Inner tegument proteins of Herpes Simplex Virus are sufficient for intracellular capsid motility in neurons but not for axonal targeting.

Upon reactivation from latency and during lytic infections in neurons, alphaherpesviruses assemble cytosolic capsids, capsids associated with enveloping membranes, and transport vesicles harboring fully enveloped capsids. It is debated whether capsid envelopment of herpes simplex virus (HSV) is completed in the soma prior to axonal targeting or later, and whether the mechanisms are the same in neurons derived from embryos or from adult hosts. We used HSV mutants impaired in capsid envelopment to test whether the inner tegument proteins pUL36 or pUL37 necessary for microtubule-mediated capsid transport were sufficient for axonal capsid targeting in neurons derived from the dorsal root ganglia of adult mice. Such neurons were infected with HSV1-ΔUL20 whose capsids recruited pUL36 and pUL37, with HSV1-ΔUL37 whose capsids associate only with pUL36, or with HSV1-ΔUL36 that assembles capsids lacking both proteins. While capsids of HSV1-ΔUL20 were actively transported along microtubules in epithelial cells and in the somata of neurons, those of HSV1-ΔUL36 and -ΔUL37 could only diffuse in the cytoplasm. Employing a novel image analysis algorithm to quantify capsid targeting to axons, we show that only a few capsids of HSV1-ΔUL20 entered axons, while vesicles transporting gD utilized axonal transport efficiently and independently of pUL36, pUL37, or pUL20. Our data indicate that capsid motility in the somata of neurons mediated by pUL36 and pUL37 does not suffice for targeting capsids to axons, and suggest that capsid envelopment needs to be completed in the soma prior to targeting of herpes simplex virus to the axons, and to spreading from neurons to neighboring cells

HSV1 outer tegument and envelope proteins are targeted to the axon independent of capsids.

<p>DRG neurons were infected after 3 div with 1 x 10⁷ pfu/mL of HSV1(17⁺)Lox-CheVP26 (A), -ΔUL36 (B), -ΔUL37 (C), or -ΔUL20 (D), fixed and permeabilized using the PHEMO protocol at 24 hpi, labeled with antibodies directed against VP13/14 (R220, ii), gB (R69, vi) or β-III-tubulin (mAb 5564, vii) and the axons were analyzed by confocal microscopy. CheVP26 (i and v), merge (iii and viii), differential interference contrast image (DIC, iv and ix). Scale bar is 5 μm.</p

HSV1(17⁺)Lox strains used in this study.

<p>HSV1(17⁺)Lox strains used in this study.</p

HSV1-pUL36, pUL37 and pUL20 are required for efficient targeting of capsids to axons.

<p>DRG neurons were infected after 3 div with 1 x 10⁷ pfu/mL of HSV1(17⁺)Lox (A and E), -ΔUL36 (B and F), -ΔUL37 (C and G), or -ΔUL20 (D and H), fixed and permeabilized using the PHEMO protocol at 26 hpi, labeled with antibodies directed against VP26 (pAb VP26_aa95-112) and VP22 (mAb 22–3, A-D) or gD (mAb DL6, E-H), and analyzed by confocal fluorescence microscopy. (i and iv) anti-VP26, (ii and v) anti-VP22 in the upper panel and anti-gD in the lower panel, (iii and vi) anti-VP26 in green and anti-VP22 in the upper panel and anti-gD in the lower panel in red. (i-iii) Cell bodies with nucleus and cytoplasm; (iv-vi) proximal axons. Scale bar, 5 μm.</p

HSV1-pUL36, pUL37 and pUL20 are required for efficient targeting of capsids to axons.

<p>DRG neurons were infected after 3 div with 1 x 10⁷ pfu/mL of HSV1(17⁺)Lox, Lox-ΔUL36, Lox-ΔUL37, or Lox-ΔUL20 (or mock treated, fixed and permeabilized using the PHEMO protocol at 24 to26 hpi, labeled with antibodies directed against VP26 (pAb VP26_aa95-112) and gD (mAb DL6, A-D) or VP22 (mAb 22–3, E-H), and analyzed by confocal fluorescence microscopy. The images were quantified with a semi-automated algorithm. Each bar represents the mean from one experiment, normalized to the parental values. (A-D) Labeling with anti-VP26 and anti-gD: (A) Total number of VP26 positive structures. (B) VP26 and gD double positive structures. (C) total gD positive structures (D) Size of gD-positive structures. (E-H) Labeling with anti-VP26 and anti-VP22: (E) Total VP26 positive structures. (F) Total number of structures labeled for VP26 and VP22. (G) Total number of structures labeled for gD. (D) Area of structures labelled for gD. The number of capsids, gD or VP22 structures, image numbers and total axon length are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006813#ppat.1006813.s013" target="_blank">S1 Table</a>.</p

HSV1(17⁺)Lox-ΔUL20 is impaired in secondary envelopment.

<p>HSV1(17⁺)Lox-ΔUL20 is impaired in secondary envelopment.</p

HSV1-pUL20 is not required for directed intracellular transport in epithelial cells.

<p>Vero cells were infected with 6.4 x 10⁶ pfu/ml (10 pfu/cell) of HSV1(17⁺)Lox-mCheVP26 (Ai), -ΔUL36 (Aii), -ΔUL37 (Aiii), or -ΔUL20 (Aiv) and movies were acquired at 8–10 hpi. (A) Stills of movies in Vero cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006813#ppat.1006813.s005" target="_blank">S1</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006813#ppat.1006813.s008" target="_blank">S4</a> Movies) and with representative track profiles (indicated by a colored line and the front of each track marked by an open circle). Scale bar, 5 μm. (B) Analysis of track profiles of HSV1(17⁺)Lox-mCheVP26 (parental), -ΔUL36, -ΔUL37, or –ΔUL20. (Bi) Track length and (Bii) maximum step velocity of tracks with a MSD_ex ≥ 1.2, (Biii) mean square displacement exponent (MSD_ex), with each dot representing one track. Box and whiskers with min and max.</p

Cytosolic capsids accumulate in the absence of pUL36 or pUL37 and pUL20 is required for secondary envelopment in neurons.

<p>DRG neurons infected with 1 x 10⁷ pfu/mL of HSV1(17⁺)Lox-CheVP26 (A), -ΔUL36 (B), -ΔUL37 (C), -ΔUL20 (D), fixed at 24 or 30 hpi and processed for conventional electron microscopy. Nucleus (N), primary enveloped virions (white stars), cytosolic capsids (white arrowheads), wrapping intermediates with capsids being closely associated with cytoplasmic membranes (black arrowheads), virions after complete secondary envelopment (black star), and extracellular virions (arrow). Scale bar is 500 nm.</p

In the absence of pUL20, HSV1 capsids associate with pUL36 and pUL37.

<p>(A) Vero cells were infected with 1.1 x 10⁷ pfu/mL (10 pfu/cell) of HSV1(17⁺)Lox-ΔUL36 (i), -ΔUL37 (ii) or -ΔUL20 (iii and iv). At 16 hpi, the cells were fixed and processed for immunoelectron microscopy with antibodies against pUL36 (ii and iii) or pUL37 (i and iv). (B) Immunogold labeling of cytoplasmic capsids was quantified after labeling with anti-pUL36 (Bi) or anti-pUL37 (Bii). The mean values and standard errors of the mean (SEM) were calculated. P-values were calculated with the Kruskal-Wallis test and Dunn’s multiple comparison test.</p

HSV1-pUL20 is required for secondary envelopment in epithelial cells.

<p>Vero cells infected with 1.9 x 10⁶ pfu/mL (10 pfu/cell) of HSV1(17⁺)Lox (A), -ΔUL20 (Bi and Bii), or -CheVP26-ΔUL20 (Biii), fixed at 14 hpi, and processed for conventional electron microscopy. Cytosolic capsids (white arrowheads), wrapping intermediates with capsids being closely associated with cytoplasmic membranes (black arrowheads), virions after complete secondary envelopment (asterisk), and extracellular virions (arrow). Scale bars are 200 nm.</p