Neurotropic alphaherpesviruses initiate infection in exposed mucosal tissues and, unlike most viruses, spread rapidly to sensory and autonomic nerves where life-long latency is established1. Recurrent infections arise sporadically from the peripheral nervous system throughout the life of the host, and invasion of the central nervous system may occur, with severe outcomes2. These viruses directly recruit cellular motors for transport along microtubules in nerve axons, but how the motors are manipulated to deliver the virus to neuronal nuclei is not understood. Here, using herpes simplex virus type I and pseudorabies virus as model alphaherpesviruses, we show that a cellular kinesin motor is captured by virions in epithelial cells, carried between cells, and subsequently used in neurons to traffic to nuclei. Viruses assembled in the absence of kinesin are not neuroinvasive. The findings explain a critical component of the alphaherpesvirus neuroinvasive mechanism and demonstrate that these viruses assimilate a cellular protein as an essential proviral structural component. This principle of viral assimilation may prove relevant to other virus families and offers new strategies to combat infection.
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Unfiltered mass spectrometry data, raw microscopy images and plasmid and virus construction details (including primer sequences) are available from the corresponding author upon request.
Smith, G. Herpesvirus transport to the nervous system and back again. Annu. Rev. Microbiol. 66, 153–176 (2012).
Lafaille, F. G. et al. Deciphering human cell-autonomous anti-HSV-1 immunity in the central nervous system. Front. Immunol. 6, 208 (2015).
Zaichick, S. V. et al. The herpesvirus VP1/2 protein is an effector of dynein-mediated capsid transport and neuroinvasion. Cell Host Microbe 13, 193–203 (2013).
Smith, G. A., Pomeranz, L., Gross, S. P. & Enquist, L. W. Local modulation of plus-end transport targets herpesvirus entry and egress in sensory axons. Proc. Natl Acad. Sci. USA 101, 16034–16039 (2004).
Antinone, S. E. & Smith, G. A. Retrograde axon transport of herpes simplex virus and pseudorabies virus: a live-cell comparative analysis. J. Virol. 84, 1504–1512 (2010).
Pernigo, S., Lamprecht, A., Steiner, R. A. & Dodding, M. P. Structural basis for kinesin-1:cargo recognition. Science 340, 356–359 (2013).
Robert, A. et al. Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport. FASEB J. 33, 388–399 (2019).
Bish, S. E., Song, W. & Stein, D. C. Quantification of bacterial internalization by host cells using a β-lactamase reporter strain: Neisseria gonorrhoeae invasion into cervical epithelial cells requires bacterial viability. Microbes Infect. 10, 1182–1191 (2008).
Heine, J. W., Honess, R. W., Cassai, E. & Roizman, B. Proteins specified by herpes simplex virus. XII. The virion polypeptides of type 1 strains. J Virol 14, 640–651 (1974).
Cavrois, M., De Noronha, C. & Greene, W. C. A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes. Nat. Biotechnol. 20, 1151–1154 (2002).
Lyman, M. G., Feierbach, B., Curanovic, D., Bisher, M. & Enquist, L. W. PRV Us9 directs axonal sorting of viral capsids. J. Virol. 81, 11363–11371 (2007).
Scherer, J. et al. A Kinesin-3 recruitment complex facilitates axonal sorting of enveloped alpha herpesvirus capsids. PLoS Pathog. 16, e1007985 (2020).
Diwaker, D., Murray, J. W., Barnes, J., Wolkoff, A. W. & Wilson, D. W. Deletion of the pseudorabies virus gE/gI–US9p complex disrupts kinesin KIF1A and KIF5C recruitment during egress, and alters the properties of microtubule-dependent transport in vitro. PLoS Pathog. 16, e1008597 (2020).
DuRaine, G., Wisner, T. W., Howard, P., Williams, M. & Johnson, D. C. Herpes simplex virus gE/gI and US9 promote both envelopment and sorting of virus particles in the cytoplasm of neurons, two processes that precede anterograde transport in axons. J. Virol. 91, e00050-17 (2017).
Engelke, M. F. et al. Engineered kinesin motor proteins amenable to small-molecule inhibition. Nat. Commun. 7, 11159 (2016).
Schipke, J. et al. The C terminus of the large tegument protein pUL36 contains multiple capsid binding sites that function differently during assembly and cell entry of herpes simplex virus. J. Virol. 86, 3682–3700 (2012).
Dohner, K. et al. Function of dynein and dynactin in herpes simplex virus capsid transport. Mol. Biol. Cell. 13, 2795–2809 (2002).
DuRaine, G., Wisner, T. W., Howard, P. & Johnson, D. C. Kinesin-1 proteins KIF5A, 5B and 5C promote anterograde transport of herpes simplex virus enveloped virions in axons. J. Virol. 92, e01269-18 (2018).
Radtke, K. et al. Plus- and minus-end directed microtubule motors bind simultaneously to herpes simplex virus capsids using different inner tegument structures. PLoS Pathog. 6, e1000991 (2010).
Diefenbach, R. J. et al. The basic domain of herpes simplex virus 1 pUS9 recruits kinesin-1 to facilitate egress from neurons. J. Virol. 90, 2102–2111 (2016).
Loret, S., Guay, G. & Lippe, R. Comprehensive characterization of extracellular herpes simplex virus type 1 virions. J. Virol. 82, 8605–8618 (2008).
Kramer, T., Greco, T. M., Enquist, L. W. & Cristea, I. M. Proteomic characterization of pseudorabies virus extracellular virions. J. Virol. 85, 6427–6441 (2011).
Miranda-Saksena, M. et al. Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons. J. Virol. 83, 3187–3199 (2009).
Smith, C. L. Culturing Nerve Cells 2nd Edn (eds Banker, G. & Goslin, K.) 261–287 (MIT Press, 1998).
Smith, G. A., Gross, S. P. & Enquist, L. W. Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons. Proc. Natl Acad. Sci. USA 98, 3466–3470 (2001).
Tanaka, M., Kagawa, H., Yamanashi, Y., Sata, T. & Kawaguchi, Y. Construction of an excisable bacterial artificial chromosome containing a full-length infectious clone of herpes simplex virus type 1: viruses reconstituted from the clone exhibit wild-type properties in vitro and in vivo. J. Virol. 77, 1382–1391 (2003).
Smith, G. A. & Enquist, L. W. Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus. J. Virol. 73, 6405–6414 (1999).
Kaufman, H. E., Ellison, E. D. & Waltman, S. R. Double-stranded RNA, an interferon inducer, in herpes simplex keratitis. Am. J. Ophthalmol. 68, 486–491 (1969).
Bohannon, K. P., Sollars, P. J., Pickard, G. E. & Smith, G. A. Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection. J. Gen. Virol. 93, 124–129 (2012).
Huffmaster, N. J., Sollars, P. J., Richards, A. L., Pickard, G. E. & Smith, G. A. Dynamic ubiquitination drives herpesvirus neuroinvasion. Proc. Natl Acad. Sci. USA 112, 12818–12823 (2015).
Richards, A. L. et al. The pUL37 tegument protein guides alpha-herpesvirus retrograde axonal transport to promote neuroinvasion. PLoS Pathog. 13, e1006741 (2017).
Stults, A. M. & Smith, G. A. The herpes simplex virus 1 deamidase enhances propagation but is dispensable for retrograde axonal transport into the nervous system. J Virol 93, e01172-19 (2019).
Tischer, B. K., Smith, G. A. & Osterrieder, N. En passant mutagenesis: a two step markerless red recombination system. Methods Mol. Biol. 634, 421–430 (2010).
Szpara, M. L. et al. A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses. PLoS Pathog. 7, e1002282 (2011).
Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image processing with ImageJ. Biophotonics Int. 11, 36–42 (2004).
Luxton, G. W. et al. Targeting of herpesvirus capsid transport in axons is coupled to association with specific sets of tegument proteins. Proc. Natl Acad. Sci. USA 102, 5832–5837 (2005).
Leelawong, M., Lee, J. I. & Smith, G. A. Nuclear egress of pseudorabies virus capsids is enhanced by a subspecies of the large tegument protein that is lost upon cytoplasmic maturation. J. Virol. 86, 6303–6314 (2012).
Lee, J. I., Luxton, G. W. & Smith, G. A. Identification of an essential domain in the herpesvirus VP1/2 tegument protein: the carboxy terminus directs incorporation into capsid assemblons. J. Virol. 80, 12086–12094 (2006).
Dodding, M. P., Mitter, R., Humphries, A. C. & Way, M. A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome. EMBO J. 30, 4523–4538 (2011).
Tombacz, D., Toth, J. S., Petrovszki, P. & Boldogkoi, Z. Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay. BMC Genomics 10, 491 (2009).
Lee, G. E., Murray, J. W., Wolkoff, A. W. & Wilson, D. W. Reconstitution of herpes simplex virus microtubule-dependent trafficking in vitro. J. Virol. 80, 4264–4275 (2006).
Kharkwal, H., Smith, C. G. & Wilson, D. W. Blocking ESCRT-mediated envelopment inhibits microtubule-dependent trafficking of alphaherpesviruses in vitro. J. Virol. 88, 14467–14478 (2014).
Howard, J. & Hyman, A. A. Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence microscopy. Methods Cell. Biol. 39, 105–113 (1993).
Shanda, S. K. & Wilson, D. W. UL36p is required for efficient transport of membrane-associated herpes simplex virus type 1 along microtubules. J. Virol. 82, 7388–7394 (2008).
Homa, F. L. et al. Structure of the pseudorabies virus capsid: comparison with herpes simplex virus type 1 and differential binding of essential minor proteins. J. Mol. Biol. 425, 3415–3428 (2013).
Huet, A. et al. Extensive subunit contacts underpin herpesvirus capsid stability and interior-to-exterior allostery. Nat. Struct. Mol. Biol. 23, 531–539 (2016).
Eng, J. K., McCormack, A. L. & Yates, J. R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass. Spectrom. 5, 976–989 (1994).
Xu, T. et al. ProLuCID: An improved SEQUEST-like algorithm with enhanced sensitivity and specificity. J. Proteomics 129, 16–24 (2015).
Cociorva, D., D, L. T. & Yates, J. R. Validation of tandem mass spectrometry database search results using DTASelect. Curr. Protoc. Bioinformatics Ch. 13, Unit 13 14 (2007).
Tabb, D. L., McDonald, W. H. & Yates, J. R., 3rd. DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1, 21–26 (2002).
UniProt, C. UniProt: a hub for protein information. Nucleic Acids Res. 43, D204–D212 (2015).
Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).
We thank V. Gelfand and K. Verhey for suggestions that helped inform the experimental design and for reagents, and A. Roberts, M. Englke, M. Way, S. Brady and G. Gunderson for reagents. D. Kirchenbuechler of the Northwestern University Cell Imaging Facility, supported by NCI CCSG P30 CA060553, provided invaluable support in the development of the automated image analysis pipeline used in this report. Some confocal imaging was performed on an Andor XDI Revolution microscope purchased through the support of NCRR 1S10 RR031680-01 at the Northwestern University Cell Imaging Facility. Sequencing services were performed at the Northwestern University Genomics Core Facility. In vitro microtubule-dependent motility assays and analyses were performed in the Analytical Imaging Facility of the Albert Einstein College of Medicine, with support from National Cancer Institute cancer center grant P30CA013330. This work was funded by NIH AI056346 (G.A.S., P.J.S. and G.E.P.), AI125244 (D.W.W.), AI141470 (D.W.) and AI148780 (J.N.S.). E.B.W. was supported by NIH NS106812. C.E.P. received support from National Science Foundation DGE-1324585 and NIH Cellular and Molecular Basis of Disease Training Grant T32GM08061.
P.J.S., G.E.P. and G.A.S. have disclosed a significant financial interest in Thyreos, Inc. In accordance with their Conflict of Interest policies, the University of Nebraska-Lincoln and Northwestern University Feinberg School of Medicine Conflict of Interest Review Committees have determined that this must be disclosed.
Peer review information Nature thanks the anonymous reviewer(s) for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Fig. 1 Transiently-expressed PRV pUL36 co-localizes with microtubules and accumulates at the cell periphery.
(A) Schematic representation of seven regions of pUL36. Regions 2 and 6 are proline rich (pink). Position of the capsid binding domain (CBD) consisting of amino acids 3033-3095 is indicated. Amino acid positions are indicated above the schematic. (B) Transiently expressed GFP-pUL36∆R7, but not GFP-pUL36 (full length), formed small punctae that moved in curvilinear trajectories (also see Supplementary Video 1) and accumulated at the periphery of Vero cells (arrowheads). The 6.9 µm x 2.4 µm boxed region is expanded as a time-lapse montage to the right of the image (n=3 independent experiments). (C) GFP-pUL36∆R7 decorates microtubules. Vero cells were transiently co-transfected with GFP-pUL36∆R7 and mCherry-tubulin. The 7.4 µm x 7.4 µm boxed region is expanded below each image. All images were captured between 24-28 h post transfection (hpt) (n=3 independent experiments). Scale bars are 20 µm.
Extended Data Fig. 2 Transiently-expressed PRV pUL36 interacts with components of the Kif5 motor complex.
(A) pUL36 region 7 is dispensable for co-immunoprecipitation (co-IP) with Kif5 heavy chain (KHC) and Kif5 light chain (KLC). HEK293 cells were lysed 16-18 hpt and GFP-pUL36 was immunoprecipitated (IP) with anti-GFP antibody and detected by western blot as indicated. Inputs are 6% of crude lysates (n=3 independent experiments) . (B) Peripheral accumulations of transiently expressed GFP-pUL36ΔR7 co-localize with endogenous cellular Kif5 components (arrows). Vero cells were fixed and immunostained for KHC or KLC at 20 hpt (n=3 independent experiments). Scale bars are 20 µm. (C) GFP-pUL36ΔR7 co-immunoprecipitates with KLC1 and KLC2 isoforms. HEK293 cells transiently expressing GFP or GFP-pUL36ΔR7 were co-transfected with HA-KLC1 and HA-KLC2. Endogenous KHC served as a positive co-immunoprecipitation control (n=3 independent experiments). (D) Transient expression mCherry-KLC1 and mCherry-KLC2 in Vero cells. Both fusion proteins show varying degrees of juxtanuclear localization and an absence of peripheral accumulation (n=3 independent experiments). (E) mCherry-KLC1 and mCherry-KLC2 redistribute with GFP-pUL36ΔR7 to the cell periphery during transient expression. Vero cells were imaged 24-28 hpt (n=3 independent experiments). Scale bars are 20 µm.
(A) Illustration of pUL36 constructs with amino- and carboxyl-terminal GFP fusions indicated (grey circles). Location of a cryptic nuclear localization signal (NLS) at the end of region 2 (vertical grey line in R2) that becomes active when region 6 is deleted is also indicated. Constructs are named for each intact region number with a dash representing deleted regions. In the case of region 6, a subregion consisting of the majority of the sequence was deleted (denoted “s” and consisting of amino acids 2087-2796). (B) pUL36 region 5 is required for interaction with the Kif5 complex (KHC, Kif5 heavy chain; KLC, Kif5 light chain; n = 3 independent experiments). (C) pUL36 region 6 is required for interaction with the Kif5 complex. Deletion of region 6 also resulted in nuclear localization of the protein due to the NLS in region 2. Mutation of the NLS (K285RRR > AAAA) restored protein localization to the cytoplasm (not shown) but did not restore Kif5 interaction (n = 3 independent experiments). (D–E) pUL36 regions 5-6, is sufficient for Kif5 binding. Interaction with a dynactin component, p150/glued (p150), was used as a positive co-immunoprecipitation control (n = 3 independent experiments each).
(A) Schematic representation of two potential bipartite kinesin-binding motifs (WD1/WD2 and WD3/WD4) in region 5 of pUL36. Corresponding regions from ten different alphaherpesvirinae are aligned. Prospective WD motifs in PRV are highlighted in red. Conserved tryptophans and/or surrounding E, D, N and Q at positions ±1 and +2 in pUL36 orthologs are in blue. The number of amino acids between the WD3 and WD4 tryptophans are indicated only when no other tryptophan was present in between (W-W); otherwise, n.a. is indicated. Table on the right summarizes mutants used for transient expression (RKB, reduced kinesin binding mutant that was subsequently introduced into PRV – see Extended Data Fig. 5 & 6). (B–D) Co-immunoprecipitations of pUL36 proteins and endogenous cellular Kif5 demonstrate that WD3 and WD4 contribute to the pUL36-Kif5 interaction. Interaction with dynactin component, p150/glued (p150) was used as a positive co-immunoprecipitation control (n = 3 independent experiments each). (E) Quantitation of cells displaying pUL36 localization at the cell periphery or juxtanuclear between 24-28 hpt. Representative images of cells and scoring are provided at top. Percentile distributions of pUL36 accumulation are illustrated by Venn diagrams (average of n = 3 independent experiments >250 cells each). Scale bars are 20 µm.
(A) Mutation of WD3 attenuated PRV spread, as measured by plaque size, whereas mutations of WD4 rendered PRV non-viable (n = 200 plaques over 3 independent experiments). (B) Mutation of the WD4 tryptophan alone also rendered PRV non-viable, whereas mutation of the surrounding acidic residues did not impair viral spread. PRV encoding both the ∆WD3 mutation and WD4(DE>AA) attenuated viral spread but could be propagated and further studied and was designated as the reduced-kinesin binding mutant (RKB; also see Figure S4). Plaque diameters were measured 43 hpt. Data are presented as scatter plots, with each dot representing a single plaque (n = 200 plaques over 3 independent experiments). Mean values ± SD are indicated. (P-values were determined by one-way analysis of variance with a post-hoc Tukey test). (C) Incoming capsids of PRV encoding the pUL36 RKB mutant accumulate juxtanuclear in explanted chick dorsal root ganglia (DRG). Representative images of neural soma (transmitted and fluorescent capsid image pairs with nuclei highlighted by dotted lines). Percentile distributions of juxtanuclear capsid accumulation are illustrated at right (n= 3 independent experiments > 500 infected cells each). DRG explants were imaged between 2.5-3 hpi. Scale bars are 20 µm. Mean values ± SD are indicated. (P values based on two-tailed unpaired t test). (D) PRV[RKB] has delayed viral gene expression. PK15 cells were infected at MOI 10 and total RNA was collected at 4 hpi. Relative IE180 mRNA levels were measured by RT-PCR first-strand DNA synthesis and qPCR amplification using primers specific for PRV IE180 and the swine ribosomal S28 rRNA (loading control). Data was normalized to S28 rRNA levels and depicted as a fold change with respect to IE180 mRNA levels during WT infection (n = independent experiments). Mean values ± SD are indicated. (P values based on two-tailed unpaired t test).
Viral particles were harvested from the cytoplasm of infected RK13 cells and allowed to bind to fluorescent microtubules in vitro. (A) Numbers of bound fluorescent viral particles were counted and are represented as a percentage of those initially added to the imaging chamber. Four independent chambers containing a total of 1204 and 805 bound particles were counted for wild-type PRV (WT) and PRV encoding the reduced kinesin binding mutant pUL36 allele [RKB]. (B) Motility was measured after addition of ATP supplemented with: no inhibitor (light grey bars), 5 μM sodium orthovanadate (a dynein inhibitor; medium grey bars) or 1 mM AMP-PNP (a kinesin inhibitor; dark grey bars). Numbers of motile virions were determined from triplicate motility chambers, and motility plotted as a percentage of the number of motile particles seen in the absence of inhibitor. For WT and RKB respectively, the numbers of individual viral particles examined for each set of conditions were as follows: no inhibitor (658, 706), sodium orthovanadate (776, 714), AMP-PNP (776, 714). Mean values ± SD are indicated (n = 3 independent experiments). (P values based on two-tailed unpaired t test).
(A) Normal human dermal fibroblasts (NHDF) cells were depleted of Kif5 isoforms by siRNA knockdown. Kif5 expression profile in SK-N-SH cells as a standard that expresses all three Kif5 isoforms (n = 1 independent experiment). (B) Control and Kif5B-depleted NHDF cells were infected with HSV-1 in the presence of 100 µM cycloheximide (n = 1 independent experiment). At 8 hpi, cells were fixed and immunofluorescence was performed. Scale bar is 20 µm.
Extended Data Fig. 8 Kif5B and Kif5C function interchangeably to rescue pUL36 peripheral accumulation in RPE∆Kif5B (KO) cells.
Quantitation of transiently-transfected cells displaying GFP-pUL36∆R7 localization at the cell periphery (n = 3 independent experiments > 150 each). Scoring methodology is provided in Extended Data Fig. 4. Mean values ± SD are indicated. (P values based on two-tailed unpaired t test).
Neuroinvasive herpesviruses capture Kif5 (conventional kinesin; kinesin-1) into the tegument of newly formed virions during infection of epithelial cells. The captured epithelial kinesin-1 is carried between cells as a structural virion component and is deposited into cells (epithelia and neurons) upon the subsequent round of infection (first blue panel). Upon entry, cytosolic capsids engage in retrograde axonal transport effected by the cytoplasmic dynein/dynactin microtubule motor. The assimilated kinesin is presumably carried in an inactive state during this step of infection (second blue panel). Dynein/dynactin-based transport directs the capsid ‘minus-ended’ along microtubules ending at the centrosome, where the virus uses assimilated kinesin to transport to nuclei (third blue panel). When viruses are attenuated for assimilated-kinesin binding (e.g., PRV[RKB]) or are produced in the absence of Kif5, capsids predominately accumulate at the centrosome and do not progress toward the nucleus despite the presence of endogenous neuronal kinesin-1 (pink panel). Nevertheless, endogenous kinesin also supports nuclear trafficking of capsids.
This file contains Supplementary Data 1 and 2, legends for Supplementary Data 3 and 4, legends for Supplementary Videos 1 and 2 and Supplementary Fig. 1.
See Supplementary Information for description.
See Supplementary Information for description.
Data derived from automated analysis of capsid localization that support Fig. 1c. The spreadsheet includes all formula for post-analysis of automated data pipeline.
Data derived from automated analysis of capsid localization that support Fig. 2d. This spreadsheet includes all formulae for post-analysis of the automated data pipeline.
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Pegg, C.E., Zaichick, S.V., Bomba-Warczak, E. et al. Herpesviruses assimilate kinesin to produce motorized viral particles. Nature 599, 662–666 (2021). https://doi.org/10.1038/s41586-021-04106-w
Nature Reviews Microbiology (2021)