Blocking ESCRT-Mediated Envelopment Inhibits Microtubule-Dependent Trafficking of Alphaherpesviruses In Vitro

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. IMPORTANCEThe alphaherpesviruses include several important human pathogens. These viruses utilize microtubule-mediated transport to travel through the cell cytoplasm; however, the molecular mechanisms of trafficking are not well understood. In this study, we have used a cell-free system to examine the requirements for microtubule trafficking and have attempted to distinguish between the movement of so-called "naked" and membrane-associated cytoplasmic alphaherpesvirus capsids. Members of the Alphaherpesvirinae subfamily include herpes simplex virus type 1 (HSV-1) and HSV-2, varicella-zoster virus, and pseudorabies virus (PRV). Like all herpesviruses, members of this subfamily replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is generally accepted that capsids then bud into the inner nuclear membrane to generate perinuclear virions that subsequently fuse with the outer nuclear membrane to release mature nucleocapsids (also termed "naked" capsids) into the cytoplasm. Naked cytoplasmic herpes capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (1-4).By performing subcellular fractionation during a single synchronized wave of HSV egress, we showed that HSV capsids bypass the cis, medial, and trans compartments of the Golgi apparatus (5) and accumulate in a buoyant membrane fraction with the biochemical and antigenic properties of the trans-Golgi network (TGN) and endosomes (5, 6)...

Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

“…3). The former resemble in size and appearance the assembly intermediates that we have described earlier, a subset of which are motile along microtubules in vitro (16). The latter, which are more abundant in Vero (Fig.…”

“…This fraction contains the organelles to which HSV capsids traffic from the nucleus during a single synchronized wave of egress and is the first compartment at which fully enveloped infectious virions accumulate in the cytoplasm (60). HSV capsids enveloping in this gradient fraction also acquire molecular motors and become capable of traffic along microtubules in vitro (16,42,63). Biochemical analyses and electron microscopy revealed that this region of the gradient contains fully enveloped HSV particles within organellar lumens, and nonenveloped capsids docked to the cytoplasmic faces of the organelles (60).…”

“…A PNS was prepared from infected cells, attached to polylysine-coated coverslips, and then incubated with DiD or DilC18(5)-DS or mock treated. Samples were then fixed and imaged in the green (VP26-GFP capsid subunit) or Cy5 (infraredemitting lipophilic dye) channels as we described earlier (16). Figure 3 shows representative fields of DilC18(5)-DS-stained PNS particles from HSV1-GS2491-infected Vero (Fig.…”

“…All imaging was performed in the Analytical Imaging Facility of the Albert Einstein College of Medicine. Immunofluorescence studies were performed as previously described (16,63). Primary antibodies were as follows: mouse monoclonal anti-Rab4 and anti-Rab5 (Transduction Laboratories), rabbit polyclonal anti-Rab7 (Santa Cruz Biotechnology), and affinity-purified sheep anti-TGN46 antiserum (Serotec).…”

“…These naked cytoplasmic capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (4)(5)(6)(7)(8)(9). Completion of cytoplasmic envelopment requires the coordinated interaction of multiple virally encoded proteins (8,(10)(11)(12)(13) and the participation of components of the cellular endosomal sorting complexes required for transport (ESCRT) machinery (14)(15)(16)(17)(18)(19).…”