Determination Of Ratio Of Short Fiber To Long Fiber On Human Adenovirus Type41and Study On Ad41Morphogenesis

Human adenovirus species F (HAdV-F) consists of adenovirus type40and41.It is one of the main causative agents of pediatric enteritis and responsible for5%-15%non-bacterial infantile diarrhea. Following characteristics make HAdV-F different from other human adenovirusesFirst, HAdV-F displays fastidious growth characteristics and cannot be propagated as efficiently as other groups. Human HAdV-F is also named fastidious adenoviruses.Second, HAdV-F is enteric adenovirus. It shows high tropism for the intestinal tract and doesn’t show any evidence of causing disease elsewhere in the body. It is resistant to low pH and digest enzymes. All these advantages make HAdV-F a promising vector candidate for intestinal gene therapy or oral vaccine.Third, HAdV-F has two fibers:the short fiber (sfiber) and the long fiber (lfiber). The genome contains two fiber genes. The expressed two fiber proteins form homotrimers, respectively, which protrude externally from different vertices of mature virions. The long one, just like fibers in other human adenoviruses except species B, can interact with Coxsackie and Adenovirus Receptor (CAR) and mediate entry of virus into host cells, while the receptor and function of the sfiber remain unknown.An Ad41E1B55K-expressing cell line,293TE7.6, was established in the laboratory previously. It can be used to amplify wild-type Ad41or package recombinant Ad41. Sufficent viruses make it possible to study Ad41in a more detailed manner. It was reported that HAdV-41contained equal number of sfiber and lfiber on its virion when being determined by method of counting under electron microscope. Here we aimed to determine the ratio of sfiber to lifiber using molecular biological methods, and the morphogenesis of Ad41was studied with techniques of electron microscopy as well.1. Determination of the ratio of fibers in Ad41virionIntact Ad41short fiber (sfiber-P) and long fiber (lfiber-P) were expressed prokaryotically. The concentration of the two purified proteins was preliminarily determined with BCA method, and further quantified by SDS-PAGE with serially diluted bovine serum albumin (BSA) as loading standards. The bands were stained with Coomassie brilliant blue R250and analyzed with software Gel-pro analyzer4.0to establish a standard curve to calculate the concentrations of sfiber-P and lfiber-P according to their bands density. The quantified sfiber-P and lfiber-P were serially diluted and loaded as standards in Western blot to determine the amount of fibers on Ad41virions, respectively. After density analysis, sfiber and lfiber in purified virions were quantified, and their ratio was then determined to be5.To further verify this result, a mutant Ad41without sfiber, Ad41DSF-GFP, was constructed, rescued from293TE7.6cells, and then purified with CsCl gradient ultracentrifugation. Relatively quantitative Western blot analysis showed that the amount of Ifiber on Ad41DSF-GFP is about6times of that on equivalent wild-type Ad41virons. Because both Ad41and Ad41DSF-GFP display equal number of total fibers on the virion (12fibers a virion), the ratio of sfiber to lfiber in Ad41should be5. These results confirmed that Ad41contains approximately five times as many short fibers as long fibers in the capsids.The reason for unequal numbers of fibers on Ad41virions was preliminarily investigated. The results of Western blot suggested that different abundance of fiber proteins in the infected cells might contribute to different number of sfiber and lfiber assembled in the virions. The differences in fiber expression were regulated at post-transcriptional level since the amount of mRNAs for both short and long fibers was comparable. Finally, the possible function of sfiber was studied. Infection experiments demonstrate that the transduction efficiency of Ad41-GFP is1.2-1.8times of that of Ad41DSF-GFP. Titration data showed that Ad41DSF-GFP VP/IU value is1.5times of that of Ad41-GFP. In addition, Ad41-GFP enters cells with a little higher speed than Ad41DSF-GFP does. Because Ad41DSF-GFP lacks sfiber, the above results indicate that short fiber facilitates Ad41infection.2. Ad41morphogenesisThe results of Ad41morphgenesis research were recorded as follows:A. Entry pathway into host cell:Ad41can adhere to cell membrane and enter into cytoplasm through three ways: clathrin-coated pit, non-clathtin-coated pit and direct penetration. The non-clathrin-coated pit passway is predominant in the three ways. Perhaps microvilli play some roles in virus entry into cell.B. Virus transportation cell:Virus could approach to nucleus through the following ways:in vacuole, in lysosome and free in cytoplasm. Virus approaches to the nuclear pore and injects nucleoprotein into nucleus through the pore.C. Virus replication:Progeny virus particles emerge and disperse in nucleus at first. The amount of viruse particles increasing gradually and then viruses arrange in form of mass or para-crystallization in nucleus. With times going on, viruses were exported into cytoplasm through unknown passway and arrange in form of mass in irregular array or para-crystallization like that of in nucleus. Two forms of inclusion bodies (IB) were induced during virus replication:fibrillous IB (FIB) and condensed IB(CIB). Immuno-EM results prove that there are Ad41components in two kinds of IBs. Short fiber and long fiber components are involved in the FIB.D. Virus release:Viruses aggregate in nuleus or cytoplasm at late stage of virus reproduction. Cell structure becomes disordered. At last, viruses egress at the site of broken cytoplasm membrane. E. Virus re-infection of cells:Viruses released from collapsed cells attach on other cell surface and enter into the cell to start a new life cycle.Compared to other adenoviruses, Ad41replication is similar to other human adenoviruses except that the FIB morphous differs from that of Ad5.Collectively, we successfully determine the ratio of short fiber to long fiber on Ad41viron is about5:1and prove that short fiber of Ad41could facilitate virus infection host cell. Besides we reveal the infection process of Ad41morphologically including:entry, transportation in cytoplasm, replication and release from the host cell. Our results will provide an important clue and experimental support for the further research on Ad41.