Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5′ poly(A) leader

Schwer, Beate; Visca, Paolo; Vos, J. Chris; Stunnenberg, Hendrik G.

doi:10.1016/0092-8674(87)90212-1

Cited by 139 publications

(114 citation statements)

References 23 publications

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“…However, a more direct analysis is required to confirm a decapping preference. VACV intermediate and late transcripts have a unique 5Ј poly(A) leader, further differentiating them from host and VACV early transcripts (5,30,31). It is known that decapping of cellular mRNAs depends on shortening of the 3Ј poly(A) tail and dissociation of the poly(A) binding protein, although the mechanism is not completely understood (26,32).…”

Section: Discussionmentioning

confidence: 99%

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

Parrish

Resch

Moss

2007

Proc. Natl. Acad. Sci. U.S.A.

114

177

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Previous studies indicated that the vaccinia virus D10 protein, which is conserved in all sequenced poxviruses, participates in the rapid turnover of host and viral mRNAs. D10 contains a motif present in the family of Nudix/MutT enzymes, a subset of which has been shown to enhance mRNA turnover in eukaryotic cells through cleavage of the 5 cap (m 7 GpppNm-). Here, we demonstrate that a purified recombinant D10 fusion protein possesses an intrinsic activity that liberates m 7 GDP from capped RNA substrates. Furthermore, point mutations in the Nudix/MutT motif abolished decapping activity. D10 has a strong affinity for capped RNA substrates (K m Ϸ 3 nm). RNAs of 24 -309 nt were decapped to comparable extents, whereas the cap of a 12-nt RNA was uncleaved. At large molar ratios relative to capped RNA substrate, competitor m 7 GpppG, m 7 GTP, or m 7 GDP inhibited decapping, whereas even higher concentrations of unmethylated analogs did not. High concentrations of uncapped RNA were also inhibitory, suggesting that D10 recognizes its substrate through interaction with both cap and RNA moieties. Thus far, poxviruses represent the only virus family shown to encode a Nudix hydrolase-decapping enzyme. Although it may seem self-destructive for a virus to encode a decapping and a capping enzyme, accelerated mRNA turnover helps eliminate competing host mRNAs and allows stagespecific synthesis of viral proteins.mRNA metabolism ͉ MutT motif ͉ Nudix hydrolase ͉ poxvirus ͉ mRNA turnover T he steady-state concentration of an mRNA is determined by synthesis and decay, allowing cells to rapidly adapt their pattern of gene expression (1). Similarly, some viruses accelerate mRNA turnover to suppress synthesis of cellular proteins and regulate expression of their own genes (2). Studies of vaccinia virus (VACV), the laboratory prototype poxvirus, indicated that viral mRNAs are relatively unstable compared with those of uninfected cells (3, 4). Thus, rapid mRNA turnover coupled with robust and sequential transcription of viral early, intermediate, and late genes allow stage-specific protein synthesis (5). Likewise, VACV infection induces the destabilization of cellular transcripts, a process thought to contribute to the shutdown of host protein synthesis (6-9). The latter may enhance viral replication by alleviating competition from cellular mRNAs for the protein synthetic machinery and by diminishing host antiviral responses. Nevertheless, the mechanisms used by VACV to regulate mRNA turnover have not been elucidated.The VACV D9 and D10 proteins were identified as putative negative regulators of gene expression during a transfectionbased DNA library screen used to isolate activators of late VACV transcription (10, 11). Both proteins are highly conserved: D10 homologs are present in all sequenced poxviruses, and D9 homologs are in all members of the chordopoxvirus subfamily. Overexpression of D10 during infection significantly reduced the amount of VACV transcripts, and overexpression of D9 had a similar but lesser effect (11). Remarkabl...

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Section: Discussionmentioning

confidence: 99%

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

Parrish

Resch

Moss

2007

Proc. Natl. Acad. Sci. U.S.A.

114

177

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“…Intermediate promoters are more prevalent in the vaccinia virus genome than previously appreciated, because many have the TAAATG motif at the start of their open reading frames, which was previously attributed to late gene promoters (X. Liu and S. S. Broyles, unpublished results). The RNA polymerase initiates on this motif within the A triplet (actually on the T triplet on the template strand) and slips repeatedly while attempting to initiate transcription (Bertholet et al, 1987;Schwer et al, 1987). The result is mRNA with a 59 end bearing a heterogeneous oligo(A) tail, averaging about 30 nt in length that is not template encoded.…”

Section: Intermediate Gene Transcriptionmentioning

confidence: 99%

Vaccinia virus transcription

Broyles

2003

Journal of General Virology

193

168

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“…plement of the three As within the motif {i.e., the three Ts on the template strandl has been proposed to be the pseudo-template for synthesis of a poly(A) head, which immediately follows the capped 5' end of the late transcripts (Bertholet et al 1986, 198L Patel andPickup 1987;Schwer et al 1987;Schwer and Stunnenberg 19881. Results from various in vitro systems Stunnenberg 1988~ Wright andMoss 1989} support the pseudo-templated polymerization hypothesis for the poly{A) head. Remarkably, the length of the poly(A) head, -3 5 As, is rather constant both in vitro and in vivo.…”

Section: T T C a T ( '~T T ' -" G G T T T T T C A A A Amentioning

confidence: 99%