Theses Doctoral

Role of Microtubule Motor Proteins in Adenoviral Infections

Scherer, Julian

Viruses have been described as a piece of nucleic acid surrounded by bad news. These bad news determine host specificity, pathogenicity, and virulence. In the case of adenovirus, a non-enveloped double-stranded DNA virus that causes self-limiting disease in healthy individuals but can cause severe and even fatal infection in immunocompromised patients, the bad news can be reduced to the viral capsid. The adenoviral capsid mainly consists of three proteins (fiber, penton base, hexon) that form a rigid shell to protect the viral genome outside of host cells. However, they are able to orchestrate a precise disassembly program initiated once the next susceptible cell is reached, leading to step-wise virus entry, controlled capsid disintegration, efficient DNA delivery, and production of progeny virus. Understanding adenovirus entry is not only beneficial for pathogenic but also therapeutic reasons since adenoviruses have become an increasingly popular vaccine and gene transfer vector due to their ability to infect a large array of dividing and post-mitotic cells, their large DNA capacity, and easy amplification. Attachment to cell surface receptors leads to cellular signaling events, some of which regulate receptor-mediated uptake into clathrin-coated pits. Conditions inside the endosome trigger escape of the virions from the organelle through membrane disruption 5-15 min post-infection and most capsids gain access to the nucleus about 30-45 min thereafter. Interestingly, adenovirus relies on the microtubule (MT) network, MT-dependent motor proteins, and virus-stimulated cAMP-activated kinase (protein kinase A, PKA) activity to traverse the cytoplasm during the critical intracellular transport phase between endosomal escape and nuclear pore complex attachment. The main virus transporter is the MT minus-end directed motor protein complex cytoplasmic dynein, which functions in organelle positioning, cell migration, cell division, and cell differentiation in uninfected cells. Cytoplasmic dynein subunits known to interact with physiological cargo also bind directly to the adenovirus capsid protein hexon, which remains with the viral genome until its delivery through the nuclear pore complex. Strikingly, for strong binding to cytoplasmic dynein, hexon requires an acidification step, indicating an additional functional role of the passage through the acidic endosomal lumen during entry, priming of hexon for cytoplasmic dynein binding. Here, we continue previous research of the hexon - dynein interaction and describe the determinants of dynein-mediated capsid transport in further detail. We show that the requirement for stimulated PKA activity on MT minus-end directed motility involves a PKA phosphorylation site in the dynein light intermediate chain 1 (LIC1). PKA phosphorylation or a phosphomimetic mutation increase hexon binding of LIC1 in vitro and RNAi rescue experiments confirm a clear role of PKA phosphorylation in adenovirus redistribution to the nucleus. To our surprise, the same phosphorylation site also plays a role in positioning of lysosomes/late endosomes (lyso/LE) a class of organelles under PKA control. However, in contrast to dynein-mediated viral cargo transport which is stimulated upon phosphorylation, lyso/LE motility in the minus-end direction is strongly reduced leading to lyso/LE dispersal into the periphery. Hence, during adenoviral infections, stimulation of PKA activity mediates two distinct functions, lyso/LE dispersal and efficient capsid transport.Remarkably, adenovirus transport is not only mediated by cytoplasmic dynein towards the cell center but also by opposite polarity motors towards the cell periphery leading to bidirectional capsid motility along MTs, similar to endogenous cargo. The motility pattern implies the involvement of members of the kinesin family, which are MT plus-end directed motor proteins regulating MT dynamics, cell division, and organelle transport in uninfected cells. We provide evidence for a direct capsid interaction with kinesin-1, which shows striking differences from the interaction between the capsid and cytoplasmic dynein. Kinesin binding appears to occur independent of low pH treatment of the virion and independent of hexon, but is very likely mediated through the capsid protein penton base. Exploring the physiological role of this interaction, subviral penton dodecahedra were purified from lysate of adenovirus infected cells and, after their specific binding to kinesin-1 but not cytoplasmic dynein was confirmed, were used for motility analysis in cultured hippocampal neurons. Dye-labeled penton dodecahedra show clear intracellular motility in live-cell microscopy. In addition, we also explored the pH dependent change in hexon affinity for cytoplasmic dynein by testing the viral capsid protein for structural changes at acidic pH conditions. In its native form inside the capsid or as soluble antigen, hexon is present as a tightly associated trimer, which is resistant to elevated temperature, high ionic strength, detergent treatment, and low pH. However, we now show that low pH treatment strongly increases the sensitivity of the trimeric structure to SDS, leading to hexon monomerization. These data indicate a subtle structural change in the hexon polypeptide upon low pH treatment, increasing dynein affinity and SDS-sensitivity. Taken together, the here presented work contributes to our understanding of adenovirus entry, especially during the cytoplasmic transport phase, and reveals mechanisms of finely orchestrated host-pathogen interactions at the evolutionary interface of viral attack and cellular host defense.



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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Vallee, Richard
Ph.D., Columbia University
Published Here
January 25, 2013