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.
Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE-US9-double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE-US9-mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific.IMPORTANCEHerpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde transport) or from cell bodies to axon tips (anterograde transport). This transit in axons is essential for alphaherpesviruses to establish latency in ganglia and then to reactivate and move back to peripheral tissues for spread to other hosts. Anterograde transport of HSV requires two membrane proteins: gE/gI and US9. Our studies reveal new mechanisms for how gE/gI and US9 initiate anterograde axonal transport. HSV mutants lacking both gE and US9 fail to properly assemble enveloped virus particles in the cytoplasm, which blocks anterograde transport of enveloped particles. In addition, there are defects in the sorting of virus particles such that particles, when formed, do not enter proximal axons.
Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, Oregon, USA.
Grayson
G
DuRaine
Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, Oregon, USA.
Todd W
TW
Wisner
Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, Oregon, USA.
Paul
P
Howard
Multiscale Microscopy Core, Oregon Health & Science University, Portland, Oregon, USA.
Melissa
M
Williams
Department of Molecular Microbiology & Immunology, Oregon Health & Science University, Portland, Oregon, USA johnsoda@ohsu.edu.
David C
DC
Johnson
D000818:Animals; D001370:Axonal Transport; D001369:Axons / Q000378:metabolism* / Q000821:virology; D002213:Capsid / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000821:virology*; D047908:Intracellular Signaling Peptides and Proteins; D016547:Kinesins / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009474:Neurons / Q000502:physiology / Q000821:virology*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D008074:Lipoproteins; D010750:Phosphoproteins; C109622:US9 protein, Human herpesvirus 1; D014759:Viral Envelope Proteins; D014764:Viral Proteins; D016547:Kinesins
anterograde; axons; envelopment; glycoproteins; kinesins; neuron