No abstract

SUMMARY Early in infection, adenovirus travels to the nucleus as a naked capsid using the microtubule motor cytoplasmic dynein. This study was initiated to address how the virus recruits dynein, and to explore the role of dynein's diverse regulatory factors in virus transport. Cytoplasmic dynein, dynactin and NudE/NudEL, but not LIS1 or ZW10, colocalized with incoming, post-endosomal adenovirus particles. Dynein alone interacted in a pH-dependent manner with the adenovirus subunit hexon, which, in turn, interacted with recombinant dynein intermediate chain and light intermediate chain 1. Interference with dynactin function had no effect on dynein colocalization with adenovirus, but reduced virus run length. Expression of hexon or injection of anti-hexon antibody inhibited virus transport without affecting Golgi distribution. These results identify hexon as a direct receptor for cytoplasmic dynein, which recruits dynein for transport to the nucleus by a mechanism both novel and distinct from that for known physiological dynein cargo forms.

Background: Cytoplasmic dynein performs a great variety of cellular functions using a diversity of regulators. Results: NudE and dynactin compete for a common site within the dynein complex. Conclusion: This mechanism prevents dual regulation by dynactin and LIS1 and suggests a major new mode of regulatory control. Significance: This is the first insight into coordination of cytoplasmic dynein regulators.

How cytoplasmic dynein is recruited to diverse organelles remains incompletely understood. Using the first subcellular localization of LIC isoforms, along with RNAi, RILP, and dynactin dominant negatives, the LIC subunits are found to recruit dynein specifically to components of the late endocytic pathway through a dynactin-independent mechanism.

PKA-mediated phosphorylation of a specific residue in the dynein light intermediate chain 1 releases the motor protein from lysosomes and late endosomes while activating its recruitment to adenovirus capsids.

Many molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based on total internal reflection fluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway.

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.

A considerable part of the herpesvirus life cycle takes place in the host nucleus. While much progress has been made to understand the molecular processes required for virus replication in the nucleus, much less is known about the temporal and spatial dynamics of these events. Previous studies have suggested that nuclear capsid motility is directed and dependent on actin filaments (F-actin), possibly using a myosin-based, ATP-dependent mechanism. However, the conclusions from these studies were indirect. They either relied on the effects of F-actin depolymerizing drugs to deduce an F-actin dependency or they visualized nuclear F-actin but failed to show a direct link to capsid motility. Moreover, no direct link between nuclear capsid motility and a molecular motor has been established. In this report, we reinvestigate the involvement of F-actin in nuclear herpesvirus capsid transport. We show for representative members of all three herpesvirus subfamilies that nuclear capsid motility is not dependent on nuclear F-actin and that herpesvirus infection does not induce nuclear F-actin in primary fibroblasts. Moreover, in these cells, three F-actin-inhibiting drugs failed to effect capsid motility. Only latrunculin A treatment stalled nuclear capsids but did so by an unexpected effect: the drug induced actin rods in the nucleus. Immobile capsids accumulated around actin rods, and immunoprecipitation experiments suggested that capsid motility stopped because latrunculin-induced actin rods nonspecifically bind nuclear capsids. Interestingly, capsid motility was unaffected in cells that do not induce actin rods. Based on these data, we conclude that herpesvirus nuclear capsid motility is not dependent on F-actin.