Processing of the L1 52/55k Protein by the Adenovirus Protease: a New Substrate and New Insights into Virion Maturation

Abstract

Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.A denovirus morphogenesis ends with a maturation step comprising proteolytic cleavage of several capsid and core precursor proteins. Without these cleavages, the immature particle lacks infectivity because of its inability to uncoat (1-3). Maturation primes the viral particle for stepwise uncoating by facilitating penton release and by loosening the condensed genome and its attachment to the icosahedral shell (4, 5). Proteolytic processing is carried out by the adenovirus protease (AVP; or L3 23K protein) (6). In human adenovirus type 2 (HAdV-2), AVP recognizes (M/I/ L)XGX-G and (M/I/L)XGG-X sequence motifs (7,8). These sequences are present in the precursor proteins pIIIa, pVI, and pVIII in the icosahedral shell, as well as in the DNA-binding polypeptides pVII, pre-, and the terminal protein. These six precursor proteins have been shown to be substrates for AVP. Another potential substrate, because it contains an AVP consensus sequence motif, is polypeptide L1 52/55k.The L1 52/55k protein in HAdV-2 is 415 residues in length, with an AVP consensus cleavage site at the 351-352 position (LAGT-G). Although the molecular mass of L1 52/55k calculated from its sequence is 47 kDa, the protein was named by its electrophoretic mobility; it moved as a doublet due to two different phosphorylation states (9). L1 52/55k is part of the genome packaging machinery, together with polypeptides IIIa, IVa2, L4 33k, and L4 22k (10-15). An L1 52/55k deletion construct produces only empty capsids (10), and a thermosensitive mutation in the L1 52/55k C-terminal region (ts369; 333-EL-336 to 333-GP-336) causes partial packaging (16). L1 52/55k binds to the viral packaging sequence in vivo and to the putative packaging ATPase IVa2 in vitro (17-19). L1 52/55k contributes to the specificity of packaging, po...

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

“…Polypeptide VI is not icosahedrally ordered, and only weak density for small fragments of VI has been observed in previous structural studies (9,11). Of these two proteins, L1 52/55k suffers the most drastic changes upon maturation, with up to 14 potential cleavages which disrupt its links with capsid and core, leading to its eventual removal from the mature particle (40). Polypeptide VI (250 residues) is cleaved at its N and C termini (33 and 11 residues, respectively) but remains in the mature particle, and its only structural change resulting from maturation detected so far is a lower degree of icosahedral ordering in the hexon-interacting region (8,63,70).…”

“…4A and B). The packaging protein L1 52/55k can form homo-oligomers and links shell (IIIa) to core (VII, dsDNA, and AVP) components (17,37,(40)(41)(42). Polypeptide VI (copy number, 360 [66]) binds to hexon and to dsDNA (67,68), and its C-terminal peptide is a cofactor for the virus protease (65).…”

“…For this reason, it is considered a putative assembly scaffold (39). L1 52/55k can form homo-oligomers and interacts with both shell and core components (17,37,(40)(41)(42). In particular, it binds to polypeptide IIIa and ⌿ in vivo, suggesting that it might act as a tether between genome and capsid shell during encapsidation (17,37).…”

“…L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation (40).…”

Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

“…The product of the 52K gene was also detected in nonnegligible amounts. In HAdV-5, the equivalent protein, L1 52/55K, is removed from the capsid during packaging and maturation (51,52). Therefore, the detection of 52K in LAdV-2 points to the presence of a minor population of immature virus particles (young virions) in the CsCl gradient heavy band.…”