Reverse Co-IP assay. Reverse Co-IP was performed with an anti-MCM5 antibody. Immunoprecipitated proteins were examined for pSTAT5, MCM2, and MCM5, respectively. (D) Co-IP of lysates treated with DNase. UT7/Epo-S1 cell lysates, either treated or untreated with DNase (750 units of Benzonase) were incubated with anti-pSTAT5(Y694) or control IgG antibodies for Co-IP assay, and immunoprecipitated proteins were examined for MCM2 by Western blot analysis. (E-H) Immunofluorescence analysis. (E&F) Mock- or B19V-infected CD36⁺ EPCs were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies, followed by (E) incubation with respective secondary antibodies, or by (F) proximal ligation assay, which produces amplified signal for labeled molecules in close proximity. (G) CD36⁺ EPCs were incubated with either DMSO or pimozide (at 30 μM) for 2 days. And then the cells were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies for proximity ligation assay. (H) Infected EPCs were stained with an anti-capsid antibody. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.</p

Failure of B19V replicative form DNA clones with STAT5-binding element mutations in replication in transfected UT7/Epo-S1 cells.

<p>(A) Diagram of the B19V full-length M20 RF genome and various half ITR-deleted N8 RF genomes with mutations at STAT5-binding elements (STAT5BEs). Red squares indicate the position of the <i>Ori</i> sequences at both ITRs, and grey squares indicate mutated Ori (<i>mOri</i>). The sequence of the <i>mOri</i> is shown with mutated nucleotides in grey in the STAT5BE. (B&C) Southern blot analysis. (B) The N8 RF DNA, or derivatives with mutations in the STAT5BE of either the left ITR (N8^mOriL), right ITR (N8^mOriR), or both (N8^mOri), were transfected into UT7/Epo-S1 cells. (C) M20, and M20^mOri, a derivative of the M20 RF DNA with STAT5BEs of both ITRs mutated, were transfected into UT7/Epo-S1 cells. At 48 h post-transfection, cells were collected for Hirt DNA extraction. And Hirt DNA samples were analyzed by Southern blotting with an M20 DNA probe. RF DNA (RF), ssDNA (ss), and Dpn I-digested DNA (shown with a line) are indicated. Mitochondrial DNA (Mito DNA) was used as a loading control (lower panels). (D) Viral protein expression of B19V DNA mutants. M20 or M20^mOri transfected UT7/Epo-S1 cells were stained with anti-NS1 or anti-capsid antibodies. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.</p

STAT5 colocalizes with B19V NS1, capsids, and the replicating B19V genome.

<p>(A&B) STAT5 colocalizes with B19V NS1 and capsids. Mock- or B19V-infected CD36⁺ EPCs were co-stained and examined with rabbit anti-STAT5 and rat anti-B19V NS1 antibodies (A) or with rabbit anti-STAT5 and mouse anti-B19V capsid antibodies (B). (C-E) Proximity ligation assay. Infected cells were co-stained with rabbit anti-STAT5 and mouse anti-B19V capsid antibodies (C), or co-stained with rabbit anti-STAT5 and mouse anti-BrdU antibodies (D), or co-stained with mouse anti-B19V capsid and rabbit anti-BrdU antibodies (E), followed by a proximity ligation assay, which produces amplified signal for labeled molecules in close proximity. (F) STAT5 colocalizes with the replicating viral genome. Mock- or B19V-infected CD36⁺ EPCs were BrdU labeled to identify replicating viral ssDNA genomes. The treated cells were co-stained with rabbit anti-STAT5 and mouse anti-BrdU antibodies, followed by incubation with secondary antibodies. Images were taken with an Eclipse C1 Plus (Nikon) confocal microscope at 100 × magnification. Nuclei were stained with DAPI.</p