Preformed hexamers of SV40 T antigen are active in RNA and origin-DNA unwinding

Abstract

Preformed hexamers of simian virus 40 (SV40) large tumor antigen (T antigen) constitute the bulk of T antigen in infected cells and are stable under physiological conditions. In spite of this they could not be assigned a function in virus replication or transformation. We report that preformed hexamers represent the active T antigen RNA helicase. Monomers and smaller oligomeric forms of T antigen were inactive due to the lack of hexamer formation under RNA unwinding conditions. In contrast to the immunologically related cellular DEAD-box protein p68, the T antigen RNA helicase is found to act in a much more processive way and it does not catalyze rearrangements of structured RNAs. Thereby, it rather seems to resemble other virus-encoded RNA helicases, like vaccinia virus NPH-II. Surprisingly, in our hands preformed hexamers also strikingly bound to and unwound the SV40 replication origin, pointing to a possible role of preformed hexamers in the initiation step of viral DNA replication. Furthermore, we have detected an extra hexamer-specific, high-affinity T antigen ATP binding site with a very slow exchange rate constant, the function of which is discussed.

Figure 1

Native gel electrophoretic analysis of T antigen complexes pre-treated under different conditions. Immunopurified T antigen (12 pmol), fixed with glutaraldehyde, is shown in lane 2. Purified hexamers and a monomer-enriched preparation (referred to as ‘monomers’; see Materials and Methods) of T antigen (6 pmol each) were either fixed with glutaraldehyde directly before electrophoresis (lanes 3 and 7, respectively) or were pre-incubated in the presence of GTP (hexamers, lane 4; monomers, lane 5) or ATP (monomers, lane 6) under RNA helicase reaction conditions at 37°C for 30 min and fixed thereafter. The gel was silver stained after electrophoresis.

Figure 2

RNA helicase activity analysis of purified hexamers. Helicase activity analysis was monitored by gel-shift electrophoresis and autoradiography. A diagram of the respective substrate used is given to the right of each part with an asterisk marking the labeled strand. The respective heat-denatured substrates (lanes 1) and control reactions without T antigen (lanes 2) were run in parallel. The positions of ds and ssRNAs are marked on the left of each gel. (A) T antigen hexamers constitute the functionally active RNA helicase. Helicase reactions were run with a ³²P-labeled 17 bp ds substrate RNA at the indicated concentrations of T antigen (hexamers, lanes 3–5; monomers, lanes 6–8; immunopurified T antigen, lanes 8–10) at 37°C for 30 min. (B) RNA unwinding by hexamers is not restricted to small dsRNA regions. Helicase reactions were run at the indicated concentrations of purified hexamers and a 123 bp ds substrate RNA as described in (A) in the absence (lane 3) or in the presence of GTP (lanes 4 and 5).

Figure 3

T antigen hexamers are unable to catalyze RNA strand exchange reactions. The design of the substrate RNAs (with asterisks marking the labeled strand) and the expected course of the strand exchange reaction are shown on top. Thick lines indicate homologous regions of RNA strands. Note that the branch migration structure is a stable reaction intermediate, which needs to be resolved in an NTP-dependent step. Strand exchange reaction mixtures contained the partially ds 76 bp RNA (with one strand ³²P-labeled) plus the respective homologous ssRNA and were incubated without (control, lane 3) or with T antigen (1 pmol, lane 6; 2 pmol, lanes 5 and 7) in the hexameric state in the absence (lane 5) or presence of GTP (lanes 3, 6 and 7) at 37°C for 30 min. As a positive control, recombinant human DEAD-box protein p72 (0.15 pmol) was used, in the absence (lane 4) or presence (lane 7) of ATP instead of GTP. The formation of the respective branch migration structure and the strand exchange activity was monitored by gel-shift electrophoresis and autoradiography. For comparison, the 76 bp dsRNA (lane 1), the ³²P-labeled RNA strand (lane 2) and the 123 bp dsRNA (lane 9) were run in parallel.

Figure 4

Preformed T antigen hexamers efficiently bind to and unwind the SV40 origin of DNA replication. Analyses were monitored by gel-shift electrophoresis and autoradiography. (A) Ori-DNA binding by preformed T antigen hexamers and purified monomers. Reactions with 2.5 pmol of T antigen (preformed hexamers, lanes 2 and 3, or purified monomers, lanes 4 and 5) were performed in the absence (lanes 3 and 5) or in the presence of ATP (lanes 2 and 4). A sample containing ATP, but no T antigen was used as a control (lane 1). The positions of free DNA (ori-DNA) as well as of hexamer (H) and double hexamer (DH) DNA complexes (DNA compl.) are indicated. (B) SV40 ori-DNA unwinding by purified monomers and preformed hexamers in the presence of different (d)NTPs. Ori-DNA-specific helicase reactions were performed without (control) or with 2.5 pmol of T antigen (purified monomers, upper part, or preformed hexamers, lower part) and in the presence of indicated nucleotides (4 mM) at 37°C for 60 min. One reaction mixture was heat denatured at 95°C prior to gel electrophoresis (denatured, lane 2).

Figure 5

Analysis of the SV40 ori-DNA binding and unwinding activity of preformed T antigen hexamers by electron microscopy. A characteristic field view of an unwinding experiment performed with preformed T antigen (12.5 pmol) hexamers and a linearized (BsaI-cut) plasmid DNA (pSV-08, containing the SV40 origin) is shown. On the left, two dsDNA molecules with ori-DNA-bound T antigen double hexamers and on the right, a partially unwound molecule (lower right) and two DNA single strands (originating from a completely unwound DNA molecule; upper center and right) are seen. The ssDNA is coated with E.coli SSB and therefore condensed more than twice relative to duplex DNA. Identical reactions (not shown) were also performed with purified monomers and without T antigen. Unwinding rates obtained are shown in the lower part of the figure and are expressed as percent of fully and partially unwound DNA molecules obtained after 1 h of incubation. The bar represents 100 nm.

Figure 6

High-affinity binding of ATP to T antigen hexamers. (A) Label ing of T antigen hexamers. Hexameric (lanes 2 and 4) or immunopurified (lanes 3 and 5) T antigen (0.85 pmol each) was incubated on ice in 3 µl of labeling buffer (see Materials and Methods) for 15 min. After removal of unbound nucleotides by gel filtration, samples were fixed with 0.01% glutaraldehyde and analyzed by native polyacrylamide gel electrophoresis followed by silver staining (lanes 1–3) or autoradiography (lanes 4 and 5). (B) Time course of ATP binding. Reactions with T antigen hexamers (0.85 pmol) were performed for the indicated time periods exactly as described above. [α-³²P]dATP bound to T antigen was determined by absorption to a nitrocellulose membrane, and the radioactivity retained on the filters was measured by liquid scintillation counting. (C) Binding of ATP to salt-stripped T antigen. Immunopurified T antigen was treated with 3.5 M magnesium acetate at room temperature for 30 min, immediately gel-filtrated on a Sephadex G50 column and then incubated with 2 mM Mg²⁺-ATP at 30°C for 1 h. The resulting hexamers were purified by sucrose gradient centrifugation and an aliquot (1.7 pmol of T antigen) was incubated with the indicated concentrations of [α-³²P]dATP as described in (A). [α-³²P]dATP bound to T antigen was determined by absorption to nitrocellulose membrane filters. (D) Bound ATP is not released from T antigen hexamers under DNA helicase reaction conditions. [α-³²P]dATP-labeled T antigen hexamers (A) were incubated under DNA helicase assay buffer conditions in the presence of the indicated concentrations of non- labeled ATP at 20°C for 10 min and analyzed by native polyacrylamide electrophoresis and autoradiography.