Fiber1, but not fiber2, is the essential fiber gene for fowl adenovirus 4 (FAdV-4)

Abstract

Fibre is the viral protein that mediates the attachment and infection of adenovirus to the host cell. Fowl adenovirus 4 (FAdV-4) possesses two different fibre trimers on each penton capsomere, and roles of the separate fibres remain elusive. Here, we attempted to investigate the function of FAdV-4 fibres by using reverse genetics approaches. Adenoviral plasmids carrying fiber1 or fiber2 mutant genes were constructed and used to transfect chicken LMH cells. Fiber1-mutated recombinant virus could not be rescued. Such defective phenotype was complemented when a fiber1-bearing helper plasmid was included for co-transfection. The infection of fiber-intact FAdV-4 (FAdV4-GFP) to LMH cells could be blocked with purified fiber1 knob protein in a dose-dependent manner, while purifed fiber2 knob had no such function. On the contrary, fiber2-mutated FAdV-4, FAdV4XF2-GFP, was successfully rescued. The results of one-step growth curves showed that proliferative capacity of FAdV4XF2-GFP was 10 times lower than that of the control FAdV4-GFP. FAdV4XF2-GFP also caused fewer deaths of infected chicken embryos than FAdV4-GFP did, which resulted from poorer virus replication in vivo. These data illustrated that fiber1 mediated virus adsorption and was essential for FAdV-4, while fiber2 was dispensable although it significantly contributed to the virulence.

Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells

“…Two unique restriction sites of Mau BI and Sbf I outside the fiber region facilitated the modification. With the help of intermediate plasmid pMD‐FAV4Fs, 31 fiber1‐modified adenoviral plasmids were constructed (Figure 1E ; see also Supporting information, Figure S1 and S2 ). Fiber2 or fiber1/fiber2 combined modifications were carried out by DNA assembly of Mau BI/ Sbf I‐digested adenoviral plasmid and the product of overlap extension PCR.…”

“… 4 , 28 , 29 HAdV‐35‐based vectors can efficiently transduce human hematopoietic cells because the virus receptor, CD46, is expressed abundantly on the membrane of these cells. 40 Because fiber1 substitution makes FAdV‐4 defective in growth and fiber2 is a non‐essential gene, 31 we replaced the knob of FAdV‐4 fiber2 with that of HAdV‐35 fiber in F1IJR. EF1a promoter moderately improved gene transfer to adherent cells: F1IJR‐EG transduced approximately 40% A549 or HEp‐2 cells at a MOI of 1000, which was two times as much as F1IJR did at the same MOI (Figures 3 and 4A, B ).…”

“…FAdV-4 in a cultured chicken cell line, 31 which implies that fiber1 may only be slightly modified, whereas fiber2 is free for radical reconstruction. Recently, chicken CAR homology was identified as a cellular receptor for FAdV-4.…”

“…32 pMD-FAV4Fs, a shuttle plasmid for FAdV-4 fiber modification, carried MauBI-SbfI fragment and short sequences flanking this region in the FAdV-4 genome. 31 pFiber5-35 carried the sequence encoding the fiber knob of HAdV-35 and was used as the template for polymerase chain reaction (PCR) cloning of HAdV-35 fiber knob. 33 Single-stranded DNA oligonucleotides were designed, synthesized and used as PCR primers (Table 1…”

“…30 We established a vector system based on FAdV-4, and found that fiber1 was the essential gene, whereas fiber2 was dispensable for FAdV-4 infection. 31 Because FAdV-4 could hardly transduce human cells, we attempt to improve the transduction of FAdV-4 vector to adherent or suspension human cells by incorporation of RGD4C oligopeptide to fibers or replacement of fiber knob.…”