Fiber Shaft-Chimeric Adenovirus Vectors Lacking the KKTK Motif Efficiently Infect Liver Cells In Vivo

Adenovirus (Ad) is a nonenveloped virus with a linear, double-stranded DNA genome. In humans, multiple serotypes of Ad cause a spectrum of illnesses in both normal and immunocompromised hosts (17, 35). There are 51 human Ad serotypes identified to date, which are divided into six species, A to F. Many of these serotypes exhibit diverse cell and tissue tropisms, which are, at least in part, responsible for the variety of observed clinical manifestations caused by Ad infection. From in vitro studies, it was found that Ad infection starts with virus binding to a high-affinity primary attachment receptor on the cell surface. This initial interaction is mediated by the fiber protein when its distal knob domain binds to a specific cellular receptor. For binding to cells, species A, C, D, E, and F Ads may utilize the coxsackievirus and Ad receptor (CAR); however, the majority of species B Ads utilize CD46 as a high-affinity cellular attachment receptor (4, 5, 9, 33, 34, 37). Fiber-mediated binding of Ad to cells is followed by binding of the viral penton base protein to integrins (50). This interaction induces integrin activation, cytoskeleton rearrangement, and the internalization of the virus particle into the cell (12, 15, 20, 21, 49). Although the early steps of Ad infection in vitro are known in great detail (reviewed in references 10, 23, 24, and 26), there is still minimal understanding of the mechanisms responsible for the efficient natural Ad infection of distal sites and tissues (such as bladder epithelial cells) in vivo. Additionally, the well-accepted two-step model of Ad cell infection fails to explain the infectivity and biodistribution of Ad-based vectors following their intravascular delivery. Specifically, in all the animal models analyzed, including mice, rats, rabbits, dogs, and nonhuman primates, the liver is the predominant organ transduced by Ad5-based vectors (6, 16, 22, 29, 30, 44). Moreover, when non-CAR-binding Ad5 vectors are injected intravenously, they transduce liver cells at levels largely indistinguishable from those observed for CAR-interacting Ads (1, 44). In recent years, significant attention has been given to the analysis of properties of an Ad5-based vector, AdS*, which has the KKTK amino acid motif within the fiber shaft domain (amino acids 91 to 94) replaced by nonhomologous amino acids GAGA (3, 18, 45, 46). When given intravenously, the AdS* vector demonstrates a remarkable reduction in liver cell transduction in mice, rats, and nonhuman primates compared to Ad5. Based on this data, it was postulated that the KKTK motif is a specific site in the Ad fiber shaft domain responsible for direct Ad attachment to hepatic heparan sulfate proteoglycans (HSPGs), thus enabling efficient Ad liver cell transduction in vivo. Although the idea of the involvement of the KKTK motif in liver infection has been widely discussed (19, 28), there is still no clear evidence that the KKTK motif binds to hepatic HSPGs and mediates liver cell transduction. Moreover, in vitro studies revealed that AdS* is very inefficient at infecting cells expressing high levels of CAR (3, 18, 45, 46). Indeed, to generate sufficient numbers of AdS* particles for in vivo analyses, the virus is propagated on cell lines which provide the fiber protein in trans to maintain virus infectivity (3). Recently, two independent groups demonstrated that AdS* cannot be used for Ad retargeting, mostly due to its inability to transduce susceptible cells (3, 18). To analyze whether the direct binding of the KKTK motif to hepatic HSPGs is the primary mechanism of liver transduction by Ad5-based vectors, in vivo studies of vectors with a mutated KKTK motif would appear to be the most informative. However, due to their attenuated cell transduction phenotype, AdS* vectors cannot be used directly for these in vivo studies. Therefore, indirect alternate strategies should be applied to analyze the contribution of the fiber shaft KKTK motif to efficient Ad transduction of hepatic cells in vivo. Here, we describe an alternate strategy to evaluate the role of the KKTK motif in liver cell transduction: the generation of fiber shaft-chimeric vectors lacking the KKTK motif. We generated viruses and conducted in vitro and in vivo infectivity studies of Ad5-based vectors possessing long Ad31- or Ad41-derived fiber shaft domains. The fiber knob domains of long-shafted Ad31 and Ad41 fibers can recognize CAR as a virus attachment receptor; however, the amino acid compositions of Ad31 and Ad41 fiber shaft domains are highly diverse and lack the KKTK motif. In contrast to AdS*, our fiber shaft-chimeric vectors were able to bind efficiently to and retain high-level infectivity toward susceptible cells in vitro. When fiber shaft-chimeric vectors were injected intravenously into mice, their accumulation in livers and the efficiency of liver cell infection were undistinguishable from those observed with unmodified Ad5. Our study demonstrates for the first time that the amino acid composition of the Ad5 fiber shaft (which is long) is not highly conserved and that this shaft can be replaced entirely with the fiber shafts from other long-shafted Ad serotypes without any reduction in overall virus infectivity. Our data also strongly suggest that the KKTK motif within the Ad5 fiber shaft plays only a minimal, if any, role in determining adenovirus infectivity toward hepatic cells in vivo. The obtained data contribute to improving our understanding of the molecular mechanisms determining Ad infectivity and biodistribution in vivo and may aid in designing efficient Ad-based vectors for application in humans.