The adenovirus L4-33K protein regulates both late gene expression patterns and viral DNA packaging

Abstract

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.

Fig 1

L4-33K mutant virus. (A) Schematic diagram of the Ad5 L4 region. Coding regions for L4-100K, L4-33K, L4-22K, and pVIII are shown. L4-33K mutant virus 33K⁻ is depicted with a stop codon introduced at amino acid position 138 of L4-33K. (B) Complementation of Ad5-WT and 33K⁻ virus in TetC4-33K cells with (OFF) or without (ON) doxycycline (1 μg/ml) in the culture medium. Infected-cell lysates were harvest at 48 hpi and subsequently titrated on TetC4-33K cells cultured without doxycycline by a plaque assay. (C) Growth curves of Ad5-WT and 33K⁻ virus in A549 cells. Infected A549 cell lysates were harvest at 6, 12, 24, and 48 hpi and subsequently titrated on TetC4-33K cells by a plaque assay.

Fig 2

L4-33K mutant virus infectivity and genome replication. (A and B) Ratios of physical particles to infectious particles of Ad5-WT and 33K⁻ virus were determined in A549 cells (particle/FFU ratio) (A) and TetC4-33K cells (particle/PFU ratio) (B). (C) Viral genome replication in Ad5-WT-, or 33K⁻-infected A549 cells. The genome copy number was determined by quantitative PCR and normalized to the GAPDH value; this value was relative to that of Ad5-WT at 6 hpi.

Fig 3

L4-33K mutant virus gene expression. (A) Northern blot analysis of Ad late gene mRNAs in Ad5-WT- or 33K⁻-infected A549 cells. Total cytoplasmic mRNAs isolated from the infected cells at 12, 24, and 48 hpi were analyzed by Northern blotting using ³²P-labeled probes for each late region. (B) RT-PCR analysis of L4-22K mRNA levels in Ad5-WT- or 33K⁻-infected A549 cells. (C) Western blot analyses of Ad early and late proteins in A549 and TetC4 cells. Representative early (E1A and DBP), intermediate (IVa2), and late (L1 to L5) gene products were analyzed by using whole-cell extracts (WCE) and nuclear extracts (NE) isolated from Ad5-WT- and 33K⁻-infected A549 cells (left) or WCE isolated from Ad5-WT- and 33K⁻-infected TetC4 cells (right) at different times (hours) postinfection (indicated at the top). Protein designations are indicated on the right; tubulin was analyzed as a loading control.

Fig 4

L4-33K mutant virus particle production. (A) CsCl density equilibrium gradient profiles of virus particles produced from Ad5-WT-, Ad5-Ψ-loxP-, or 33K⁻-infected TetC4 (Cre recombinase-positive), 293, or A549 cells. The arrow and arrowhead indicate empty capsids (EC) and mature virions (MV), respectively. Ad5-Ψ-loxP contains loxP sites flanking the PS and thus produces only EC in TetC4 cells due to cleavage of the PS from the genome by Cre recombinase. (B to F) Electron microscopy examination of the MV and EC isolated in panel A.

Fig 5

Protein composition of the empty capsids generated by 33K⁻ virus. (A) Silver staining of protein components of 2 μg of MV and EC isolated from virus particles indicated in Fig. 4A. Protein designations are indicated on the left. M, molecular weight markers. (B) Western blot analysis of protein components of 2 μg of MV and EC isolated from virus particles indicated in panel A. Protein designations are indicated on the right. Virus particles used in these analyses were banded twice by CsCl equilibrium centrifugation. Two independent batches of 33K⁻ EC samples from A549 cells were prepared and included in the analysis (adjacent lanes).

Fig 6

ChIP assays for packaging protein binding to the PS in vivo. A549 cells were infected with Ad5-WT or 33K⁻, and L4-22K-, L4-33K-, IVa2-, L1-52/55K-, and IIIa-specific antibodies were used for ChIP to quantify binding to the PS. The results are presented as normalized fold enrichment by dividing the copy number of PS pulled down specifically by the antibody-antigen complex to the copy number of the E4-ORF6 fragment that was pulled down nonspecifically by the antibody. The fold enrichment value of each antibody was normalized to that of the preimmune serum negative control.