The Adenovirus L4-22K Protein Has Distinct Functions in the Posttranscriptional Regulation of Gene Expression and Encapsidation of the Viral Genome

Abstract

The adenovirus L4-22K protein is multifunctional and critical for different aspects of viral infection. Packaging of the viral genome into an empty capsid absolutely requires the L4-22K protein to bind to packaging sequences in cooperation with other viral proteins. Additionally, the L4-22K protein is important for the temporal switch from the early to late phase of infection by regulating both early and late gene expression. To better understand the molecular mechanisms of these key functions of the L4-22K protein, we focused our studies on the role of conserved pairs of cysteine and histidine residues in the C-terminal region of L4-22K. We found that mutation of the cysteine residues affected the production of infectious progeny virus but did not interfere with the ability of the L4-22K protein to regulate viral gene expression. These results demonstrate that these two functions of L4-22K may be uncoupled. Mutation of the histidine residues resulted in a mutant with a similar phenotype as a virus deficient in the L4-22K protein, where both viral genome packaging and viral gene expression patterns were disrupted. Interestingly, both mutant L4-22K proteins bound to adenovirus packaging sequences, indicating that the paired cysteine and histidine residues do not function as a zinc finger DNA binding motif. Our results reveal that the L4-22K protein controls viral gene expression at the posttranscriptional level and regulates the accumulation of the L4-33K protein, another critical viral regulator, at the level of alternative pre-mRNA splicing.H uman adenoviruses (Ad) consist of a nonenveloped icosahedral capsid with a linear double-stranded viral genome of ϳ36,000 bp. Many questions remain about the basic biology of Ad infection. In particular, virus assembly for complex eukaryotic viruses is relatively poorly understood. Understanding the molecular mechanisms of the viral life cycle is critical for development of strategies for treatment of Ad infections and for the use of Ad as a gene therapy vector. The Ad5 genome contains five early transcription units (E1A, E1B, E2, E3, and E4), which encode ϳ25 proteins that are expressed before viral DNA replication, and a set of delayed mRNAs, which encode proteins IX and IVa2, synthesized at the onset of DNA replication. These early and intermediate transcripts encode proteins with various roles during infection, including transcriptional regulation, viral DNA replication, inhibition of cellular antiviral responses, and inhibition of immune responses (1-4). Following DNA replication, a single major late transcription unit (MLTU) is transcribed and includes five different groups of mRNAs (L1 to L5) that encode capsid structural proteins and proteins that promote virus assembly, direct Ad genome packaging, and serve regulatory functions (2). The Ad major late promoter (MLP) drives transcription from the MLTU regions L1 to L5, producing all late mRNAs by alternative splicing and polyadenylation of a primary transcript. Prior to DNA replication, the MLP is active at low leve...

The Human Adenovirus 5 L4 Promoter Is Activated by Cellular Stress Response Protein p53

“…However, we also found that the expression of some late gene products, particularly L2 pV and L3 pVI, was disproportionately reduced upon p53 depletion in comparison to the L3 hexon and L2 penton. Studies using L4-22K- and L4-33K-deficient viruses have demonstrated that the expression of both L2 pV and L3 pVI is dependent on the expression of L4-22K and L4-33K (13–15) and that an L4P mutant genome, late protein expression from which can be partially restored upon complementation in trans with L4-22K, requires further addition of L4-33K for expression of L3 pVI (16). The lack of L2 pV and L3 pVI expression during p53 depletion is therefore consistent with L4-22K and L4-33K being absent or expressed at low levels, as would be expected if L4P activity was impaired.…”

“…Upon activation of the MLP, L4-22K and L4-33K additionally contribute to the correct expression of the full repertoire of adenovirus late proteins by influencing the splicing of the MLTU pre-mRNA (10–12); L4-22K also cooperates with IVa2 to promote packaging of viral DNA into nascent capsids (7). Both L4-22K- and L4-33K-deficient viruses display defects in late gene expression and efficiency of packaging (13–15), emphasizing the importance of these proteins for efficient replication at multiple levels.…”

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

“…AdV genome packaging starts from the left end of the genome (30) and requires the coordinated interaction of virus proteins IIIa, L1 52/55k, L4 33k, L4 22k, and IVa2 among themselves and with the virus DNA packaging sequence, ⌿ (17,22,26,(33)(34)(35)(36)(37)(38). One of these proteins, L1 52/55k, is present in empty capsids (50 to 100 copies) but must be released upon genome entry as it is absent from the final virion.…”

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

“…Both the full-length and 40-kDa bands disappear and are replaced by higher-molecular-mass species in electrophoresis performed in the absence of ␤-mercaptoethanol, suggesting the formation of disulfide-linked homodimers mediated by the only Cys residue (Cys24) in L1 52/55k (9,22). The 34-kDa band has been proposed to originate by an additional cleavage at the N terminus, because it does not react with antibodies against either residues 9 to 22 (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22) or 402-415 in L1 52/55k, and its electrophoretic mobility is not sensitive to nonreducing conditions, a finding consistent with the absence of Cys24 (9,22).…”

“…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)(11)(12)(13)(14)(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).…”