The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription

Abstract

A characteristic feature of the infectious cycles of viruses with DNA genomes is DNA synthesis-dependent activation of transcription of viral late genes. In the case of human adenoviruses, such as adenovirus type 5 (Ad5), replication of the viral genome initiates a two-step transcriptional cascade. Transcription of the viral IVa 2 gene is first activated as a result of viral DNA synthesis-dependent titration of a cellular transcriptional repressor that binds to the IVa 2 promoter (10,27,36). Synthesis of the IVa 2 protein in infected cells then leads to maximally efficient transcription from the major late (ML) promoter, which controls expression of the coding sequences for all but one of the viral structural proteins (58). Entry into the late phase is accompanied by several changes in ML transcription. During the early phase, the ML and other early promoters are utilized with similar efficiencies (57), but ML transcription terminates at multiple sites within a large region in the middle of the transcription unit (1, 2, 28, 57). In contrast, termination occurs close to the right end of the viral genome during the late phase of infection (17). The difference in how much of the ML unit is transcribed, in conjunction with differences in the posttranscriptional processing of ML premRNAs, results in production of only the L1 52/55-kDa protein early in infection but at least 15 ML mRNAs late in infection (15,58). It has also been reported that the processivity of ML transcription beyond approximately position ϩ1,000 increases late in infection (33). Finally, the efficiency of ML transcription increases by a factor of 20 to 30 once viral DNA synthesis has commenced (57).The basal ML promoter comprises a typical TATA sequence, an initiator, GC-rich sequences near the initiator, and binding sites for the cellular proteins USF and CBF located upstream of the TATA sequence (8,11,42,48,50,51,55). Late phase-specific stimulation of ML transcription in vitro and in infected cells requires additional, intragenic sequences, termed DE1 (positions ϩ86 to ϩ96) and DE2 (positions ϩ101 to ϩ116) (29, 34, 41). These DE sequences are recognized by proteins present only in extracts of Ad5-infected cells harvested during the late phase of infection (29,34,44). Previous biochemical studies identified DEF-B, which binds to the DE2 sequence shown in Fig. 1, as a dimer of the IVa 2 protein (38,60). This interaction was shown to stimulate ML transcription in transient-expression assays (60). The DE1 and DE2a sequences of the ML promoter ( Fig. 1) are recognized by a second infected-cell-specific protein, termed DEF-A (30, 43). Initial efforts to purify DEF-A were not successful, although it was reported that the IVa 2 protein is also a component of this . In addition to binding to the ML promoter, the IVa 2 protein recognizes repeated, redundant sequences termed A repeats (20,21,56) within the viral packaging signal (65) and is required for packaging of the viral genome during assembly (46,66,67). This protein is a component of infected-cell-...

Adenovirus Serotype 5 L4-22K and L4-33K Proteins Have Distinct Functions in Regulating Late Gene Expression

“…The importance of these factors is most apparent in the absence of activation via upstream promoter elements (32). DEF-A is a heterodimer of IVa2 and another protein, and there is published evidence for this protein being either L4-33K or L4-22K (2,31). Our data do not bear directly on this issue, since no studies of protein interactions with the DEs were performed.…”

“…L4-22K has also been shown to bind, with IVa2, to the downstream elements (downstream element 1 [DE1] and DE2) of the MLP (31), which have been shown previously to bind IVa2 as both a homodimer (DEF-B) (25,40) and in complex (DEF-A) with an unidentified viral protein (25,28), suggesting that L4-22K, in conjunction with IVa2, may play a role in activating the MLP (31). However, DEF-A has also been shown to contain L4-33K, and IVa2 plus L4-33K activated a MLP reporter (2). Thus, it is uncertain what the relative contributions of these two L4 proteins are to regulation of the MLP.…”

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k

“…For Western blotting, proteins resolved by SDS-PAGE were transferred to a nitrocellulose membrane and probed with the required antibodies. The following antibodies were used: mouse monoclonal anti-V (52), rabbit anti-VI serum (53), rabbit anti-L1 52/55k serum (36), and mouse anti-IVa2 hybridoma supernatants (55). Primary antibodies were detected with the appropriate horseradish peroxidase-conjugated secondary antibody.…”

Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells

“…Ad6 also induced higher levels of IVa2 and E4orf3 than did Ad26 at this early time point. Interestingly all four proteins play a role in the shift from early to late transcription (35,39,40,62,63). This suggests that the transition from early to late transcription occurs earlier in Ad6 infection than in Ad26 infection.…”