Activation of adenoviral gene expression by protein IX is not required for efficient virus replication

Abstract

The adenovirus (Ad) protein IX (pIX) is a minor component of the Ad capsid and is in part responsible for virion stability; virions lacking pIX are heat labile and lose their infectivity if the DNA content is greater than approximately 35 kb. More recently, pIX has been identified as a transcriptional activator and, in transient-transfection assays, was shown to enhance expression from the E1A, E4, and major late Ad promoters by as much as 70-fold. In this study, we examined the role of pIX's ability to activate transcription during Ad replication. In transient-transfection assays, pIX had a minimal effect on expression from the E1A promoter, increasing expression by only 1.4-fold. We used helper-dependent Ad vectors, which had all Ad protein coding sequences deleted with the exception of E1A and which had capsids that either contained or lacked pIX, to show that pIX derived from decapsidation of the infecting virion does not influence expression of E1A. Similarly, expression of pIX from the Ad genome did not alter the expression levels of E1A. Viruses that had pIX deleted showed a threefold reduction in virus yield and expression of late genes compared to those of a similar virus which encoded pIX. This phenotype could not be rescued by growing the virus in cells which constitutively express pIX. Our results indicate that, although pIX can affect transcription from a variety of viral promoters, it does not appear to play a significant role in activation of Ad promoters during normal Ad replication.

FIG. 1.

Simplified transcription map of the Ad5 genome. Also shown is an enlargement of the left end of the Ad genome and the regions utilized or deleted in the various constructs used in this study. ITR, inverted terminal repeat.

FIG. 2.

Transcriptional enhancement mediated by pIX. A549 cells were cotransfected with a pIX expression plasmid and a reporter plasmid containing the luciferase gene under the control of the E1A promoter. Twenty-four hours later, crude protein extracts were prepared and assayed for luciferase activity. The data have been normalized for transfection efficiency relative to a control MCMV-lacZ expression cassette included in the transfection. Shown is a data set representative of at least two independent transfections.

FIG. 3.

Expression of E1A from hdAd. (A) Structure of hdAdE1A, which encodes a lacZ expression cassette and expresses the E1A gene under the control of its endogenous promoter and encodes no other Ad proteins. (B) A549 cells were infected with E1A-expressing hdAd with virions that contained pIX (hdAdE1ApIX⁺) or lacked pIX (hdAdE1ApIX⁻). These hdAd vectors do not encode pIX; the pIX present in the viral capsid is provided by the helper virus during vector propagation with a helper virus that encodes or does not encode pIX. At various times postinfection (p.i.), protein samples were collected, separated by SDS-PAGE, and analyzed by immunoblotting with an E1A- or tubulin-specific antibody. Due to multiple splicing events and posttranscriptional modification, proteins derived from the E1A region range in size from ∼25 to 50 kDa. Numbers at left show molecular masses in kilodaltons. HPRT, hypoxanthine phosphoribosyltransferase.

FIG. 4.

Growth of Ad vector lacking pIX. A549 cells were infected at an MOI of 1 with AdRP2233 (pIX⁺), AdRP2234 (pIX⁻), or AdRP2234 that had been grown on a pIX-complementing cell line for virions which contained pIX (AdRP2234pIX⁺). At various times postinfection, the medium and cells were collected and their titers were determined on 293 cells.

FIG. 5.

Expression of late protein from Ad vector with pIX deleted. A549 cells were infected at an MOI of 10 with AdRP2233 or AdRP2234. At various times postinfection (in hours, shown above the gels), the medium was removed and the cells were lysed with SDS-PAGE loading buffer. The protein samples were separated by SDS-10% PAGE, transferred to a PVDF membrane, and immunoblotted with antibody to Ad5 fiber or tubulin. Numbers at left are molecular masses in kilodaltons.

FIG. 6.

Growth of Ad vector with pIX deleted in 293pIXc4 cells. (A) The level of pIX expression in 293pIXc4 cells was verified by Western blot analysis with 293 cells infected with Ad at various MOIs as a standard. (B) 293pIXc4 cells were infected at an MOI of 1 with AdRP2233 or AdRP2234, the medium and cells were harvested 24 h later, and their titers were determined on 293 cells. (C) Protein samples were prepared in duplicate from cells infected with the indicated virus and analyzed by immunoblotting with fiber- or tubulin-specific antibody.

FIG. 7.

Growth of Ad vector with pIX deleted in 293 cells. (A) 293 cells were infected at an MOI of 1 with AdRP2233 or AdRP2234, the medium and cells were harvested 24 h later, and their titers were determined on 293 cells. (B) Protein samples were prepared in duplicate from cells infected with the indicated virus and analyzed by immunoblotting with fiber- or tubulin-specific antibody. (C) RNA was isolated from 293 or A549 cells and subjected to RT-PCR analysis with synthetic oligonucleotides specific for pIX. The resulting samples were separated on an 0.8% agarose gel. As a control, we performed RT-PCR analysis for β-actin.