The HSV-1 pUL37 protein promotes cell invasion by regulating the kinesin-1 motor

Abstract

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), recruit microtubule motor proteins to invade cells. The incoming viral particle traffics to nuclei in a two-step process. First, the particle uses the dynein-dynactin motor to sustain transport to the centrosome. In neurons, this step is responsible for long-distance retrograde axonal transport and is an important component of the neuroinvasive property shared by these viruses. Second, a kinesin-dependent mechanism redirects the particle from the centrosome to the nucleus. We have reported that the kinesin motor used during the second step of invasion is assimilated into nascent virions during the previous round of infection. Here, we report that the HSV-1 pUL37 tegument protein suppresses the assimilated kinesin-1 motor during retrograde axonal transport. Region 2 (R2) of pUL37 was required for suppression and functioned independently of the autoinhibitory mechanism native to kinesin-1. Furthermore, the motor domain and proximal coiled coil of kinesin-1 were sufficient for HSV-1 assimilation, pUL37 suppression, and nuclear trafficking. pUL37 localized to the centrosome, the site of assimilated kinesin-1 activation during infection, when expressed in cells in the absence of other viral proteins; however, pUL37 did not suppress kinesin-1 in this context. These results indicate that the pUL37 tegument protein spatially and temporally regulates kinesin-1 via the amino-terminal motor region in the context of the incoming viral particle.

Keywords: HSV-1; axonal transport; centrosome; herpes simplex virus; kinesin.

Fig. 1.

Overview of virus production in the presence or absence of kinesin-1 (KIF5B) and three KIF5B mutant isoforms. (A) Schematic of kinesin-1 (KIF5) heavy chain highlighting three regions contributing to motor autoinhibition. (B) Western blot of hTERT-RPE cells expressing endogenous KIF5B (WT), knocked out for KIF5B (KO), and KO expressing a double (H, I), triple (C, H, I), or truncated (561) KIF5B mutant. (C) Diagram of HSV-1 production. Producer hTERT-RPE cells are infected with HSV-1 that is either wild type (WT) or R2 mutant (R2*). The harvested virus either contains or lacks a kinesin-1 motor based on the producer cell type. These virus stocks are used to infect normal sensory neurons or epithelial cells for further study.

Fig. 2.

The HSV-1 pUL37 R2 effector promotes capsid retrograde axonal transport by suppressing assimilated kinesin-1. (A) Example retrograde axonal transport kymographs of WT HSV-1 produced from WT RPE cells (Left) and R2* HSV-1 produced from WT RPE cells (Right). Distance (d) and time (t) axis are indicated in the Right panel. (B) Frequency of capsid motion in axons: % time retrograde (light), % time anterograde (medium), and % time stopped (dark). (C) Average number of interruptions (reversals and stops) per retrograde moving capsid during a 20-s period. (D) Net displacement of capsids after 20 s. Positive values indicate movement toward neuronal soma (retrograde displacement). Error bars are SD; ****P < 0.0001.

Fig. 3.

Inhibiting dynein does not promote HSV-1 anterograde axonal transport. (A) Experiment timeline. (B) Frequency of capsid motion in axons: % time retrograde (light), % time anterograde (medium), and % time stopped (dark). No significant differences in the anterograde component of motion were observed. (C) Net displacement of capsids after 10 s. Positive values indicate movement toward neuronal soma (retrograde displacement). Error bars are SD; ** P<0.01.

Fig. 4.

pUL37 intrinsically associates with the centrosome but is insufficient for kinesin-1 suppression. (A) hTERT-RPE cells were stained with anti-GFP, anti-pericentrin (centrosome), and DAPI (nucleus) to document protein localization in cells transduced with the indicated GFP constructs. Arrows indicate centrosome locations based on pericentrin fluorescence. Values overlaid on the merged images indicate the percentage of cells with enriched GFP emissions at the centrosome. (B) Quantitation of centrosomal GFP intensities (Top) and corresponding pericentrin fluorescence intensities (Bottom). Data were extracted from samples used to produce representative images in panel (A). Error bars are SD; ***P < 0.001. (C) hTERT-RPE cells were stained with anti-GFP, anti-vimentin, and DAPI (nucleus) to document kinesin-1 activity in cells. (Scale bar, 10 µm.)

Fig. 5.

HSV-1 R2 mutant capsids lacking packaged kinesin accumulate at the centrosome in neurons and epithelial cells. (A) Live imaging of dorsal root ganglion sensory neurons infected with either WT or R2* HSV-1 encoding pUL25/mCherry (capsid) and counterstained with Hoechst (nucleus). Images were captured 4 hpi. (Scale bar, 10 µm.) (B) Immunofluorescence microscopy of hTERT-RPE cells fixed 4 hpi following infection with either WT or R2* HSV-1 and stained with anti-VP5 (capsid), DAPI (nucleus), and anti-pericentrin (centrosome). (Scale bar, 10 µm.) Magnified regions centered on centrosomes are shown in the corresponding panels below. (C) Automated analysis of capsid distribution in fixed hTERT-RPE cells. Capsids were scored as either at the nuclear rim (dark), centrosome (medium), or cytoplasmic away from the nucleus and centrosome (light). ***P < 0.0001.

Fig. 6.

Model of HSV-1 retrograde transport. (Top panel) WT HSV-1 capsid with assimilated KIF5. pUL37 R2 suppresses the KIF5 activity during retrograde transport. (Second panel) Kinesin-less WT HSV-1 capsid. Removing KIF5 allows capsid to transport retrogradely. (Third panel) R2* HSV-1 capsid with assimilated KIF5. pUL37 R2* fails to suppress the KIF5 activity, which prohibits continuous retrograde movement. (Fourth panel) Kinesin-less R2* HSV-1 capsid. R2 effector function is dispensable in the absence of KIF5 during the retrograde transport step.