Characterization of species C human adenovirus serotype 6 (Ad6)

Abstract

Adenovirus serotype (Ad5) is the most studied Ad. Ad1, 2, and 6 are also members of species C Ad and are presumed to have biologies similar to Ad5. In this work, we have compared the ability of Ad1, 2, 5, and 6 to infect liver and muscle after intravenous and intramuscular injection. We found that Ad6 was surprisingly the most potent at liver gene delivery and that Ad1 and Ad2 were markedly weaker than Ad5 and 6. To understand these differences, we sequenced the Ad6 genome. This revealed that the Ad6 fiber protein is surprisingly three shaft repeats shorter than the others which may explain differences in virus infectivity in vitro, but not in the liver. Comparison of hexon hypervariable regions (HVRs) suggests that the higher transduction by Ad5 and 6 as compared to Ad1 and 2 may be related to differences in charge and length.

Figure 1

In vivo luciferase activity induced by HD-Adenovirus. The HD-Ad genome expressing eGFP fused to luciferase was packaged by serotype 1, 2 5, and 6 helper-viruses. Groups of 5 mice were administered 10¹⁰ vp of the indicated vectors by the intramuscular or intravenous route. 24 hours later the animals were anesthetized, injected with luciferin, and imaged for luciferase activity.

Figure 2

Quantitation of in vivo luminescence. Groups of 5 mice were injected with 10¹⁰ vp of the indicated HD-Ad by the intramuscular (A) or intravenous (B) route. Sum luminescence intensities were determined using the Lumazone imaging system software. Error bars indicate standard error.

Figure 3

Effect of FX on binding and transduction on HD-Ad1, 2, 5 and 6 viruses. HD-Ad viruses were incubated with and without FX for 1 hr. at 4 C to determine the effect of FX on HD-Ad cell binding (A). Total DNA was extracted and measured using qPCR. The effect of FX on HD-Ad virus was determined using two cell lines HepG2 (B) and SKOV3 (C). Cells and HD-Ad viruses were incubated with and without FX at 37 °C for 3h. The cells were washed twice and luminescence was determined after 72 hrs. Error bars indicate standard error.

Figure 4

Bayesian Inference Phylogeny. Markov chain Monte Carlo (MCMC) analysis of 35 complete Adenovirus genome sequences. Complete genome sequences were downloaded from Genbank and aligned using ClustalX. The alignment was inspected visually and all gaps were removed prior to phylogenetic analysis using MrBayes v3.1. The posterior probability of phylogenetic trees (and other parameters of the substitution model) cannot be determined analytically. Instead, MCMC is used to approximate the posterior probabilities of trees by drawing (dependent) samples from the posterior distribution.

Figure 5

Identification of comparative differences by sequence alignment analysis. Adenovirus serotypes 5 and 6 were aligned using a Pustell DNA Matrix Analysis tool in MacVector v8.1.1 (A). The complete nucleotide sequence of Ad6 and Ad5 are shown on the X and Y axis, respectively. The diagonal line represents homology and gaps represent non-homologous regions. The relative genomic location of the non-homologous regions is represented below (B).

Figure 6

Fiber sequence comparison. Ad1, 2, 5, and 6 fiber proteins were aligned using ClustalW in macVector v8.1.1. A neighbor-joining tree shows the phylogenetic relationships of the Ad fibers (A). The aligned fiber protein sequences are shown below (B). Boxes over the alignment indicate the location of the fiber tail, fiber shaft repeat motifs, and the fiber knob.

Figure 7

Penton base sequence comparison. Ad1, 2, 5, and 6 penton proteins were aligned using ClustalW in MacVector v8.1.1. A neighbor-joining tree shows the phylogenetic relationships of the proteins (A). The aligned penton base sequences are shown below (B). The box identifies the RGD motif in each.

Figure 8

Hexon sequence comparison. Ad1, 2, 5, and 6 hexon proteins were aligned using ClustalW in MacVector v8.1.1. A neighbor-joining tree shows the phylogenetic relationships of the Ad hexons (A). The aligned hexon protein sequences are shown below (B). Boxes over the alignment indicate the location of the 7 hypervariable regions (HVR1-7).

Figure 9

Gross differences in the genetic relationships of the HVRs between viruses. Neighbor-joining phylogenetic analyses for each HVR were performed. Seven phylogenetic trees represent the genetic distances at each HVR.