In vitro evolution of high-titer, virus-like vesicles containing a single structural protein.
Self-propagating, infectious, virus-like vesicles (VLVs) are generated when an alphavirus RNA replicon expresses the vesicular stomatitis virus glycoprotein (VSV G) as the only structural protein. The mechanism that generates these VLVs lacking a capsid protein has remained a mystery for over 20 years. We present evidence that VLVs arise from membrane-enveloped RNA replication factories (spherules) containing VSV G protein that are largely trapped on the cell surface. After extensive passaging, VLVs evolve to grow to high titers through acquisition of multiple point mutations in their nonstructural replicase proteins. We reconstituted these mutations into a plasmid-based system from which high-titer VLVs can be recovered. One of these mutations generates a late domain motif (PTAP) that is critical for high-titer VLV production. We propose a model in which the VLVs have evolved in vitro to exploit a cellular budding pathway that is hijacked by many enveloped viruses, allowing them to bud efficiently from the cell surface. Our results suggest a basic mechanism of propagation that may have been used by primitive RNA viruses lacking capsid proteins. Capsids may have evolved later to allow more efficient packaging of RNA, greater virus stability, and evasion of innate immunity.
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510; and.
Nina F
NF
Rose
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510; and.
Linda
L
Buonocore
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510; and.
John B
JB
Schell
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510; and.
Anasuya
A
Chattopadhyay
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510; and.
Kapil
K
Bahl
Center for Cellular and Molecular Imaging, Yale University School of Medicine, New Haven, CT 06510.
Xinran
X
Liu
Department of Pathology, Yale University School of Medicine, New Haven, CT 06510; and john.rose@yale.edu.
John K
JK
Rose
D000524:Alphavirus / Q000737:chemistry / Q000235:genetics*; D020816:Amino Acid Motifs; D005075:Biological Evolution; D066298:In Vitro Techniques; D046529:Microscopy, Electron, Transmission; D015678:Viral Structural Proteins / Q000737:chemistry*
D015678:Viral Structural Proteins
SFV replicon; VSV glycoprotein; evolution; late domain