Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

William N.D Gao,Geoffrey L Smith,Helen A Ewles,Stacey‐Ann Lee,David C.J Carpentier

doi:10.1111/tra.12494

Abstract

Vaccinia virus (VACV) utilizes microtubule‐mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin‐1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin‐1, yet in its absence VACV egress still occurs on microtubules. During a co‐immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C‐terminal tail of KLC2, to a region that overlaps the binding site of cellular 14‐3‐3 proteins. F12/E2 displaces 14‐3‐3 from KLC and, unlike 14‐3‐3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N‐terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co‐operatively enhance A36 association with KLC, particularly when using a KLC1‐KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co‐operatively associate with kinesin‐1.

Highlights

Microtubule (MT)-mediated intracellular trafficking is exploited at several stages of the vaccinia virus (VACV) replication cycle.[1]
VACV proteins A36, F12 and E2 all associate with kinesin-1 during intracellular enveloped virions (IEVs) egress and influence IEV egress efficiency
Of these proteins only A36 is associated directly with the IEV particle via a transmembrane domain, yet deletion of A36 is less detrimental to virus egress than deletion of either F12 or E2, and IEVs lacking A36 are still transported in a MT-dependent manner.[41]

Summary

| INTRODUCTION

Microtubule (MT)-mediated intracellular trafficking is exploited at several stages of the vaccinia virus (VACV) replication cycle.[1]. The region of KLC2 responsible for the enhanced association of F12/E2 has been mapped using KLC1/KLC2 chimaeras to the Cterminal tail of KLC2.40 To map this interaction more accurately, a series of mutants of KLC2 were made lacking the C-terminal 16 (KLC2ΔC16), 46 (KLC2ΔC46) or 88 (KLC2ΔC88) amino acids (Figure 3B) Precipitation of these mutants from cells infected with vF12-HA showed that only the last 16 amino acids were dispensable for F12/E2 binding and further truncation resulted in F12 coprecipitation levels equivalent to those with KLC1 (Figure 3D). Virus infection of cells expressing a KLC1/2 chimaera (Flag-KLC1/2 A) that bound both A36 and F12/E2, so that all components may be present in the same complex, showed reduced A36-KLC interaction in the absence of F12 or E2 (Figure 7B) This suggests that there is co-operativity in the association of the different components of the IEV trafficking complex, especially if all components are able to efficiently associate

| DISCUSSION

| MATERIALS AND METHODS