Adenovirus virus-associated RNA is processed to functional interfering RNAs involved in virus production

Abstract

Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression.

FIG. 1.

Predicted structure of VAI RNA from adenovirus type 2. The sequences that hybridize to oligonucleotides 5′VA, 3′VAn, 3′VApe, 5′apVA, and 3′apVA are indicated. The structure can be divided into a terminal stem, a central domain, and an apical stem. The sequence of VAI RNA from adenovirus type 5 is identical except for two nucleotides in the apical stem (A66G and G72U).

FIG. 2.

svaRNAs are found in adenovirus-infected 293 cells.(A) Northern blotting was carried out with RNA isolated from 293 cells that were either mock infected or infected with AdWT or AdΔVA. (B) 293 cells were mock infected or infected with wild-type adenovirus, and Northern blotting was done with RNAs isolated at different hours postinfection (hpi) as indicated at the top. The Northern blots were hybridized to oligonucleotide 3′VAn (upper panels), oligonucleotide 5′VA (middle panels), or a U6 snRNA oligonucleotide as a loading control (lower panels). The number of nucleotides is indicated on the left. The positions of 3′svaRNA, 5′svaRNA, and U6 snRNA are indicated.

FIG. 3.

svaRNAs are produced in the absence of viral proteins or adenovirus replication. (A) Nonpermissive 3T3 cells were infected as for Fig. 2B, and Northern blotting was carried out accordingly. (B) 293 or HeLa cells were either mock transfected, transfected with pAdvantage (T), or infected (only 293 cells) with wild-type adenovirus (I). RNA was isolated and analyzed by Northern blotting by hybridization to oligonucleotide 3′VAn, 5′VA, or U6 snRNA as indicated. The number of nucleotides is indicated on the sides. The positions of 3′svaRNA, 5′svaRNA, and U6 snRNA are indicated.

FIG. 4.

Characterization of svaRNAs. (A) Efficiency of svaRNA processing. RNA isolated from mock-infected or infected (I) 293 cells was analyzed by Northern blotting by hybridization to oligonucleotide 3′VAn. Single and double (×2) amounts of RNA were used to ensure proper quantitation. Positions of intact 160-nucleotide VAI RNA and 3′svaRNA are indicated. Note that the lower panel shows a 10-times-longer exposure. (B) Sequence and localization of svaRNAs. RNA was isolated from total, nuclear, or cytoplasmic fractions of HeLa cells that were either mock transfected or transfected with pAdv. Total RNA was separated in a polyacrylamide gel, and 20- to 30-nucleotide small RNAs were purified. Buffer (Oligo) (lanes 1, 5, and 8), total RNA (lanes 2 to 4), small RNAs (lanes 6 and 7), nuclear RNA (lane 9), or cytoplasmic RNA (lane 10) was analyzed by primer extension with oligonucleotide 3′VApe (upper panel) or 5′VA (middle panel) or with a U6 snRNA oligonucleotide as a loading control (lower panel). A double amount of RNA was used where indicated (×2). The primer extension corresponding to 3′svaRNA, 5′svaRNA, and U6 snRNA is indicated. The number of nucleotides is given on the left. (C) Schematic representation of svaRNAs. Sequences of 5′ and 3′ svaRNAs are shown together with the oligonucleotides used for primer extension.

FIG. 5.

svaRNAs coimmunoprecipitate with Ago2. (A) Flag-tagged Ago2 protein can be efficiently immunoprecipitated with an anti-Flag antibody. Extracts from cells transfected with pAdv or cotransfected with pAdv and a plasmid that encodes a Flag-tagged Ago2 protein (pAdvFAgo2) were immunoprecipitated with a control antibody (αMock) or with an anti-Flag antibody (αFlag). Input, bound, and unbound fractions were separated by SDS-polyacrylamide gel electrophoresis, and Western blotting was carried out with anti-Flag antibody. The position of Flag-tagged Ago2 (FAgo2) is indicated. (B) Ago2 binds svaRNAs. RNA was isolated from the fractions described for panel A, and a primer extension reaction was carried out. The oligonucleotides used revealed let-7 miRNA (upper panel), 3′svaRNA (middle panel), or 5′svaRNA (lower panel). Ub, unbound fractions; B, bound fractions; I, input; O, oligonucleotide-only reaction. The primer extension corresponding to let-7, 3′svaRNA, and 5′svaRNA is indicated. (C) Characterization of HeLa clones expressing Flag-tagged Ago2. Extracts from HeLa cells or cells transfected with a plasmid expressing FAgo2 transiently (FAgo2) or stably (clones C1 to C4) were immunoprecipitated with αFlag antibody. Total extracts (upper panel) or extracts bound to αFlag (lower panel) were analyzed by Western blotting with an αFlag antibody. The position of FAgo2 is indicated. (D) svaRNAs from adenovirus-infected cells bind Ago2. Extracts from HeLa cells transfected with pAdv and pFAgo2 (pAdvFAgo2) or from adenovirus-infected HeLa clones 1 (C1 + AdWT) and 4 (C4 + AdWT) that express Ago2 were immunoprecipitated with an αFlag or a control antibody. RNA was isolated from the input, bound, and unbound fractions of the immunoprecipitates, and a primer extension reaction was carried out.

FIG. 6.

Expression of RL mRNAs that bind to VAI RNA terminal sequences is inhibited in cells expressing VAI RNA. (A) Schematic representation of the reporter genes used for this study. VAI RNA (diagramed at the top) sequences were expressed in the 3′ UTR of RL. Sequences chosen were VA internal sequences (VAint) and either the sense (s) or antisense (as) 5′ (5VA) or 3′ (3VA) end. Note that the sense sequence of one end of VAI RNA contains the antisense sequence of the other end with two mutations that disrupt base pairing (Mut). Sequences were inserted once or twice (x2). (B) HeLa cells were cotransfected with the RL constructs shown and a control plasmid (Mock) or pAdv, and luciferase activity was measured 2 days posttransfection. (C) HeLa cells transfected with the RL constructs shown in panel A were infected with AdWT or AdΔVA. Luciferase activity was measured 3 days posttransfection. Percentages of RL expression relative to the expression of the unmodified RL construct are indicated. Error bars, standard deviations. Results shown are averages from four different experiments.

FIG. 7.

Blocking of svaRNAs affects adenovirus viability. (A) svaRNA-mediated inhibition can be blocked with antisense 2′-O-methyl oligonucleotides. Cells were transfected with pAdv and RLas3VA either alone (Mock) or in combination with 2′-O-methyl oligonucleotides with sense (2omeMut) or antisense (2omeAS3′) sequences of 3′svaRNA. Luciferase expression, measured 3 days posttransfection, is plotted as the percentage of activity relative to an unmodified RL construct. (B) Cells transfected with 2′-O-methyl oligonucleotides with 3′svaRNA sense sequences (2omeMut), with 5′svaRNA (2omeAS5′) or 3′svaRNA (2omeAS3′) antisense sequences, or with mutated 5′svaRNA (2omeAS5′Mut) or 3′svaRNA (2omeAS3′Mut) antisense sequences were infected with wild-type adenovirus. Cell supernatants were collected at 3 days postinfection, and virus titers were evaluated and plotted. Error bars, standard deviations. Results shown are averages from three different infections.