Blocking ESCRT-mediated envelopment inhibits microtubule-dependent trafficking of alphaherpesviruses in vitro

Himanshu Kharkwal, Caitlin G. Smith, Duncan W. Wilson

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Herpes simplex virus (HSV) and, as reported here, pseudorabies virus (PRV) utilize the ESCRT apparatus to drive cytoplasmic envelopment of their capsids. Here, we demonstrate that blocking ESCRT-mediated envelopment using the dominant-negative inhibitor Vps4A-EQ (Vps4A in which glutamate [E] at position 228 in the ATPase active site is replaced by a glutamine [Q]) reduced the ability of HSV and PRV particles to subsequently traffic along microtubules in vitro. HSV and PRV capsid-associated particles with bound green fluorescent protein (GFP)-labeled Vps4A-EQ were readily detected by fluorescence microscopy in cytoplasmic extracts of infected cells. These Vps4A-EQ-associated capsid-containing particles bound to microtubules in vitro but were unable to traffic along them. Using a PRV strain expressing a fluorescent capsid and a fluorescently tagged form of the envelope protein gD, we found that similar numbers of gD-positive and gD-negative capsid-associated particles accumulated in cytoplasmic extracts under our conditions. Both classes of PRV particle bound to microtubules in vitro with comparable efficiency, and similar results were obtained for HSV using anti-gD immunostaining. The gD-positive and gD-negative PRV capsids were both capable of trafficking along microtubules in vitro; however, motile gD-positive particles were less numerous and their trafficking was more sensitive to the inhibitory effects of Vps4A-EQ. We discuss our data in the context of microtubule-mediated trafficking of naked and enveloped alphaherpesvirus capsids.

Original languageEnglish (US)
Pages (from-to)14467-14478
Number of pages12
JournalJournal of Virology
Volume88
Issue number24
DOIs
StatePublished - 2014

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capsid
Suid Herpesvirus 1
Capsid
Suid herpesvirus 1
Microtubules
microtubules
herpes simplex
Simplexvirus
viruses
virion
traffic
Virion
extracts
Green Fluorescent Proteins
In Vitro Techniques
Cell Extracts
fluorescence microscopy
Glutamine
green fluorescent protein
Fluorescence Microscopy

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Blocking ESCRT-mediated envelopment inhibits microtubule-dependent trafficking of alphaherpesviruses in vitro. / Kharkwal, Himanshu; Smith, Caitlin G.; Wilson, Duncan W.

In: Journal of Virology, Vol. 88, No. 24, 2014, p. 14467-14478.

Research output: Contribution to journalArticle

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