Dynein- and Microtubule-Mediated Translocation of Adenovirus Serotype 5 Occurs after Endosomal Lysis

Abstract

Modified viruses are used as gene transfer vectors because of their ability to transfer genetic material efficiently to the nucleus of a target cell. To better understand intracellular translocation of adenovirus serotype 5 (Ad), fluorophores were covalently conjugated to Ad capsids, and movement of fluorescent Ad within the cytoplasm was observed during the first hour of infection of a human lung epithelial carcinoma cell line (A549). Ad translocation was characterized with respect to its ability to achieve nuclear envelope localization as well as directed movement in the cytoplasm. Whereas Ad achieved efficient nuclear localization 60 min after infection of A549 cells under control conditions, depolymerization of the microtubule cytoskeleton by addition of 25 microM nocodazole reversibly inhibited development of nuclear localization. In contrast, depolymerization of microfilaments by addition of 1 microM cytochalasin D had no effect on nuclear localization. Direct video observation of Ad motility showed that nocodazole, but not cytochalasin D, caused a reversible decrease in rapid linear translocations of Ad in the cytoplasm of A549 cells. Microinjection of function-blocking antibodies against the microtubule-dependent motor protein, cytoplasmic dynein, but not kinesin, blocked nuclear localization of Ad, consistent with net minus end-directed motility indicated by accumulation of Ad at mitotic spindles. Fluorescence ratio imaging revealed a neutral pH in the environment of translocating Ad, leading to a model in which the interaction of Ad with an intact microtubule cytoskeleton and functional cytoplasmic dynein occurs after escape from endosomes and is a necessary prerequisite to nuclear localization of adenovirus serotype 5.

Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer

“…[22][23][24][25][26] In these studies, significant accumulation of viral particles near the nucleus was seen within 1-2 h. The actin network can also act as a transport conduit, as demonstrated by bacculovirus, which uses the actin network exclusively for transport, 27 and HIV, which uses both actin and microtubule networks for cytoplasmic trafficking. 22 Nonviral vectors, depending on their mode of transfection, can also use cytoskeletal machinery to travel toward the nucleus.…”

Adenoviral capsid modulates secretory compartment organization and function in acinar epithelial cells from rabbit lacrimal gland

“…Ad capsid proteins are known to interact with MTs and cytoplasmic dynein during transit to the nucleus. 40,41 We have previously shown that inhibition of cytoplasmic dynein activity in lacrimal acini severely impairs the formation of rab3D-enriched secretory vesicles. 27 However, direct dynein inhibition also prevents the recruitment of VAMP2-enriched secretory transport vesicles to the apical membrane in response to CCH, while this response is unaffected by Ad (data not shown), suggesting that some dynein-mediated traffic to the apical membrane can proceed normally in Ad-treated acini.…”

Oncolytic viral therapies

“…[72][73][74][75][76][77][78] Receptor binding leads to internalization of the virus by the cell through receptormediated endocytosis, lysis of the endosomal membrane, escape to the cytosol, trafficking along microtubules towards the nucleus, binding the nuclear envelope, and insertion of the viral genome into the nucleus. [79][80][81][82][83][84][85][86][87][88] The adenoviral reproductive cycle is a highly orchestrated process, and gene modification strategies, which enable selectivity, often reduce replication capacity. At the molecular level, completion of the infectious cycle relies on timely expression of a set of viral regulatory proteins that interact with essential endogenous cellular pathways that determine cell viability.…”