Association of the adenovirus DNA-binding protein with RNA both in vitro and in vivo.

Abstract

The multifunctional DNA-binding protein (DBP) encoded by human adenovirus binds RNA. The association of purified DBP with RNA in vitro was demonstrated by using either a gel filtration or a filter binding assay. This association is sensitive to ionic strength and exhibits no apparent sequence specificity. DBP also interacts with RNA in vivo; it can be crosslinked to polyadenylylated RNA by UV-irradiation of intact cells during the late phase of adenovirus infections. The 46-kDa carboxyl-terminal domain ofDBP binds RNA in vitro and was found to be associated with polyadenylylated RNA in vivo. This is the same domain that interacts with DNA. However, the differences in sensitivity of DBP to trypsin when bound to RNA versus DNA suggest that RNA and DNA either bind at different sites within this domain or induce different conformational changes within the protein.The early region 2A gene (E2A) of human adenovirus types 2 and 5 (Ad2 and AdS) encodes a 72-kDa DNA-binding phosphoprotein (DBP) (1, 2), which is synthesized both early and late during an infectious cycle (3,4). This protein has a number of purported functions in infected cells. Analysis of DBP mutants has implicated the protein in viral DNA replication, viral early and late gene expression, and virion assembly.Studies of a DBP temperature-sensitive (ts) mutant, Ad5ts125, provides evidence of the protein's involvement in DNA replication. This mutant encodes a DBP that is thermolabile in its ability to bind to single-stranded (ss) DNA in vitro (5) and, at the nonpermissive temperature, is defective for viral DNA synthesis in vivo (6) and in vitro (7).The role of DBP in the regulation of Ad early gene expression is more controversial. DBP has been implicated in stimulating expression of early region 1B (EIB) (8) and early region 4 (E4) (9), while negatively influencing expression of early genes at intermediate and late times (10, 11). The purported negative regulation of early genes mediated by wild-type (wt) DBP appears to operate at the transcription level for E4 (12,13) and at the level of mRNA turnover for regions EIA, EIB, and probably E2A (14). However, studies by Rice and Klessig (15) (21-24). The ts, DNA replication-minus mutations are located in the 3' two-thirds of the gene, while the hr mutations map in the 5' portion ofthe gene. These functional domains appear to correspond to physical domains because digestion of purified DBP with a variety of proteases generates a C-terminal fragment of =44-46 kDa and an N-terminal fragment of -24-26 kDa (2, 25). The C-terminal fragment binds ss DNA (2) and can substitute for intact DBP in an in vitro DNA replication system (26). The N-terminal region also appears to function independently because DBP encoded by the hr mutants is able to overcome the block to late gene expression in monkey cells, even when its DNA replication function is perturbed by a ts mutation in the C-terminus (27).How the-DBP accomplishes this varied set of purported functions is still poorly understood. No enzymatic functions have...

In vivo analysis of the promoter structure of the influenza virus RNA genome using a transfection system with an engineered RNA.

“…We also examined the protein specificity in step I (Table 1). Adenovirus DNA binding protein or calf thymus histones, both of which can bind to RNA (21,22), substituted for NP to a limited extent. This suggests that the specific complex formed by NP and RNA is recognized by RNA polymerase.…”

SV40 stimulates expression of the transacting factor Sp1 at the mRNA level.

“…In this regard, it is of particular interest that T antigen may be an RNA-binding protein [Khandjian et al 1982;Carroll et al 1988). Another papovavirus protein, the 72-kD product of the adenovirus E2A gene, has been shown to contribute to the post-transcriptional regulation of mRNA (Cleghon and Klessig 1986;Lazardis et al 1988). It is noteworthy that <3 kb of the 8.2-kb Spl message corresponds to protein-coding sequences.…”

The Adenovirus E1B 55-Kilodalton and E4 Open Reading Frame 6 Proteins Limit Phosphorylation of eIF2α during the Late Phase of Infection

“…In both of these circumstances, VAI RNA is present but evidently nonfunctional. Although it is a DNA-binding protein, DBP also binds RNA during the late phase of infection (15). Perhaps the abundance of DBP during E1B-55K and E4orf6 mutant virus infections and during treatment with 2-aminopurine inactivates VAI RNA.…”