Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged M protein allows dynamic imaging of M protein and virus uncoating in infected cells

Subash C. Das, Debasis Panda, Debasis Nayak, Asit K Pattnaik

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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-ΔM-Mtc and VSV-ΔM-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-ΔM- 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-δM-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.

Original languageEnglish (US)
Pages (from-to)2611-2622
Number of pages12
JournalJournal of virology
Volume83
Issue number6
DOIs
StatePublished - Mar 1 2009

Fingerprint

Virus Uncoating
Vesiculovirus
Vesicular Stomatitis
image analysis
Viruses
viruses
virion
Virion
Proteins
proteins
Nucleocapsid
nucleocapsid
cells
plasma membrane
Cell Membrane
dyes
Coloring Agents
Rhabdoviridae
green fluorescent protein
Microtubules

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Biarsenical labeling of vesicular stomatitis virus encoding tetracysteine-tagged M protein allows dynamic imaging of M protein and virus uncoating in infected cells. / Das, Subash C.; Panda, Debasis; Nayak, Debasis; Pattnaik, Asit K.

In: Journal of virology, Vol. 83, No. 6, 01.03.2009, p. 2611-2622.

Research output: Contribution to journalArticle

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