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. 2008 Sep;9(9):1458-70.
doi: 10.1111/j.1600-0854.2008.00782.x. Epub 2008 Jun 28.

Two viral kinases are required for sustained long distance axon transport of a neuroinvasive herpesvirus

Kelly E Coller  1 , Gregory A Smith
Affiliations

Two viral kinases are required for sustained long distance axon transport of a neuroinvasive herpesvirus

Kelly E Coller et al. Traffic. 2008 Sep.

Abstract

Axonal transport is essential for the successful establishment of neuroinvasive herpesvirus infections in peripheral ganglia (retrograde transport) and the subsequent spread to exposed body surfaces following reactivation from latency (anterograde transport). We examined two components of pseudorabies virus (US3 and UL13), both of which are protein kinases, as potential regulators of axon transport. Following replication of mutant viruses lacking kinase activity, newly assembled capsids displayed an increase in retrograde motion that prevented efficient delivery of capsids to the distal axon. The aberrant increase in retrograde motion was accompanied by loss of a viral membrane marker from the transported capsids, indicating that the viral kinases allow for efficient anterograde transport by stabilizing membrane-capsid interactions during the long transit from the neuron cell body to the distal axon.

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Figures

Figure 1
Figure 1. Mutagenesis of the viral kinases
(A) Illustration of UL13 and US3 mutations made in the PRV genome (PRV-GS847: strain Becker previously modified to express mRFP1 fused to the VP26 capsid protein). (B) Western blot analysis of extracellular purified virions. Viral kinases were detected with anti-UL13 or anti-US3 antibodies. A revertant of the double-kinase-null virus (PRV-GS1555) in which both kinase deletion alleles were repaired (rev) expressed both kinases similar to the wild type (wt). Representative proteins of each component of the virus were also examined for structural incorporation: VP5 (capsid), UL37 (inner tegument), VP22 (outer tegument), gD (envelope). (C) Kinase mutants propagate with reduced kinetics. PK15 cells were infected at MOI=5, and the cells and supernatants were harvested at the indicated times post infection. Viral titers were quantitated by plaque assay.
Figure 2
Figure 2. Capsid transport dynamics in axons
(A) The frequency of retrograde transport during the egress stage of infection for viruses encoding wild-type (wt and rev) or mutated kinase genes are shown. Transport of individual RFP-capsids was imaged in axons of cultured DRG sensory neurons between 12–15 hours post infection at 20 frames/s. The number of capsids undergoing retrograde events are presented as a percentage of all capsids observed actively transporting in either direction. Parental and revertant wild-type viruses are indicated (wt and rev), and reproduced findings from a previous study (2). Error bars indicate standard error (of the proportions), and asterisks designate values statistically distinct from the wild type (p < 0.0001). (n, number of capsids tracked) (B) Frequency of stalls in capsid transport during the egress stage of infection for viruses encoding wild-type (wt and rev) or mutated kinase genes. Stalls in capsid transport were scored when actively transported capsids ceased motion and failed to recover during the duration of a recording (500 frames). Infections and imaging was as described in panel A. Error bars are standard error (of the proportions). Asterisks indicate values that show deviation from the wild type (p < 0.01). (C) Retrograde and anterograde capsid transport velocities and run lengths in axons during the entry and egress phases of infection. RFP-capsid images were acquired continuously with 50 ms exposures (20 frames/s). Recordings of entry transport were acquired within the first hour post infection, while egress recordings were made between 12–14 hours post infection. Horizontal bars indicate the mean for each sample.
Figure 3
Figure 3. Transport of capsids to axon terminals
(A) DRG sensory neurons were infected with the wild-type and revertant viruses (wt and rev), the double-kinase-null virus (Δ/Δ), and the virus in which both kinases were inactivated by a single amino acid substitution (D>A/D>A). Neurons were fixed with parafomaldehyde at 14 hours post infection and axon terminal growth cones were imaged by differential interference contrast (DIC) and fluorescence microscopy. Scale bars = 10 microns. (B) Average number of capsids per growth cone (n, number of growth cones examined for each sample). Error bars are standard error (of the means) and asterisks indicate significant deviation from the wild type (p < 0.001).
Figure 4
Figure 4. Increased frequency of retrograde motion is coupled with a loss of viral membrane association
DRG sensory neurons were infected with dual-fluorescent viruses expressing mRFP1-capsids and GFP fused to the viral membrane protein, gD, and encoding either wild-type or mutated kinase genes. De novo assembled progeny virus particles were imaged and tracked in axons for red and green emissions between 12–15 hours post-infection using automated filter wheels and sequential capture of each emission spectra. This resulted in an artificial spatial shift of red and green fluorescence due to movement of the dual-fluorescent particles between red and green imaging. (A) Example of two viral particles in one axon with the double-kinase-null virus. One red capsid particle exhibits retrograde motion and lacks detectable GFP fluorescence, while the second capsid moves anterograde and is associated with GFP emissions, indicating the presence of the gD viral transmembrane protein. The frames were captured over a period of 6 seconds and are each 5.6 × 17.4 microns. (B) Quantification of mRFP1-capsid transport direction and gD association. (n, number of capsids tracked)
Figure 5
Figure 5. Minus-end motion is not dependent upon the virus fusion machinery
(A) Illustration of two possible origins of unenveloped capsids that undergo aberrant retrograde transport in axons during the egress stage of infection. In the lower model, membrane fusion is required to release the enveloped capsid into the cytosol to initiate retrograde transport; the upper model does not require a fusion event to release the capsid from the associated membrane. (B) Cultured DRG sensory neurons were infected with gB-null viruses, and de novo assembled progeny viral particles were imaged and tracked in axons between 12–15 hours post infection. Frequencies of retrograde transport events are shown. The data support the upper model in panel A (see text). Error bars are standard error (of the proportions). (n, number of capsids tracked)
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