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Journal of Virology, May 2008, p. 5089-5092, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.00040-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Beverly P. Wohl and Patrick Hearing^*

Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11794

Received 7 January 2008/ Accepted 29 February 2008

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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.

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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 5'-TTTG-N₈-CG-3' (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^–) (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. pTG3602 (2) contains the wild-type Ad5 genome, from which these mutants were derived, and was used as a positive control in all experiments.

N52.E6 cells (20) were transfected with pTG3602-pm8002 (2.5 µg) alone or cotransfected with pTG3602-pm8002 and a pcDNA3-derived (Roche) plasmid (2.5 µg) expressing either the Ad5 IVa2 or the Ad17 IVa2 protein or an empty expression vector (pcDNA3-Ad5-IVa2, pcDNA3-Ad17-IVa2, or pcDNA3, respectively). Virus rescue was measured by a plaque assay in three independent experiments. Plasmid pTG3602-pm8002 alone or cotransfected with pcDNA3 produced no plaques, as expected. In contrast, both the Ad5 and the Ad17 IVa2 proteins were able to complement the growth of the Ad5 IVa2 mutant pm8002 (Table 1). pTG3602-L4 22K^– was transfected alone or cotransfected with a plasmid expressing either the Ad5 L4-22K or the Ad17 L4-22K protein or an empty expression vector (pcDNA3-Ad5-22K, pcDNA3-Ad17-22K, or pcDNA3, respectively). Plasmid pTG3602-L4 22K^– alone or cotransfected with pcDNA3 produced no plaques, as expected. In contrast, both the Ad5 and the Ad17 L4-22K proteins were able to complement the growth of the Ad5 L4-22K mutant (Table 1), although the Ad17 protein was 4-fold less efficient in this assay. Multiple plaques were analyzed for their abilities to be propagated under noncomplementing conditions to determine if recombination took place between the mutant viral genomes and sequences in the expression vectors. Marker rescue assays indicated that none of the viruses isolated from plaques were able to grow under noncomplementing conditions, demonstrating that complementation of loss of the original mutations in the IVa2 or L4 22K gene did not occur.

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TABLE 1. Complementation of Ad5 IVa2, L4-22K, and L4-33Kmutantsa

The Ad5 L4-33K and L4-22K proteins share the N-terminal 105 amino acids and differ only in their C termini (124 unique amino acids for L4-33K and 91 unique amino acids for L4-22K). Complementation experiments were performed to examine if the Ad5 L4-22K and L4-33K proteins could functionally substitute for one another by virtue of their shared sequences. The pTG3602-L4 22K^– and pTG3602-L4 33K^– infectious clones, containing termination codons in the regions unique to the L4-22K and L4-33K proteins, respectively, were transfected alone or cotransfected with expression plasmids that contained cDNA clones corresponding to the Ad5 L4-22K or Ad5 L4-33K protein or an empty expression vector (pcDNA3-Ad5-L4-22K, pcDNA3-Ad5-L4-33K, or pcDNA3, respectively). These results (Table 1) revealed that the expression of each L4 protein could complement the growth of only the corresponding L4 mutant. In complementary experiments, an expression plasmid that contains an Ad5 L4 genomic DNA fragment corresponding to the L4 22K and L4 33K open reading frames was used along with expression plasmids that contain genomic fragments from the L4 22K and L4 33K mutant viruses (pcDNA3-L4-WT, pcDNA3-L4-22K^–, and pcDNA3-L4-33K^–, respectively). Once again, each L4 protein was able to complement the growth of only the corresponding L4 mutant (Table 1). These results demonstrate that the Ad5 L4 22K and L4 33K proteins perform distinct functions within infected cells.

The analysis of complementation of the Ad5 L1-52/55K mutant revealed a different result. When pTG3602-pm8001 was cotransfected with expression plasmids for either the Ad5 L1-52/55K or the Ad17 L1-52/55K protein, only the Ad5 L1-52/55K protein was able to complement the L1-52/55K mutant (Table 2). The results of plaque assays were confirmed by measuring infectious-virus yield, using a fluorescence focus assay (Table 2). Fluorescence focus assays were performed as described previously (18), using a monoclonal antibody against DNA binding protein (19). These results support a role for the L1-52/55K protein in the serotype specificity of Ad DNA packaging. Western blot analysis showed that comparable levels of the Ad5 and Ad17 L1-52/55K proteins were expressed in these experiments (Fig. 1). Western blot analyses were performed using antibodies as described previously (15).

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TABLE 2. Complementation of Ad5 L1 52/55K mutant virusa

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FIG. 1. Western blot analysis of Ad5 and Ad17 L1 52/55K proteins. N52.E6 cells were infected with wild-type Ad5 or Ad17 (200 particles/cell) or mock infected (lanes 1 to 3, respectively) and were untransfected (–) or transfected with pcDNA3, pcDNA3 plus Ad5-L1, or pcDNA3 plus Ad17-L1 (lanes 4 to 8), without (lane 4) or with (lanes 5 to 8) cotransfection of pTG3602-pm8001, as indicated above the lanes. Molecular weight markers are indicated on the left.

The L1-52/55K proteins from Ad5 and Ad17 share considerable amino acid sequence homology throughout most of the protein, with the only major differences found within the first 60 amino acids at the N terminus and within the C-terminal region (Fig. 2A). Three Ad5 and Ad17 L1-52/55K chimeric proteins were constructed. Ad5/17-1 L1 and Ad17/5 L1 were generated to fuse the N-terminal half of the L1-52/55K protein of one serotype to the C-terminal half of the L1-52/55K protein of the alternative serotype (Fig. 2B, Ad5/17-1 and Ad17/5). The site where the Ad5 and Ad17 amino acid sequences were identical was chosen for the junction of the chimeric proteins. The complementation assay revealed that the Ad5/17-1 L1 chimeric protein could rescue the L1-52/55K mutant virus, whereas the Ad17/5 L1 chimera could not (Table 2). A third chimera, containing the N-terminal 60 amino acids of the Ad5 L1-52/55K protein fused to the remainder of the Ad17 L1 52/55K protein, was created (Fig. 2B, Ad5/17-2 L1). This chimeric protein, however, was unable to complement the growth of the L1 52/55K mutant virus (Table 2). Western blot analysis demonstrated that all of the wild-type and chimeric L1 52/55K proteins were produced at comparable levels (Fig. 2C). We conclude that the L1-52/55K protein contributes to the serotype specificity of Ad DNA packaging and that the region involved in this activity is contained within the N-terminal half of this protein, specifically between amino acids 1 and 191 of Ad5.

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FIG. 2. Ad5 and Ad17 L1-52/55K proteins. (A) An alignment (CLUSTAL-N) of the Ad5 and Ad17 L1-52/55K proteins is shown. Asterisks indicate sequence identity, and one- and two-dot symbols indicate sequence similarity. The arrows above the amino acid sequence indicate the sites of the junctions of the Ad5/17 chimeric proteins. (B) Schematic diagram of the Ad5/Ad17 L1-52/55K chimeric proteins. Ad5 sequences are indicated with solid lines; Ad17 sequences are indicated with hatched lines. Amino acid numbers of the chimeric proteins are given above the lines. (C) Western blot analysis of Ad5/17 chimeric proteins. N52.E6 cells were cotransfected with pTG3602-pm8001 and expression vectors for wild-type Ad5 and Ad17 L1-52/55K proteins or Ad5/17 chimeric proteins (lanes 1 to 5). Molecular weight markers are indicated to the left and adjacent to lane 5.

The mechanism of packaging of Ad DNA into the capsid is unknown but likely involves targeting of the packaging domain, and DNA-bound packaging proteins, to an immature empty capsid termed the prohead (reviewed in reference 13). The packaging of Ad DNA of one viral serotype into the capsid of another Ad serotype, pseudopackaging, is inefficient unless the two serotypes are closely related (14, 24). Our results demonstrate that the IVa2 and L4-22K proteins of Ad5 and Ad17 each complement the loss of the respective gene product in the background of Ad5 (Table 1). Thus, these two proteins are interchangeable in the context of Ad pseudopackaging. In contrast, the Ad5 L1-52/55K protein was able to complement an Ad5 L1-52/55K mutant, but the Ad17 L1-52/55K protein was not (Table 2). These results are consistent with a previous report that showed that neither Ad7, Ad12, nor Ad17 wild-type viruses were able to complement pm8001, the Ad5 L1-52/55K viral mutant used in this study (24). This same published report also indicated that the IVa2 protein contributes to the serotype specificity of Ad packaging (24), in contrast to the results obtained in our experiments, where the IVa2 protein of either Ad5 or Ad17 could complement an Ad5 IVa2 mutant. These published experiments utilized an Ad5/7 chimeric virus that contained the Ad5 inverted terminal repeats and the Ad5 packaging domain in an otherwise Ad7 background. The chimeric virus was unable to produce virus on its own but showed evidence of virus growth, as evidenced by a cytopathic effect when the Ad5 IVa2 protein was provided in trans. However, the extent of complementation was not measured directly. Our conclusion may differ with the published report due to differences in the experimental design whereby we assessed the abilities of Ad17 proteins to complement viral mutants in a complete Ad5 background and/or due to differences in the properties of the packaging proteins of Ad7 (24) and Ad17 (this report).

Experiments using Ad5 and Ad17 chimeric L1-52/55K proteins demonstrated that the region responsible for conferring serotype specificity of packaging is located among the first 191 amino acids of the Ad5 L1-52/55K protein (Table 2). Perez-Romero et al. recently reported that the amino-terminal 173 amino acids of the Ad5 L1-52/55K protein are necessary for interaction with the IVa2 protein (16). A model consistent with these published results and our results would be that the Ad5 L1-52/55K protein is able to interact with both the Ad5 and the Ad17 IVa2 proteins, whereas the Ad17 L1 52/55K protein is capable of binding only to the Ad17 IVa2 protein.

Viral mutants of the L1-52/55K protein assemble immature virus particles that either are devoid of Ad DNA or contain minimal DNA sequences from the left end of the genome (7, 9), indicating that the L1-52/55K protein is required for efficient viral DNA encapsidation but not for prohead assembly. We speculate that the L1-52/55K protein may act as a bridge between the viral DNA and the capsid and thus contribute to the serotype specificity of Ad DNA packaging.

Finally, we have shown that the L4-22K and L4-33K proteins perform distinct functions in the Ad lytic cycle (Table 1). Both proteins are involved during the late stages of viral infection in the production of infectious virions (5, 6, 10, 12). Both proteins have been suggested to bind to regulatory sequences in the packaging domain and the Ad major late promoter, indicating that these proteins may have functional redundancy (1, 12). Yet, this clearly is not the case. Future experimentation will be required to resolve the roles that each protein plays in the regulation of Ad late gene expression and infectious-virus production.

 
We thank Gudrin Schiedner and Stefan Kochanek for the N52.E6 cell line and Michael Imperiale for mutants pm8001 and pm8002. We thank Mary Anderson and Ilana Shoshani for excellent technical help and members of our laboratory for informed discussions.

This work was supported by NIH grant AI041636.

 
* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794. Phone: (631) 632-8813. Fax: (631) 632-8891. E-mail: phearing{at}ms.cc.sunysb.edu

Published ahead of print on 12 March 2008.

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Journal of Virology, May 2008, p. 5089-5092, Vol. 82, No. 10
0022-538X/08/$08.00+0     doi:10.1128/JVI.00040-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wohl, B. P. Articles by Hearing, P. Search for Related Content PubMed PubMed Citation Articles by Wohl, B. P. Articles by Hearing, P.