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

Abstract

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.

Parvovirus B19 integration into human CD36+ erythroid progenitor cells

“…Gel shift assays have shown NS1 binding to AGGGCGGA octanucleotide GC elements upstream of the P6 promoter (Gareus et al, 1998; Raab et al, 2001; Raab et al, 2002). Additionally, within the B19V ITR, NS1 binding motifs have been identified as GC rich imperfect palindromic repeats of CCGGCGGC (Ganaie et al, 2017; Guan et al, 2009; Sanchez et al, 2016). Recent studies have found NS1 can complex with STAT5 to facilitate replication (Ganaie et al, 2017).…”

“…Additionally, within the B19V ITR, NS1 binding motifs have been identified as GC rich imperfect palindromic repeats of CCGGCGGC (Ganaie et al, 2017; Guan et al, 2009; Sanchez et al, 2016). Recent studies have found NS1 can complex with STAT5 to facilitate replication (Ganaie et al, 2017). This genomic configuration of NS1 interacting with DNA and cellular factors is similar to that of AAV Rep binding domains (McCarty et al, 1994).…”

“…Replication of the B19V 5.5 KB single stranded DNA genome utilizes the basic mechanism established for other members of the parvovirus family (Chen et al, 2011; Ganaie et al, 2017; Guan et al, 2009; Luo and Qiu, 2015). The viral inverted terminal-repeat elements present at both ends of the linear genome (Figure 1D) serve as replication primers for cellular DNA polymerase.…”

Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development

“…Protein expression was induced by addition of IPTG (isopropyl-␤-Dthiogalactopyranoside) to the bacterial culture at 1 mM. Protein was purified with Ni-nitrilotriacetic acid (NTA) affinity agarose (Qiagen) as described previously (42).…”

“…The minimal replication origin (Ori) of B19V DNA has been identified to be 67 nucleotides (nt), which includes a STAT5-binding element (STAT5BE), a trs or nicking site, two copies of the NS1-binding element (NS1BE), and the putative cellular factorbinding site (CFBE) ( Fig. 2A) (25,27,41,42). Specific cleavage of the ssDNA Ori has been demonstrated in an in vitro nicking assay using the purified B19V NS1 N terminus (aa 1 to 176) and a 5=-end 32 P-labeled oligonucleotide that contain the trs (28).…”