Fusion of mApple and Venus fluorescent proteins to the Sindbis virus E2 protein leads to different cell-binding properties

Virus Res. 2013 Nov 6;177(2):138-46. doi: 10.1016/j.virusres.2013.07.014. Epub 2013 Jul 31.


Fluorescent proteins (FPs) are widely used in real-time single virus particle studies to visualize, track and quantify the spatial and temporal parameters of viral pathways. However, potential functional differences between the wild type and the FP-tagged virus may specifically affect particular stages in the virus life-cycle. In this work, we genetically modified the E2 spike protein of Sindbis virus (SINV) with two FPs. We inserted mApple, a red FP, or Venus, a yellow FP, at the N-terminus of the E2 protein of SINV to make SINV-Apple and SINV-Venus. Our results indicate that SINV-Apple and SINV-Venus have similar levels of infectivity and are morphologically similar to SINV-wild-type by negative stain transmission electron microscopy. Both mutants are highly fluorescent and have excellent single-particle tracking properties. However, despite these similarities, when measuring cell entry at the single-particle level, we found that SINV-Apple and SINV-Venus are different in their interaction with the cell surface and FPs are not always interchangeable. We went on to determine that the FP changes the net surface charge on the virus particles, the folding of the spike proteins, and the conformation of the spikes on the virus particle surface, ultimately leading to different cell-binding properties between SINV-Apple and SINV-Venus. Our results are consistent with recent findings that FPs may alter the biological and cellular localization properties of bacterial proteins to which they are fused.

Keywords: Alphaviruses; Fluorescent proteins; Surface properties.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural

MeSH terms

  • Alphavirus Infections / virology*
  • Cell Line
  • Humans
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Protein Binding
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sindbis Virus / genetics
  • Sindbis Virus / physiology*
  • Viral Envelope Proteins / genetics
  • Viral Envelope Proteins / metabolism*
  • Virus Internalization


  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • Viral Envelope Proteins