Abstract
Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85α−/−β−/−) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85α−/−β−/− cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses.
Highlights
Orthopoxviruses, including vaccinia virus (VV), monkeypox (MPX), and variola (VarV), are large dsDNA viruses that cause characteristic umbilicated vesiculo-pustular skin lesions (‘‘pox’’)
phosphatidylinositol 3-kinase (PI3K) regulate plaque morphology To identify host kinases utilized by VV, we screened a small directed library, which included approximately 350 novel and previously described inhibitors, for those that decreased the size of plaques
We define the involvement of PI3Ks in orthopoxvirus morphogenesis using PI3K inhibitors and cell lines deficient in the regulatory subunit of Type I PI3Ks, p85
Summary
Orthopoxviruses, including vaccinia virus (VV), monkeypox (MPX), and variola (VarV), are large dsDNA viruses that cause characteristic umbilicated vesiculo-pustular skin lesions (‘‘pox’’). Infection is initiated upon entry of either of two forms of the virus. The first, called the Intracellular Mature Virus (‘‘IMV’’, and called Mature Virion ‘‘MV’’), consists of a brick-shaped viral core surrounded by one or two lipid bilayers derived from an ER-golgi intermediate compartment (ERGIC) [5,6,7,8]. A second infectious form of the virus, called the extracellular enveloped virus (‘‘EEV’’, and called Enveloped Virus ‘‘EV’’) [13], is released from the cell surface and consists of an IMV enveloped in additional host-cell derived membranes. After delivery of the viral core to the cytoplasm, early viral gene transcription and translation initiates replication and morphogenesis
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