Dependence of the Encapsidation Function of the Adenovirus L1 52/55-Kilodalton Protein on Its Ability To Bind the Packaging Sequence

The packaging of adenovirus (Ad) DNA into virions is dependent upon cis-acting sequences and trans-acting proteins. We studied the involvement of Ad packaging proteins in the serotype specificity of packaging. Both Ad5 and Ad17 IVa2 and L4-22K proteins complemented the growth of Ad5 IVa2 and L4-22K mutant viruses, respectively. In contrast, the Ad5 L1-52/55K protein complemented an Ad5 L1-52/55K mutant virus, but the Ad17 L1-52/55K protein did not. The analysis of chimeric proteins demonstrated that the N-terminal half of the Ad5 L1-52/55K protein mediated this function. Finally, we demonstrate that the L4-33K and L4-22K proteins have distinct functions during infection.The adenovirus (Ad) packaging domain is a cis-acting region located at the left end of the genome that directs viral DNA encapsidation (reviewed in reference 13). The packaging domain consists of seven A repeats, each sharing the consensus motif 5Ј-TTTG-N 8 -CG-3Ј (13). The Ad5 protein IVa2 binds to the CG portion of A repeats (15,17,22). The IVa2 protein is necessary for both Ad virion assembly and viral DNA packaging (23, 24). The IVa2 protein binds to the Ad5 L1-52/55 K protein (8), which also is involved in Ad5 DNA packaging (7, 9). The L1-52/55K protein is recruited to the packaging domain in vivo, although this does not require the IVa2 protein (15,17). It is unclear how this protein is recruited to the packaging domain. The Ad5 L4-22K and L4-33K proteins are splice variants of an Ad late region 4 transcript (11). The L4-33K protein was found to be involved in the early-to-late switch of the infectious cycle (4, 21) and also is necessary for virus assembly (5, 6, 10). The Ad5 L4-22K protein binds to the TTTG portion of A repeats (3,12). Binding of the IVa2 protein is a prerequisite for the specific interaction of the L4-22K protein with DNA (3,12). An Ad5 L4-22K viral mutant displays a late defect in the production of infectious virus (12).Although Ad DNA packaging is largely serotype specific (24), it is possible to pseudopackage the DNA from one Ad serotype into the capsid of a different serotype (14). These results imply that recognition exists between the viral proteins bound to the packaging domain and the capsid of the virus. Here, we provide evidence that the L1-52/55K protein is involved in the serotype specificity of Ad packaging. In contrast, the IVa2 and L4-22K proteins of two different Ad serotypes have interchangeable functions. The amino-terminal half of the L1-52/55K protein mediates this function. Finally, we demonstrate that the Ad5 L4-33K and L4-22K proteins have distinct functions during viral infection.We analyzed the Ad packaging proteins to determine which protein(s) may be implicated in the serotype specificity of DNA packaging. Viral mutants that are unable to express functional IVa2 (pm8002) (23), L1-52/55K (pm8001) (7), or L4-22K (v22K Ϫ ) (12) proteins were used in complementation experiments to determine if these mutants could be rescued when the corresponding proteins of Ad5 or Ad17 were provided in trans. pTG36...

supporting

confidence: 82%

Role for the L1-52/55K Protein in the Serotype Specificity of Adenovirus DNA Packaging

Wohl

Hearing

2008

“…Finally, the region in the L1-52/55K protein that interacts with the IVa2 protein is located within the N-terminal 173 amino acids (20). A comparison between these sequences of the Ad5 and Ad17 L1-52/55K proteins shows that the major differences are located within the N-terminal 60 amino acids of Ad5, which are truncated to 35 amino acids in Ad17.…”

Section: Discussionmentioning

confidence: 99%

Adenovirus Structural Protein IIIa Is Involved in the Serotype Specificity of Viral DNA Packaging

Hearing

2011

The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.

“…2B). These include the only cysteine residue in the polypeptide chain, the two phosphorylation sites (38), part of the IVa2 binding domain (12), part of the region required for interaction with the viral packaging sequence (39), and the region containing residues mutated in the thermosensitive mutant ts369 (16).…”

Section: Processing Of Proteins In Mildly Disrupted Ts1 Particles By mentioning

confidence: 99%

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

Pérez-Berná

Mangel

McGrath

et al. 2014

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...