Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged m protein allows dynamic imaging of m protein and virus uncoating in infected cells.
A recombinant vesicular stomatitis virus (VSV-PeGFP-M-MmRFP) encoding enhanced green fluorescent protein fused in frame with P (PeGFP) in place of P and a fusion matrix protein (monomeric red fluorescent protein fused in frame at the carboxy terminus of M [MmRFP]) at the G-L gene junction, in addition to wild-type (wt) M protein in its normal location, was recovered, but the MmRFP was not incorporated into the virions. Subsequently, we generated recombinant viruses (VSV-PeGFP-DeltaM-Mtc and VSV-DeltaM-Mtc) encoding M protein with a carboxy-terminal tetracysteine tag (Mtc) in place of the M protein. These recombinant viruses incorporated Mtc at levels similar to M in wt VSV, demonstrating recovery of infectious rhabdoviruses encoding and incorporating a tagged M protein. Virions released from cells infected with VSV-PeGFP-DeltaM-Mtc and labeled with the biarsenical red dye (ReAsH) were dually fluorescent, fluorescing green due to incorporation of PeGFP in the nucleocapsids and red due to incorporation of ReAsH-labeled Mtc in the viral envelope. Transport and subsequent association of M protein with the plasma membrane were shown to be independent of microtubules. Sequential labeling of VSV-DeltaM-Mtc-infected cells with the biarsenical dyes ReAsH and FlAsH (green) revealed that newly synthesized M protein reaches the plasma membrane in less than 30 min and continues to accumulate there for up to 2 1/2 hours. Using dually fluorescent VSV, we determined that following adsorption at the plasma membrane, the time taken by one-half of the virus particles to enter cells and to uncoat their nucleocapsids in the cytoplasm is approximately 28 min.
Das
Subash C
SC
Department of Veterinary and Biomedical Sciences and the Nebraska Center for Virology, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0900, USA.
Panda
Debasis
D
Nayak
Debasis
D
Pattnaik
Asit K
AK
D000818:Animals; D001151:Arsenic / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003116:Color; D006224:Cricetinae; D005453:Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
C094460:M protein, Vesicular stomatitis virus; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; D001151:Arsenic