title: Direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized L2-interacting protein A30.5.
abstract: Crescents consisting of a single lipoprotein membrane with an external protein scaffold comprise the initial structural elements of poxvirus morphogenesis. Crescents enlarge to form spherical immature virions, which enclose viroplasm consisting of proteins destined to form the cores of mature virions. Previous studies suggest that the L2 protein participates in the recruitment of endoplasmic reticulum (ER)-derived membranes to form immature virions within assembly sites of cytoplasmic factories. Here we show that L2 interacts with the previously uncharacterized 42-amino-acid A30.5 protein. An open reading frame similar in size to the one encoding A30.5 is at the same genome location in representatives of all chordopoxvirus genera. A30.5 has a putative transmembrane domain and colocalized with markers of the endoplasmic reticulum and with L2. By constructing a complementing cell line expressing A30.5, we isolated a deletion mutant virus that exhibits a defect in morphogenesis in normal cells. Large electron-dense cytoplasmic inclusions and clusters of scaffold protein-coated membranes that resemble crescents and immature virions devoid of viroplasm were seen in place of normal structures. Crescent-shaped membranes were continuous with the endoplasmic reticulum membrane and oriented with the convex scaffold protein-coated side facing the lumen, while clusters of completed spherical immature-virion-like forms were trapped within the expanded lumen. Immature-virion-like structures were more abundant in infected RK-13 cells than in BS-C-1 or HeLa cells, in which cytoplasmic inclusions were decorated with scaffold protein-coated membrane arcs. We suggest that the outer surface of the poxvirus virion is derived from the luminal side of the ER membrane.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.
authors.forename: Liliana
authors.initials: L
authors.lastname: Maruri-Avidal
authors.forename: Andrea S
authors.initials: AS
authors.lastname: Weisberg
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism / Q000821:virology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000821:virology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D014615:Vaccinia / Q000378:metabolism* / Q000821:virology; D014616:Vaccinia virus / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism*; D014771:Virion / Q000502:physiology*; D019065:Virus Assembly; D014779:Virus Replication
chemical_list: D014764:Viral Proteins
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