The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons

Abstract

The alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used an in vitro pulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1.

Importance: Herpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.

FIG 1

Key domains of pUS9 are conserved across the Alphaherpesvirinae subfamily. pUS9 is a type II transmembrane protein consisting of an acidic domain and a basic domain within the cytoplasmic tail, in addition to a transmembrane (TM) domain and a short C-terminal extracellular domain (ED). The encoded pUS9 sequences were obtained from the following herpesviral genome sequence accession numbers: HSV-1 strain 17, X14112; HSV-1 strain F, GU734771; HSV-2, Z86099; simian agent 8 (SA8), AY714813; herpesvirus B (HVB), AF533768; BHV-1, AJ004801; BHV-5, AY261359; PrV, BK001744; EHV-1, AY665713; equine herpesvirus 4 (EHV-4), AF030027; and VZV, X04370. Clustal W alignments were generated using Lasergene (version 11.1) MegAlign from DNAStar. The kinesin-1 binding domain (residues 56 to 60 are underlined for HSV-1) determined in this study maps within the basic domain of pUS9. The consensus residue is shown above the alignments when at least 5 residues of one type only match between aligned sequences; otherwise, residues are indicated by dots or dashes. Consensus strength is shown above the alignments as vertical bars (red, 11 residues match; orange, 9 residues match; green, 7 residues match; light blue, 5 residues match; dark blue, <5 residues match).

FIG 2

Basic domain of HSV-1 pUS9 contains a kinesin-1 binding domain. (A) Summary of C-terminal truncations of the cytoplasmic tail (corresponds to residues 1 to 64) of HSV-1 pUS9 and their ability to bind kinesin-1. The kinesin-1 binding domain maps within the basic domain of pUS9. (B) In vitro pulldown assay showing His₆-tagged kinesin-1 C-terminal domain and not His₆-tagged dynein light-chain roadblock (DYNLRB) binds preferentially to GST-tagged pUS9 1-64. (Upper) Immunoblots (anti-His₆ tag) to detect His₆-tagged proteins eluted from boiled GST Bind beads. (Lower) Total protein stains with Coomassie blue to confirm the presence of GST-tagged proteins eluted from boiled GST Bind beads.

FIG 3

Further definition of the kinesin-1 binding domain within the basic domain of HSV-1 pUS9. (A) Summary of C-terminal truncations of the cytoplasmic tail (corresponds to residues 1 to 64) of HSV-1 pUS9 and their ability to bind kinesin-1. The kinesin-1 binding domain maps to pUS9 residues 56 to 60 within the basic domain. (B) In vitro pulldown assay showing His₆-tagged kinesin-1 C-terminal domain and not His₆-tagged dynein light-chain roadblock (DYNLRB) binds preferentially to GST-tagged pUS9 1-64 and 1-60. (Upper) Immunoblots (anti-His₆ tag) to detect His₆-tagged proteins eluted from boiled GST Bind beads. (Lower) Total protein stains with Coomassie blue to confirm the presence of GST-tagged proteins eluted from boiled GST Bind beads.

FIG 4

Confirmation that the pUS9 kinesin binding domain (KBD) is essential for binding kinesin-1. (A and B) In vitro pulldown assay was undertaken with a GST-tagged HSV-1 pUS9 cytoplasmic tail (residues 1 to 64) containing a substitution mutation. This mutation, KBDM, consisted of pUS9 residues 57 to 61 with the sequence RRRRR mutated to NNNNN. Binding of either the His₆-tagged kinesin-1 C-terminal domain (A) or native kinesin-1 (derived from rat brain synaptosomes) (B) was abolished for the GST-tagged pUS9 KBDM but not the GST-tagged pUS9 WT. This also illustrates that HSV-1 pUS9 binds native kinesin-1. The upper images in each case are immunoblots (anti-His₆ tag [A] or anti-kinesin-1 [B]) to detect either His₆-tagged proteins or kinesin-1 eluted from boiled GST Bind beads. The lower images in panels A and B are total protein stains with Coomassie blue to confirm the presence of GST-tagged proteins eluted from Bind GST Bind beads.

FIG 5

Characterization of HSV-1pUS9KBDM and HSV-1pUS9KBDR (rescuant), generated by BAC recombination. Vero cell lysates harvested at 24 h p.i. and immunoblotted with the indicated antibodies are shown. Relative to those of HSV-1Fparental (F strain), HSV-1pUS9KBDM and HSV-1pUS9KBDR showed similar expression of major viral structural proteins. Only in the case of HSV-1pUS9KBDM was there enhanced expression of pUS9.

FIG 6

HSV zosteriform disease in the mouse flank infection model as a means to assess viral neuronal spread. Anesthetized, 6-week-old female adult C57BL/6 mice were scarified on the flank within a 2-mm² area using an 18-gauge needle and then topically infected with 1 × 10⁶ PFU/mouse of HSV-1pUS9KBDR or HSV-1pUS9KBDM. Mice were scored for HSV disease at the primary skin site (measure of primary infection) and the secondary skin site (measure of ganglionic neuronal spread) per the lesion scoring scheme given in Materials and Methods. (A) Mean skin lesion scores (± SEM) at selected times (n = 5 mice/time point) from days 1 to 28 p.i. for HSV-1pUS9KBDM and HSV-1pUS9KBDR. Similar primary skin lesion scores were observed. A significant decrease in secondary skin lesion score (*, P < 0.05 by Wilcoxon signed-rank test), which peaked at day 8 p.i., was observed for HSV-1pUS9KBDM compared to that of HSV-1pUS9KBDR. In each case HSV-1pUS9KBDR behaved the same as parental strain F (not shown). (B) Illustration of the progression of zosteriform disease at both primary and secondary skin sites in the mouse flank model using HSV-1pUS9KBDM and HSV-1pUS9KBDR. Similar disease progression was observed at the primary (P) site for both viruses with a peak decrease at the secondary (S) site at day 8 p.i. for HSV-1pUS9KBDM. (C) qPCR analysis of total DNA extracted from mouse DRG tissue (n = 5 mice/virus) at day 4 p.i. Levels of total viral genome copy number present in DRG tissue from infected mice were not significantly different between HSV-1pUS9KBDM and HSV-1pUS9KBDR. This confirms that there were no defects in the retrograde transport of HSV-1pUS9KBDM. The standard curve was based on a plasmid containing the HSV-1 UL35 gene. Samples were normalized to mGAPDH.

FIG 7

Analysis of anterograde axonal transport of HSV-1 capsids. Neonatal rat DRG neurons were dissociated, pelleted through a 35% Percoll gradient, and plated into the somal compartment of three-chamber microfluidic devices. Cultures were incubated for 5 to 6 days to allow axons to grow into the axonal compartment via the middle grooves. Vero cells were added to the axonal compartment 24 h prior to the addition of either HSV-1pUS9KBDM or HSV-1pUS9KBDR (5 PFU/cell) to the somal compartment. Foscarnet (100 μg/ml) was added to the axonal compartment 8 h p.i. to prevent secondary virus spread in Vero cells. The cultures were fixed at 22 h p.i., immunostained for C capsids (PTNC), and examined using a Leica SP5 confocal microscope. (A) Representative confocal micrographs of axons (indicated by arrows) emerging from the middle grooves (visible on the left side of the lower images) into the axonal compartment. These axons were counted from 10 randomly selected fields of view and scored as positive or negative for the presence of HSV-1 capsid (red in the upper images). Scale bars, 25 μm. (B) The percentage of capsid-positive (+ve) axons versus capsid-negative (−ve) axons in either case is shown for three separate experiments. A significant reduction in capsid transport along axons was observed for HSV-1pUS9KBDM compared to that of HSV-1pUS9KBDR.

FIG 8

HSV-1 spread from axons to nonneuronal Vero cells. Neonatal rat DRG neurons were dissociated, pelleted through a 35% Percoll gradient, and plated into the somal compartment of three-chamber microfluidic devices. Cultures were incubated for 5 to 6 days to allow axons to grow in the axonal compartment. Vero cells were added to the axonal compartment 24 h prior to addition of either HSV-1pUS9KBDM or HSV-1pUS9KBDR (5 PFU/cell) to the somal compartment. Foscarnet (100 μg/ml) was added to the axonal compartment 8 h p.i. to prevent secondary virus spread in Vero cells. The cultures were fixed at 22 h p.i., immunostained for HSV-1 immediate-early protein ICP27, and examined using a Leica SP5 confocal microscope. (A) Representative confocal micrographs of Vero cells in the axonal compartment. (Lower) The presence of axons innervating the Vero cells was confirmed for each virus. (Upper) Vero cells were counted from 10 randomly selected fields of view and scored as positive or negative for the presence of HSV-1 ICP27 (blue). Scale bars, 25 μm. (B) The total number of ICP27-positive Vero cells in the axonal compartment was counted for each virus in three separate experiments (Exp). A significant reduction in virus spread from axons to Vero cells was observed for HSV-1pUS9KBDM compared to that of HSV-1pUS9KBDR.