Formation of a Multiple Protein Complex on the Adenovirus Packaging Sequence by the 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, ⌬IVa2, 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.

mentioning

confidence: 99%

Characterization of Empty Adenovirus Particles Assembled in the Absence of a Functional Adenovirus IVa2 Protein

Ostapchuk

Almond

Hearing

2011

“…Three Ad proteins, IVa2, L1-52/55K, and L4-22K, are known to associate with the packaging domain in vitro and/or in vivo, and each is required for viral DNA packaging (5, 7, 9, 16, 19-21, 26, 29-32). The IVa2 protein binds to the CG motif and the L4-22K protein binds the TTTG motif of the packaging A repeats in vitro and in vivo (5,19,26,29). The L1-52/55K protein has only been found to be associated with the packaging domain in vivo, and the L1-52/55K protein associates with IVa2 in vitro (8,19,21).…”

mentioning

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.

“…Viruses containing mutations in the 52/55-kDa gene are either partially or fully defective for encapsidation of the genome, although they assemble capsids in a manner similar to that of the wild-type virus (15,18). On the basis of this interaction and knowing that the IVa2 protein is a sequence-specific DNA binding protein in its role as an activator of the viral major late promoter (24), we asked whether IVa2 binds to the packaging sequence in vitro and determined that it does indeed bind to the A-repeat consensus sequence (40) and forms multimeric structures on the entire packaging sequence (35). Chromatin immunoprecipitation experiments have demonstrated interactions of the IVa2 and 52/55-kDa proteins with the packaging sequence in infected cells (29,31).…”

mentioning

confidence: 99%

Presence of the Adenovirus IVa2 Protein at a Single Vertex of the Mature Virion

Christensen¹,

Byrd²,

Walker

et al. 2008

Self Cite

Assembly of adenovirus particles is thought to be similar to that of bacteriophages, in which the doublestranded 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.