Identification of Adenovirus Serotype 5 Hexon Regions

24 Most of an intravenous dose of species C adenovirus serotype 5 (Ad5) is 25 destroyed by liver Kupffer cells. In contrast, another species C virus, Ad6, evades these 26 cells to mediate more efficient liver gene delivery. Given that this difference in Kupffer 27 cell interaction is mediated by the hypervariable (HVR) loops of the virus hexon protein, 28 we genetically modified each of the seven HVRs of Ad5 with a cysteine residue to 29 enable conditional blocking of these sites with polyethylene glycol (PEG). We show that 30 these modifications do not affect in vitro virus transduction. In contrast, after 31 intravenous injection, targeted PEGylation at HVRs 1, 2, 5 and 7 increased viral liver 32 transduction up to 20 fold. Elimination or saturation of liver Kupffer cells did not 33 significantly affect this increase in the liver transduction. . In vitro, PEGylation blocked 34 uptake of viruses via the Kupffer cell scavenger receptor SRA-II. These data suggest 35 that HVR1, 2, 5, and 7 of Ad5 may be involved in Kupffer cell recognition and 36 subsequent destruction. These data also demonstrate that this conditional genetic37 chemical mutation strategy is a useful tool to investigate the interactions of viruses with 38 host tissues. 39 40 on O cber 2, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Introduction 41 Adenovirus serotype 5 (Ad5) has proven to be one of the most potent in vivo 42 gene delivery vectors for liver-directed gene therapy. As much as 95 to 98% of an 43 intravenously (i.v.) injected dose of Ad5 is trafficked to the liver (12). The adenovirus 44 capsid is comprised of three major proteins: hexon, penton base, and fiber (reviewed in 45 (7)). Based on numerous in vitro studies, Ad fiber and penton base have been shown to 46 be cellular attachment proteins. The fiber of Ad serotype 5 (Ad5) binds coxsackie and 47 adenovirus receptor (CAR) (3), which triggers binding of penton base to αv integrins via 48 an RGD motif (35, 36) and results in viral cell internalization. Although necessary for 49 providing specificity of the virus in vitro, modifications to the fiber have little effect on 50 vector tropism in vivo (25). Instead, increasing evidence suggests that hexon plays a 51 large role in the natural liver tropism of Ad5. 52 Hexon is the most abundant viral capsid protein with 720 monomers per virion. 53 Hexon organizes into trimers so that three hexon monomers and their loops wrap tightly 54 around each other to create a tower-like structure with a central depression (20, 28). 55 Each hexon monomer has seven flexible, serotype-specific loops, named hypervariable 56 regions (HVR) (23) that are predicted to be located on the surface of the hexon trimer 57 and the virion (29). This location allows the HVRs of hexon to interact with neutralizing 58 antibodies, receptors, proteins, and cells. Considering there are 5,040 (720 x 7 =5040) 59 HVRs per virion, these represent a complex, exposed surface for many interactions. 60 After intravenous (i.v.) injection, Ad5 exhibits the greatest transduction within liver 61 hepatocytes (12). Despite this robust in vivo gene delivery, approximately 90% of the 62 injected dose is sequestered and destroyed by resident liver macrophages called 63 on O cber 2, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Kupffer cells (1). These antigen-presenting cells not only destroy the virus, but are 64 themselves destroyed. This cellular necrosis plays a role in inflammation and innate and 65 adaptive immune responses to the virus (21). Previous studies indicate that Kupffer 66 cells take up Ad5 via scavenger receptors (13, 37). It is hypothesized that scavenger 67 receptors on these cells recognize the virus by interacting with the highly charged HVRs 68 on Ad5 (1, 37). In particular, HVR1 is thought to be a good ligand for scavenger 69 receptors (1), since it is the largest HVR and also has a number of negatively charged 70 amino acids (18)). Results shown here indicate that hexon and its HVRs are important 71 surfaces involved in Kupffer cell recognition. By evading such cells, adenoviruses 72 should be able to more readily transduce downstream sites. 73 After evading the reticuloendothelial system (which includes both Kupffer cells 74 and liver sinusoidal endothelial cells), Ad5 enters the liver parenchyma through 75 fenestrations in the vessel walls (32). If Ad can enter the parenchyma, robust 76 hepatocyte infection is dependent not only on cell binding ligands evolved by the virus, 77 but also on host blood proteins. Evidence shows that Ad5 hexon binds to vitamin K 78 dependent coagulation factors, such as factor X (FX), with high affinity, and this 79 interaction markedly increases hepatocyte infection (26, 31). Structural and mutational 80 analysis has revealed that FX binds to the top of the hexon trimer depression, with 81 predicted interactions with HVR5, HVR7, and possibly HVR3 (16, 33). These data 82 implicate the HVRs of the Ad5 hexon in two pharmacologic bottlenecks of the virus after 83 i.v. injection: 1) binding or evasion of Kupffer cells and 2) binding of FX and retargeting 84 to hepatocytes. 85 on O cber 2, 2017 by gest http/jvi.asm .rg/ D ow nladed fom We recently compared the biology of Ad5 with another species C adenovirus, 86 Ad6 (18, 34). In this work, we found that native Ad6 mediates higher liver transduction 87 than Ad5 after i.v. injection (34). To identify the underlying molecular basis for this 88 difference, the HVRs of Ad5 were replaced with those of Ad6 producing a virus called 89 Ad5/6 (Fig. 1A and (18)). When Ad5 and Ad5/6 were compared, the Ad5/6 virus 90 mediated 10-fold increases in hepatocyte transduction after i.v. injection. This effect 91 appeared to be due to reduced interactions of Ad5/6 with macrophages and Kupffer 92 cells. 93 These data suggest that particular surfaces of the Ad5 hexon are involved in 94 Kupffer cell recognition. Given this and the pivotal role of Kupffer cell depletion during 95 systemic therapy, we evaluated the roles of each of the seven HVRs by conditionally 96 mutating them using genetic and chemical engineering techniques (18, 19). Single 97 cysteine residues were inserted into each of the seven HVRs of Ad5 hexon individually. 98 These “silent” mutations were then “activated” by specifically modifying the site by 99 reaction with cysteine-reactive polyethylene glycol (PEG) to conditionally block 100 interactions with each HVR. By this approach, we probed the role of each HVR in 101 Kupffer cell recognition. 102