Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site

2012

A variety of cellular and viral processes are coordinately regulated during adenovirus (Ad) infection to achieve optimal virus production. The Ad late gene product L4-22K has been associated with disparate activities during infection, including the regulation of late gene expression, viral DNA packaging, and infectious virus production. We generated and characterized two L4-22K mutant viruses to further explore L4-22K functions during viral infection. Our results show that L4-22K is indeed important for temporal control of viral gene expression not only because it activates late gene expression but also because it suppresses early gene expression. We also show that the L4-22K protein binds to viral packaging sequences in vivo and is essential to recruit two other packaging proteins, IVa2 and L1-52/55K, to this region. The elimination of L4-22K gave rise to the production of only empty virus capsids and not mature virions, which confirms that the L4-22K protein is required for Ad genome packaging. Finally, L4-22K contributes to adenovirus-induced cell death by regulating the expression of the adenovirus death protein. Thus, the adenovirus L4-22K protein is multifunctional and an integral component of crucial aspects of infection.

Section: Discussionsupporting

confidence: 87%

The Adenovirus L4-22K Protein Is Multifunctional and Is an Integral Component of Crucial Aspects of Infection

Orozco

2012

“…The conserved histidine residues also were not required for DNA binding, indicating that it is highly unlikely that the cysteine and histidine residues are part of a zinc finger motif involved in binding of L4-22K to the packaging domain. A role for L4-22K as a transcription factor stimulating major late transcription through direct binding to DNA sequences located downstream of the MLP start site was previously proposed (7,11,13). We cannot rule out the possibility that the conserved cysteine and histidine residues are part of a DNA binding motif necessary for L4-22K to bind to the DE and activate the MLP.…”

Section: Discussionmentioning

confidence: 81%

“…The specific activation of the MLP requires binding of Ad-infected cell-specific transcription factor complexes to the downstream element (DE) of the MLP (6). The Ad L4-22K protein is thought to be the minimal factor required for MLP activation, but additional components, such as IVa2, are required to obtain the maximum activation observed at late times of infection (7). Following DNA replication, the MLP is fully activated, and transcription continues to the L4 and L5 regions.…”

mentioning

confidence: 99%

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

Guimet

2013

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...

“…At this stage, the L4-22K protein is transcribed from a novel, internal L4 promoter (L4P) embedded in the open reading frame of L4-100K (4). L4-22K activates the expression of the full panel of Ad late genes at the transcriptional level by binding to the downstream element of the major late promoter (MLP) and at the posttranscriptional level by an unknown mechanism (5)(6)(7). At the same time, L4-22K also suppresses early gene expression, presumably to redistribute the cellular transcriptional/translational machinery from early genes to late genes in order to maximize progeny virus production (5).…”

mentioning

confidence: 99%

The Adenovirus L4-33K Protein Regulates both Late Gene Expression Patterns and Viral DNA Packaging

Guimet

2013

The adenovirus (Ad) L4-33K protein has been linked to disparate functions during infection. L4-33K is a virus-encoded alternative RNA splicing factor which activates splicing of viral late gene transcripts that contain weak 3= splice sites. Additionally, L4-33K has been indicated to play a role in adenovirus assembly. We generated and characterized an Ad5 L4-33K mutant virus to further explore its function(s) during infection. Infectivity, viral genome replication, and most viral gene expression of the L4-33K mutant virus are comparable to those of the wild-type virus, except for a prominent decrease in the levels of the late proteins IIIa and pVI. The L4-33K mutant virus produces only empty capsids, indicating a defect in viral DNA packaging. We demonstrate that L4-33K does not preferentially bind to viral packaging sequences in vivo, and mutation of L4-33K does not interfere with the binding of the known viral packaging proteins IVa2, L4-22K, L1-52/55K, and IIIa to the packaging sequences in vivo.Collectively, these results demonstrate that the phenotype of an Ad5 L4-33K mutant virus is complex. The L4-33K protein regulates the accumulation of selective Ad late gene mRNAs and is involved in the proper transition of gene expression during the late phase of infection. The L4-33K protein also plays a role in adenovirus morphogenesis by promoting the packaging of the viral genome into the empty capsid. These results demonstrate the multifunctional nature of the L4-33K protein and its involvement in several different and critical aspects of viral infection.