This discontinuous activity is not surprising as often Kinase Inhibitor Library vesicles moving in one direction can apparently invert their motion by jumping on near microtubules tracks or through the coordination activity of a macromolecular complex. As a consequence, the final transport may result from several short movements that can occur in both directions. In PRV, US9 has been shown to interact with KIF1A, a microtubule-dependent kinesin-3 motor, and the interaction is dependent on the presence of other still unidentified viral factor. The same report demonstrates that HSV-1 US9 cannot be co-purified with KIF1A, launching the quest for a HSV US9 motor partner. Irrespective of the identity of that molecule, our data suggest that HSV US9 can engage the cellular transport machinery, and that its dynamic behavior occurs in the absence of other viral factors. It is possible that US9-dependent axonal transport of HSV particles requires additional proteins, as suggested by the PRV data, with glycoproteins E and I being good, but not exclusive, candidates to play such a role. Nevertheless and not considering virion transport, US9-containing vesicles traffic occurs with no additional factors. In primary neurons, the GFP-US9 localization could be clearly appreciated and may result extremely helpful to understand the molecular basis of US9 activity. In these cells, GFP-US9 fluorescent vesicles localize both at cell body and throughout neurites, frequently in close proximity to microtubules. Identical distribution and microtubules proximity were seen in neurons infected with a recombinant virus expressing GFP-US9, showing no differences with the behavior of the viral protein expressed in the absence of other viral factors. By using different fluorescent tags to distinguish stand alone-, and virus-encodedUS9, we were able to precisely assess the extent of co-localization. The two differently tagged versions of US9 co-expressed in the same cell mostly localize in the same vesicles, in both proximal and distal cellular regions. Few but constantly present individually tagged vesicles with no preferential sub-localization were detected in all the experiments, and this reproducible distribution can be interpreted with the equivalence between the two differently tagged US9. As well established, due to the peculiar way viruses replicate themselves, there is no difference between stand alone RFP-US9 and virus-encoded GFP-US9, once they are co-expressed in the same infected cell. The presence of few green or red, and many yellow vesicles simply demonstrates US9 incorporation in these transport vesicles. In agreement with our hypothesis of an autonomous transport activity is the observation that ablations of two phosphorylation sites in the cytosolic tail of US9 do not affect US9 localization. PRV transneuronal spread depends on the presence of two tyrosines and two serines in a conserved acidic cluster. Results presented here, showing co-localization of wild type US9 with both mutant proteins, indirectly support the idea that no other viral factors are required for the US9 transport function. This interpretation implies that US9 is able to autonomously traffic inside the cells, while other modifications and/or viral factors are required to make the virus be transported along.