Study of adenovirus packaging
Abstract
Adenovirus (AdV) assembly is a multistep process that begins with the accumulation of viral structural proteins and DNA in the nucleus of the infected cell. After empty or precursor capsids are assembled from these structural proteins, the recognition and packaging of the viral genome into the empty capsids follows. Specific recognition of viral genome is dependent on the presence of a packaging domain located between 220 and 400 nucleotides from the left end of the genome. The human Ad serotype 5 (HAdV5) packaging domain consists of seven A repeats, designated as A1 to A7. The IVa2 protein is the first factor to bind at the A repeats thus making it essential for genome recognition. Apart from recognition, IVa2 is essential for genome packaging since IVa2 deletion mutants fail to package the genome. IVa2 is also thought to be the DNA packaging ATPase. Despite these identified facts, the complete mechanistic details of AdV assembly and packaging are not known. Attempting to discover other novel proteins involved in AdV assembly and packaging, we found that the viral proteins L4 33K and DNA binding protein (DBP) interact with IVa2. The IVa2, L4 33K and DBP proteins form a complex in virus-infected cells, co-localize even in the absence of other viral proteins, and are located at single sites on viral particles, implying that IVa2, 33K and DBP are possibly co-localized at a unique vertex and together participate in genome packaging. IVa2 and DBP are present in both empty and mature capsids, whereas 33K is present only in empty capsids. As anticipated for proteins involved in genome packaging, the purified IVa2 and 33K formed ring-like oligomers and their oligomers were also detected in purified virus. Using recombinant proteins expressed and purified from soluble fraction of E. coli, ATPase activity of IVa2 was demonstrated in the presence of 33K and the viral genome in an in vitro assay. These results provide compelling evidence for role of 33K as small terminase equivalent for AdV assembly.
Degree
Ph.D.
Advisors
Mittal, Purdue University.
Subject Area
Virology
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