Analysis of the interaction of the adenovirus L1 52/55-kilodalton and IVa2 proteins with the packaging sequence in vivo and in vitro

We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.     

Dependence of the encapsidation function of the adenovirus L1 52/55-kilodalton protein on its ability to bind the packaging sequence

The adenovirus IVa2 and L1 52/55-kDa proteins are involved in the assembly of new virus particles. Both proteins bind to the packaging sequence of the viral chromosome, and the lack of expression of either protein results in no virus progeny: the absence of the L1 52/55-kDa protein leads to formation of only empty capsids, and the absence of the IVa2 protein results in no capsid assembly. Furthermore, the IVa2 and L1 52/55-kDa proteins interact with each other during adenovirus infection. However, what is not yet clear is when and how this interaction occurs during the course of the viral infection. We defined the domains of the L1 52/55-kDa protein required for interaction with the IVa2 protein, DNA binding, and virus replication by constructing L1 52/55-kDa protein truncations. We found that the N-terminal 173 amino acids of the L1 52/55-kDa protein are essential for interaction with the IVa2 protein. However, for both DNA binding and complementation of the pm8001 mutant virus, which does not express the L1 52/55-kDa protein, the amino-terminal 331 amino acids of the L1 52/55-kDa protein are necessary. These results suggest that the production of infectious virus particles depends on the ability of the L1 52/55-kDa protein to bind to DNA.     

Role for the adenovirus IVa2 protein in packaging of viral DNA

Although it has been demonstrated that the adenovirus IVa2 protein binds to the packaging domains on the viral chromosome and interacts with the viral L1 52/55-kDa protein, which is required for viral DNA packaging, there has been no direct evidence demonstrating that the IVa2 protein is involved in DNA packaging. To understand in greater detail the DNA packaging mechanisms of adenovirus, we have asked whether DNA packaging is serotype or subgroup specific. We found that Ad7 (subgroup B), Ad12 (subgroup A), and Ad17 (subgroup D) cannot complement the defect of an Ad5 (subgroup C) mutant, pm8001, which does not package its DNA due to a mutation in the L1 52/55-kDa gene. This indicates that the DNA packaging systems of different serotypes cannot interact productively with Ad5 DNA. Based on this, a chimeric virus containing the Ad7 genome except for the inverted terminal repeats and packaging sequence from Ad5 was constructed. This chimeric virus replicates its DNA and synthesizes Ad7 proteins, but it cannot package its DNA in 293 cells or 293 cells expressing the Ad5 L1 52/55-kDa protein. However, this chimeric virus packages its DNA in 293 cells expressing the Ad5 IVa2 protein. These results indicate that the IVa2 protein plays a role in viral DNA packaging and that its function is serotype specific. Since this chimeric virus cannot package its own DNA, but produces all the components for packaging Ad7 DNA, it may be a more suitable helper virus for the growth of Ad7 gutted vectors for gene transfer.     

Requirement of the adenovirus IVa2 protein for virus assembly

The adenovirus L1 52/55-kDa protein is required for viral DNA packaging and interacts with the viral IVa2 protein, which binds to the viral packaging sequence. Previous reports suggest that the IVa2 protein plays a role in viral DNA packaging and that this function of the IVa2 protein is serotype specific. To further examine the function of the IVa2 protein in viral DNA packaging, a mutant virus that does not express the IVa2 protein was constructed by introducing two stop codons at the beginning of the IVa2 open reading frame in a full-length bacterial clone of adenovirus type 5. The mutant virus, pm8002, was defective for growth in 293 cells, although it replicated its DNA and produced early and late viral proteins. Electron microscopic and gradient analyses revealed that the mutant virus did not assemble any viral particles in 293 cells. In 293-IVa2 cells, which express the IVa2 protein, infectious viruses were produced, although the titer of the mutant virus was lower than that of the wild-type virus, indicating that these cells may not fully complement the mutation. The mutant viral particles produced in 293-IVa2 cells were heterogeneous in size and shape, less stable, and did not traffic efficiently to the nucleus. Marker rescue experiments with a wild-type IVa2 DNA fragment confirmed that the only mutations present in pm8002 were in the IVa2 gene. The results indicate that the IVa2 protein is required for adenovirus assembly and suggest that virus particles may be assembled around the DNA rather than DNA being packaged into preformed capsids.     

Functional interaction of the adenovirus IVa2 protein with adenovirus type 5 packaging sequences

Adenovirus type 5 (Ad5) DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent on the cis-acting packaging domain located between nucleotides 230 and 380. Seven AT-rich repeats that direct packaging have been identified within this domain. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Both cellular and viral proteins that interact with adenovirus packaging elements in vitro have been identified. In this study, we characterized a group of recombinant viruses that carry site-specific point mutations within a minimal packaging domain. The mutants were analyzed for growth properties in vivo and for the ability to bind cellular and viral proteins in vitro. Our results are consistent with a requirement of the viral IVa2 protein for DNA packaging via a direct interaction with packaging sequences. Our results also indicate that higher-order IVa2-containing complexes that form on adjacent packaging repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Chromatin immunoprecipitation was used to study proteins that bind directly to the packaging sequences. These results demonstrate site-specific interaction of the viral IVa2 and L1 52/55K proteins with the Ad5 packaging domain in vivo. These results confirm and extend those previously reported and provide a framework on which to model the adenovirus assembly process.     

Presence of the adenovirus IVa2 protein at a single vertex of the mature virion

Assembly of adenovirus particles is thought to be similar to that of bacteriophages, in which the double-stranded DNA genome is inserted into a preformed empty capsid. Previous studies from our and other laboratories have implicated the viral IVa2 protein as a key component of the encapsidation process. IVa2 binds to the packaging sequence on the viral chromosome in a sequence-specific manner, alone and in conjunction with the viral L4 22K protein. In addition, it interacts with the viral L1 52/55-kDa protein, which is required for DNA packaging. Finally, a mutant virus that does not produce IVa2 is unable to produce any capsids. Therefore, it has been proposed that IVa2 nucleates capsid assembly. A prediction of such a model is that the IVa2 protein would be found at a unique vertex of the mature virion. In this study, the location of IVa2 in the virion has been analyzed using immunogold staining and electron microscopy, and the copy number of IVa2 in virions was determined using three independent methods, quantitative mass spectrometry, metabolic labeling, and Western blotting. The results indicate that it resides at a unique vertex and that there are approximately six to eight IVa2 molecules in each particle. These findings support the hypothesis that the IVa2 protein plays multiple roles in the viral assembly process.     

Adenovirus structural protein IIIa is involved in the serotype specificity of viral DNA packaging

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.     

Role for the L1-52/55K protein in the serotype specificity of adenovirus DNA packaging

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.     

Ternary complex formation on the adenovirus packaging sequence by the IVa2 and L4 22-kilodalton proteins

Assembly of infectious adenovirus particles requires seven functionally redundant elements at the left end of the genome, termed A repeats, that direct packaging of the DNA. Previous studies revealed that the viral IVa2 protein alone interacts with specific sequences in the A repeats but that additional IVa2-containing complexes observed during infection require the viral L4 22-kDa protein. In this report, we purified a recombinant form of the 22-kDa protein to characterize its DNA binding properties. In electrophoretic mobility shift assay analyses, the 22-kDa protein alone did not interact with the A repeats but it did form complexes on them in the presence of the IVa2 protein. These complexes were identical to those seen in extracts from infected cells and had the same DNA sequence dependence. Furthermore, we provide data that the 22-kDa protein enhances binding of the IVa2 protein to the A repeats and that multiple binding sites in the packaging sequence augment this activity. These data support a cooperative role of the IVa2 and 22-kDa proteins in packaging and assembly.     

Characterization of Empty adenovirus particles assembled in the absence of a functional adenovirus IVa2 protein

The molecular mechanism for packaging of the adenovirus (Ad) genome into the capsid is likely similar to that of DNA bacteriophages and herpesviruses-the insertion of viral DNA through a portal structure into a preformed prohead driven by an ATP-hydrolyzing molecular machine. It is speculated that the IVa2 protein of adenovirus is the ATPase providing the power stroke of the packaging machinery. Purified IVa2 binds ATP in vitro and, along with a second Ad protein, the L4 22-kilodalton protein (L4-22K), binds specifically to sequences in the Ad genome that are essential for packaging. The efficiency of binding of these proteins in vitro was correlated with the efficiency of packaging in vivo. By utilizing a virus unable to express IVa2, pm8002, it was reported that IVa2 plays a role in assembly of the empty virion. We wanted to address the question of whether the ATP binding, and hence the putative ATPase activity, of IVa2 was required for its role in virus assembly. Our results show that ATPase activity was not required for the assembly of empty virus particles. In addition, we present evidence that particles were assembled in the absence of IVa2 by using two viruses null for IVa2-a deletion mutant virus, ΔIVa2, and the previously described mutant virus, pm8002. Empty virus particles produced by these IVa2 mutant viruses did not contain detectable viral DNA. We conclude that the major role of IVa2 is in viral DNA packaging. A characterization of the empty particles obtained from the IVa2 mutant viruses compared to wild-type empty particles is presented.     

Interaction of the adenoviral IVa2 protein with a truncated viral DNA packaging sequence

Adenoviral (Ad) infection typically poses little health risk for immunosufficient individuals. However, for immunocompromised individuals, such as AIDS patients and organ transplant recipients, especially pediatric heart transplant recipients, Ad infection is common and can be lethal. Ad DNA packaging is the process whereby the Ad genome becomes encapsulated by the viral capsid. Specific packaging is dependent upon the packaging sequence (PS), which is composed of seven repeated elements called A repeats. The Ad protein, IVa2, which is required for viral DNA packaging, has been shown to bind specifically to synthetic DNA probes containing A repeats I and II, however, the molecular details of this interaction have not been investigated. In this work we have studied the binding of a truncated form of the IVa2 protein, that has previously been shown to be sufficient for virus viability, to a DNA probe containing A repeats I and II. We find that the IVa2 protein exists as a monomer in solution, and that a single IVa2 monomer binds to this DNA with high affinity (K_d <  10 nM), and moderate specificity, and that the trIVa2 protein interacts in a fundamentally different way with DNA containing A repeats than it does with non-specific DNA. We also find that at elevated IVa2 concentrations, additional binding, beyond the singly ligated complex, is observed. When this reaction is modeled as representing the binding of a second IVa2 monomer to the singly ligated complex, the K_d is 1.4 ± 0.7 µM, implying a large degree of negative cooperativity exists for placing two IVa2 monomers on a DNA with adjacent A repeats.     

Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.     

Adenovirus L1 52- and 55-kilodalton proteins are present within assembling virions and colocalize with nuclear structures distinct from replication centers.

Analysis of a temperature-sensitive mutant, Ad5ts369, had indicated that the adenovirus L1 52- and 55-kDa proteins (52/55-kDa proteins) are required for the assembly of infectious virions. By using monoclonal antibodies directed against bacterially produced L1 52-kDa protein, the L1 52/55-kDa proteins were found to be differentially phosphorylated forms of a single 48-kDa polypeptide. Both phosphoforms were shown to be present within all suspected virus assembly intermediates (empty capsids, 50 to 100 molecules; young virions, 1 to 2 molecules) but not within mature virions. The mobilities of these proteins in polyacrylamide gels were affected by reducing agents, indicating that the 52/55-kDa proteins may exist as homodimers within the cell and within assembling particles. Immunofluorescence analysis revealed that the 52/55-kDa proteins localize to regions within the infected nucleus that are distinct from viral DNA replication centers, indicating that replication and assembly of viral components likely occur in separate nuclear compartments. Immunoelectron microscopic studies determined that the 52/55-kDa proteins are found in close association with structures that appear to contain assembling virions. These results are consistent with an active but transient role for the L1 products in assembly of the adenovirus particle, perhaps as scaffolding proteins.     

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

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.     

Formation of a multiple protein complex on the adenovirus packaging sequence by the IVa2 protein

During adenovirus virion assembly, the packaging sequence mediates the encapsidation of the viral genome. This sequence is composed of seven functional units, termed A repeats. Recent evidence suggests that the adenovirus IVa2 protein binds the packaging sequence and is involved in packaging of the genome. Study of the IVa2-packaging sequence interaction has been hindered by difficulty in purifying the protein produced in virus-infected cells or by recombinant techniques. We report the first purification of a recombinant untagged version of the adenovirus IVa2 protein and characterize its binding to the packaging sequence in vitro. Our data indicate that there is more than one IVa2 binding site within the packaging sequence and that IVa2 binding to DNA requires the A-repeat consensus, 5'-TTTG-(N(8))-CG-3'. Furthermore, we present evidence that IVa2 forms a multimeric complex on the packaging sequence. These data support a model in which adenovirus DNA packaging occurs via the formation of a IVa2 multiprotein complex on the packaging sequence.