Adenovirus late-phase infection is controlled by a novel L4 promoter

Abstract

During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.

FIG. 1.

(A) Ad5 transcription map showing immediate-early (light gray), early (black), intermediate (white), and major late transcription units (MLTU; dark gray arrows), which are expressed during infection in a temporal cascade (bottom). All transcription units except IX and IVa2 produce multiple mRNAs and protein products by alternative RNA processing. MLTU mRNAs each comprise the three exons of the tripartite leader (TPL) spliced to 1 of ∼15 possible acceptor sites within regions L1 to L5. (B) Organization of the L4 region of the MLTU showing L4-100K, L4-22K, L4-33K, and pVIII ORFs, with 3′ splice sites for their expression from the major late promoter. 22K and L4-33K share the same N-terminal sequence but have distinct C termini.

FIG. 2.

Differences in control of L4-22K, L4-33K, and L4-100K expression. (A) Schematic representing the method for generating stable L4 protein cell lines. Target cells express GFP under doxycycline control, and this gene then is replaced with L4 sequences by Cre-mediated recombination with a promoterless shuttle plasmid. (B) 100/22/33KFLAG cells containing the L4 region linked to a tetracycline-regulated promoter or parental 293TETOFF cells were grown in the presence (+) or absence (−) of doxycycline for 3 days with daily medium changes. L4-100K and L4-33KFLAG in total cell lysates were detected by Western blot analysis. (C) 22/33KFLAG cells (a to d) or parental 293TETOFF cells (e to h) were grown in the presence (a, b, e, and f) or absence (c, d, g, and h) of doxycycline for 3 days with daily medium changes. Cells were fixed and stained for FLAG-tagged 33K (red) and for nuclear DNA (DAPI; blue), and images were collected sequentially using a Leica SP2 confocal microscope to avoid cross-talk between the fluors. FLAG and DAPI images were overlaid using Leica software (b, d, f, and h); scale bar, 20 μm. (D) Schematic representation of the Ad5 sequences present in pShuttle plasmids. Brown, MLTU exon sequences (rightward transcription); blue, E2A exon (leftward transcription); pale brown, L4 ORFs as indicated. (E) Transient expression from promoterless L4 shuttle plasmids. 293TETOFF cells were transfected with pShuttle100/22/33KFLAG (a and b), pShuttle22/33KFLAG (c and d), or pShuttle22/33KFLAG ORF (e and f), or they were mock transfected (g and h). Cells were fixed and stained 48 h later and were imaged as described for panel C; scale bar, 20 μm. (F) 293TETOFF cells were mock transfected or transfected with L4-22K⁻ genome together with either pShuttle100/22/33KFLAG or pShuttle22/33KFLAG. 33KFLAG protein in total cell lysates was detected by Western blot analysis. Protein molecular mass markers migrated to the positions shown on the left of panels B and F (in kDa).

FIG. 3.

Defining sequences important for L4 promoter activity. (A) A schematic representation of the L4 sequences included in luciferase reporter constructs analyzed in panels B to D. (B and C) 293 cells were mock transfected or transfected with luciferase reporter constructs containing various lengths of L4 sequence as the promoter. Firefly luciferase activity, corrected for transfection efficiency using β-galactosidase expression from an independent control plasmid, is expressed as the fold difference from the activity of the promoterless reporter plasmid (pGL3Basic) (B) or as the percentage of the activity of the full L4 promoter, construct F (C). (D) HeLa cells (black bars) or 293 cells (white bars) were transfected with construct D plus either empty vector, E1A expression plasmid, or the Ad5 WT genome (wt gen). Firefly luciferase activity, corrected as described for panels B and C, is expressed as the fold difference from the activity of construct D in the presence of empty vector, which was set as 1. Each panel shows the mean values from biological triplicates within a single experiment (error bars indicate standard deviations) and is representative of at least three independent experiments.

FIG. 4.

L4-22K expression from L4 promoter is functionally significant. (A to D) 293 cells were mock transfected or transfected with L4-22K⁻ genome alone or with either pA-22KFLAG (A and C) or pA-22/33KFLAG (B and D). Immunoprecipitated FLAG-tagged 22K (A) or 33K (B), or Ad5 late proteins in total cell lysates (C and D), were detected by Western blot analysis. (E) Ad5 wild-type (Wt), L4-22K⁻, or L4P⁻ genomes were transfected into 293 cells with combinations of expression plasmids for individual L4 proteins 100K, 22K, and 33K as indicated. Total cell extracts were analyzed as described for panels A and B for specific viral proteins indicated to the right of the panel. Where a blot with a given antibody is shown in segments, these all were taken from the same exposure of the same blot, with lanes rearranged for clarity of explanation. The positions to which proteins of known molecular mass migrated are indicated for all panels (in kDa).

FIG. 5.

Activation of L4P by Ad5 genome. (A) 293 cells were mock transfected or transfected with pA-22KFLAG together with undigested Ad5 WT genome (wt gen.), Ad5 WT genome digested with either BglII/HindIII (digest 1) or HaeII (digest 2), or an E1A expression plasmid, and 22KFLAG was detected as described for Fig. 4. (B) 293 cells were transfected with L4 promoter luciferase reporter construct D (Fig. 3A), with or without L4-22K⁻ genome and/or treatment with 10 mM hydroxyurea (HU) from 5 h posttransfection. Luciferase expression, corrected for transfection efficiency, is expressed as the fold increase above the activity of core promoter alone, set as 1. Error bars show the standard deviations from three replicate determinations. The graph is representative of two experiments. (C) As described for panel A, except using digested salmon sperm DNA as an L4P activator in comparison with intact Ad5 genome. (D) 293 cells were mock treated or pretreated with 3 mM caffeine for 3 h and then were mock transfected or transfected with pA25887-22KFLAG together with intact Ad5 WT genome or digest 1. From 5 h posttransfection, cells were maintained in DMEM or DMEM supplemented with 3 mM caffeine (Caf) or 10 mM hydroxyurea (HU).

FIG. 6.

Viral proteins activate the L4 promoter. (A, C, and D) 293 cells were mock transfected or transfected with pA-22KFLAG or pA-22/33KFLAG, together with viral protein expression plasmids as indicated, or with WT Ad5 genome. Immunoprecipitated 22KFLAG protein or proteins from unfractionated cell lysates were detected by Western blot analysis as indicated at the right of each panel. The positions of molecular mass marker proteins are shown on the left (in kDa). (B) 293 cells were mock transfected or transfected with L4 promoter luciferase reporter construct D or F (Fig. 3A), with or without IVa2 expression plasmid. Firefly luciferase activity, corrected for transfection efficiency, is expressed as the fold difference from the activity of the relevant promoter reporter alone, which was set as 1. Error bars show the standard deviations from three replicate determinations. (E) As described for panel B, except that cells were transfected with L4 promoter reporter construct D, with or without either E4-Orf3 or E4-Orf6 expression plasmid. The graphs are representative of two experiments.