Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end–directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end–directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1–10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end–directed vesicular transport, significantly reduced the extent and the frequency of minus end–directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end–directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end– directed cytoplasmic dynein and an unknown plus end– directed activity.

M.Suomalainen and M.Y.Nakano contributed equally to this workNuclear targeting of adenovirus is mediated by the microtubule-dependent, minus-end-directed motor complex dynein/dynactin, in competition with plusend-directed motility. We demonstrate that adenovirus transiently activates two distinct signaling pathways to enhance nuclear targeting. The ®rst pathway activates integrins and cAMP-dependent protein kinase A (PKA). The second pathway activates the p38/MAP kinase and the downstream MAPKAP kinase 2 (MK2), dependent on the p38/MAPK kinase MKK6, but independent of integrins and PKA. Motility measurements in PKA-inhibited, p38-inhibited or MK2-lacking (MK2 ±/± ) cells indicate that PKA and p38 stimulated both the frequency and velocity of minus-end-directed viral motility without affecting the perinuclear localization of transferrincontaining endosomal vesicles. p38 also suppressed lateral viral motilities and MK2 boosted the frequency of minus-end-directed virus transport. Nuclear targeting of adenovirus was rescued in MK2 ±/± cells by overexpression of hsp27, an MK2 target that enhances actin metabolism. Our results demonstrate that complementary activities of PKA, p38 and MK2 tip the transport balance of adenovirus towards the nucleus and thus enhance infection.

Disassembly is a key event of virus entry into cells. Here, we have investigated cellular requirements for the first step of adenovirus type 2 (Ad2) disassembly, the release of the fibers. Although fiber release coincides temporally with virus uptake, fiber release is not required for Ad2 endocytosis. It is, however, inhibited by actin-disrupting agents or soluble RGD peptides, which interfere with integrin-dependent endocytosis of Ad2. Fiber release occurs at the cell surface. Actin stabilization with jasplakinolide blocks Ad2 entry at extended cell surface invaginations and efficiently promotes fiber release, indicating that fiber release and virus endocytosis are independent events. Fiber release is not sufficient for Ad2 escape from endosomes, since inhibition of protein kinase C (PKC) prevents Ad2 escape from endosomes but does not affect virus internalization or fiber release. PKC-inhibited cells accumulate Ad2 in small vesicles near the cell periphery, indicating that PKC is also required for membrane trafficking of virus. Taken together, our data show that fiber release from incoming Ad2 requires integrins and filamentous actin. Together with correct subcellular transport of Ad2-containing endosomes, fiber release is essential for efficient delivery of virus to the cytosol. We speculate that fiber release at the surface might extend the host range of Ad2 since it is associated with the separation of a small fraction of incoming virus from the target cells.Adenoviruses (Ads) are among the best-characterized viral systems (24, 49). Both past and ongoing Ad studies have significantly contributed to concepts in molecular and cellular biology and have facilitated the development of Ad vectors for preclinical and clinical trials (3). Ads are nonenveloped icosahedral particles of about 90 nm in diameter (8). The main capsid component is the facette-associated hexon protein, which is assembled and stabilized by various minor proteins. Hexon largely protects a double-stranded linear DNA genome, which is packed inside the capsid together with additional viral proteins including the cysteine protease L3/p23. The bases of the capsid vertices are built from the penton base protein from which fibers emanate. Of the six Ad subgroups, comprising almost 50 serotypes, the subgroup C viruses such as Ad2 and Ad5 have been studied the most extensively. It was realized relatively early that subgroup C Ad entry requires two receptors, a primary receptor, CAR (coxsackie-adenovirus receptor), for attachment (4, 56) and secondary receptors, ␣ v ␤ 5 or ␣ v ␤ 3 integrins, for internalization (62). While the primary receptor determines virus tropism and is a major aim in current retargeting studies of Ad (for a recent review, see reference 12), the secondary receptor plays more subtle but less well understood roles in the infection process. Besides facilitating virus internalization, most probably via clathrin-coated pits (41, 58), ␣ v ␤ 5 integrins contribute to Ad-dependent permeabilization of the plasma membrane (61), activation ...

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