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, 13 (2), 193-203

The Herpesvirus VP1/2 Protein Is an Effector of Dynein-Mediated Capsid Transport and Neuroinvasion

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The Herpesvirus VP1/2 Protein Is an Effector of Dynein-Mediated Capsid Transport and Neuroinvasion

Sofia V Zaichick et al. Cell Host Microbe.

Abstract

Microtubule transport of herpesvirus capsids from the cell periphery to the nucleus is imperative for viral replication and, in the case of many alphaherpesviruses, transmission into the nervous system. Using the neuroinvasive herpesvirus, pseudorabies virus (PRV), we show that the viral protein 1/2 (VP1/2) tegument protein associates with the dynein/dynactin microtubule motor complex and promotes retrograde microtubule transport of PRV capsids. Functional activation of VP1/2 requires binding to the capsid protein pUL25 or removal of the capsid-binding domain. A proline-rich sequence within VP1/2 is required for the efficient interaction with the dynein/dynactin microtubule motor complex as well as for PRV virulence and retrograde axon transport in vivo. Additionally, in the absence of infection, functionally active VP1/2 is sufficient to move large surrogate cargoes via the dynein/dynactin microtubule motor complex. Thus, VP1/2 tethers PRV capsids to dynein/dynactin to enhance microtubule transport, neuroinvasion, and pathogenesis.

Figures

Figure 1
Figure 1. The pUL25 Capsid Protein Unmasks VP1/2 Nuclear Membrane Localization
(A) Schematic representation of seven regions of VP1/2. Regions 2 and 6 are proline rich (black). Position of the capsid binding domain (CBD) consisting of amino acids 3034–3095 is indicated. Amino acid positions based on GenBank JF797219.1 are indicated above the schematic (Szpara et al., 2011). (B) Transiently expressed mCherry-VP1/2 or GFP-pUL25 were diffuse in Vero cells, with mCherry-VP1/2 often enriched in the nucleus. (C) Coexpressed mCherry-VP1/2 and GFP-pUL25 were enriched in the perinuclear region and nuclear rim. (D) VP1/2 localized to the nuclear rim in the absence of pUL25 when the capsid/pUL25 binding domain was removed (ΔR7). Images were captured 18–24 hr posttransfection (hpt). Scale bars are 20 µm.
Figure 2
Figure 2. VP1/2 Promotes Microtubule Transport and Is Active Under Load
(A) Transiently expressed GFP-VP1/2ΔR7 moved in curvilinear trajectories and accumulated adjacent to the nucleus in Vero cells. The 3.0 µm × 7.8 µm region denoted (a) is expandedasa time-lapse montage below the image. The 5.6 mm linescan labeled (b) was used to produce the kymograph right of the image. Motion traces are shown below the kymograph (d, distance; t, time). (B) Anchoring of VP1/2 to the surface of mitochondria promoted organelle redistribution. GFP-VP1/2ΔR7 fused to a membrane targeting sequence that directs association with the outer surface of mitochondria (GFP-ΔR7-MTS) or coexpression of mCherry-VP1/2 and pUL25-GFP-MTS fusions resulted in redistribution of mitochondria to the perinuclear region of Vero cells (yellow arrowheads). A GFP-MTS fusion served as a negative control and a GFP-BICD-N-MTS fusion as a positive control. Cells were stained with MitoTracker Red to visualize mitochondria. (C) Summary of mitochondria redistribution results. Values are the percentage of cells with clustered mitochondria ± standard error of the proportion (SEP). Asterisks indicate statistically significant difference from GFP-MTS (***p < 0.0001, **p < 0.001) as determined by Student’s t test. All images (A–C) were captured between 18 and 24 hpt. Scale bars are 20 mm. See also Figure S1 and Movie S1.
Figure 3
Figure 3. VP1/2 Interacts with Components of the Dynein/Dynactin Complex
(A) Illustration of VP1/2 constructs used for mapping the dynein/dynactin interaction. Locations of proline-rich region, CBD, and cryptic nuclear localization signal (NLS) are indicated at top. Constructs are named for each intact region number with a dash indicating removed regions. The in-frame deletion of a subregion of region 6 is indicated by a red colored lowercase ‘‘s’’ (deletion of amino acids 2087–2796). Gray dots indicate position of the GFP tag for each construct. (B) Dynein intermediate chain (DIC) and dynactin (p150/glued) coimmunoprecipitated with VP1/2. (C) VP1/2 region 7 was dispensable for p150/glued coimmunoprecipitation. (D) Removal of regions 1–5 did not impair dynactin (p50/dynamitin and p150/glued) interaction with VP1/2ΔR7. (E) VP1/2 region 6S was required for efficient interaction with dynactin. Mutation of the NLS (K285RRR > AAAA change) did not restore the interaction. (F) VP1/2 regions 6 and 7 were sufficient for dy-nactin interaction, but regions 1–4 were required for optimal binding. Densitometry plots show normalized p150/glued and p50/dynamitin binding efficiencies. Values in bar graphs (A–F) are averages ± SD. Number of repetitions is indicated (n). Asterisks indicate statistically significant difference from full-length VP1/2-GFP as determined by Student’s t test (***p < 0.0001, **p < 0.001). For all experiments, HEK293 cells were lysed 16–18 hr posttransfection, and VP1/2 was immunoprecipitated with anti-GFP antibody. Coimmunoprecipitated endogenous proteins were detected by western blot. INP, input (6% of crude lysate). See also Figure S2.
Figure 4
Figure 4. The Proline-Rich Sequences in VP1/2 Region 6 Contribute to Efficient PRV Propagation, Spread, and Virulence
(A) Single-step growth kinetics of PRV encoding either wild-type VP1/2 (WT), VP1/2ΔR6S (MUT), or repaired VP1/2 (REP). All viruses encode a mRFP1-VP26 fusion (red capsids). Plaque-forming units were quantified from Vero cells (cells) and tissue culture supernatants (media). (B) Plaque diameters of the MUT and REP viruses were measured in side-by-side experiments with WT virus. A total of 30–70 plaques were analyzed per virus per experiment. Error bars are SD based on three independent experiments. p values were determined by Student’s t test. (C) Mice were infected by intranasal instillation of the WT, MUT, and REP viruses. Each triangle on the scatterplot represents one mouse. Error bars are SD. Asterisks indicate statistically significant difference from WT (***p < 0.001) as determined by Tukey’s test. See also Figure S3.
Figure 5
Figure 5. Capsid Transport Dynamics in Sensory Axons
(A–F) DRG explants were infected with the WT, MUT, or REP viruses at 7.0 3 106 pfu/coverslip. During the first hour postinfection, moving fluorescent capsids were imaged at 10 frames per second. (A) DRG neuron infected with RFP-capsid tagged virus is illustrated at top. Middle panel is the first frame from a time-lapse recording with the corresponding kymograph aligned below (d, distance; t, time). Scale bar, 10 mm. (B) Profile of capsid retrograde transport velocities. Each data point is an average velocity of a capsid run (uninterrupted period of unidirectional transport). Data were combined from three replicate experiments (n > 80 capsid recordings per experiment). (C) Profile of run lengths. Analyses in (B) and (C) are from the same data set. (D and E) Average velocity and length of retrograde runs longer than 0.5 mm based on data presented in (B) and (C). (F) Frequency of anterograde runs ≥ 0.5 mm per particle. Error bars are SEM based on three replicate experiments per sample. p values were determined by one-way analysis of variance and a post hoc Tukey’s test.
Figure 6
Figure 6. Mutant Virus Was Less Efficient Than WT Virus in Reaching the Nucleus of Explanted DRG
(A–D) DRG were coinfected with equal pfu of two viruses with contrasting fluorophores (mCherry or GFP) fused to pUL25. Illustration of the assay is shown in (A) along with examples of equal distribution of color at the nuclear rim (RFP = GFP) and unequal distribution (RFP > GFP). (B–D) Distribution of capsids at the nuclear rim for WT-MUT (B and C) and WT-WT (D) coinfections. Numbers of analyzed nuclei are indicated (n). Scale bars are 10 µm.
Figure 7
Figure 7. Deletion of VP1/2 Proline-Rich Sequence Delays Neuroinvasion in Retrograde Circuits
(A) Illustration of the rat eye injection model. Virus injected into the anterior chamber is exposed to the ciliary body and iris, which receive sympathetic and parasympathetic input. The sympathetic circuit (top) consists of the superior cervical ganglion (SCG), which receives input from the intermediolateral nucleus (IML), which in turn receives input from the paraventricular nucleus (PVN). The parasympathetic circuit (bottom) consists of the ciliary ganglion (CG), which receives input from the Edinger-Westphal (EW) nucleus. (B–D) Male Long-Evans rats were injected with PRV encoding either VP1/2ΔR6S (MUT) or repaired VP1/2 (REP). All viruses encoded the mRFP1-VP26 fusion (capsid reporter). Each circle on the scatterplot represents a single animal. Animals were sacrificed and fluorescent cells were counted at times indicated. (B), SCG; (C), PVN; (D), EW. Average values are indicated by horizontal dashes and connected to show trends in the data for the mutant virus (red) and the repaired virus (blue).

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