A beta-herpesvirus with fluorescent capsids to study transport in living cells.
Fluorescent tagging of viral particles by genetic means enables the study of virus dynamics in living cells. However, the study of beta-herpesvirus entry and morphogenesis by this method is currently limited. This is due to the lack of replication competent, capsid-tagged fluorescent viruses. Here, we report on viable recombinant MCMVs carrying ectopic insertions of the small capsid protein (SCP) fused to fluorescent proteins (FPs). The FPs were inserted into an internal position which allowed the production of viable, fluorescently labeled cytomegaloviruses, which replicated with wild type kinetics in cell culture. Fluorescent particles were readily detectable by several methods. Moreover, in a spread assay, labeled capsids accumulated around the nucleus of the newly infected cells without any detectable viral gene expression suggesting normal entry and particle trafficking. These recombinants were used to record particle dynamics by live-cell microscopy during MCMV egress with high spatial as well as temporal resolution. From the resulting tracks we obtained not only mean track velocities but also their mean square displacements and diffusion coefficients. With this key information, we were able to describe particle behavior at high detail and discriminate between particle tracks exhibiting directed movement and tracks in which particles exhibited free or anomalous diffusion.
Max von Pettenkofer-Institute, Ludwig Maximilians University, Munich, Germany.
Jens B
JB
Bosse
Rudolf
R
Bauerfeind
Leonhard
L
Popilka
Lisa
L
Marcinowski
Martina
M
Taeglich
Christophe
C
Jung
Hannah
H
Striebinger
Jens
J
von Einem
Ulrike
U
Gaul
Paul
P
Walther
Ulrich H
UH
Koszinowski
Zsolt
Z
Ruzsics
D000595:Amino Acid Sequence; D000818:Animals; D018141:Betaherpesvirinae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport / Q000187:drug effects; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D023061:Gene Order; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D018146:Muromegalovirus / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D050257:Tubulin Modulators / Q000494:pharmacology; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure
D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D050257:Tubulin Modulators; D049452:Green Fluorescent Proteins; D015739:Nocodazole