A baculovirus-expressed dicistrovirus that is infectious to aphids

Abstract

Detailed investigation of virus replication is facilitated by the construction of a full-length infectious clone of the viral genome. To date, this has not been achieved for members of the family Dicistroviridae. Here we demonstrate the construction of a baculovirus that expresses a dicistrovirus that is infectious in its natural host. We inserted a full-length cDNA clone of the genomic RNA of the dicistrovirus Rhopalosiphum padi virus (RhPV) into a baculovirus expression vector. Virus particles containing RhPV RNA accumulated in the nuclei of baculovirus-infected Sf21 cells expressing the recombinant RhPV clone. These virus particles were infectious in R. padi, a ubiquitous aphid vector of major cereal viruses. The recombinant virus was transmitted efficiently between aphids, despite the presence of 119 and 210 vector-derived bases that were stably maintained at the 5' and 3' ends, respectively, of the RhPV genome. The maintenance of such a nonviral sequence was surprising considering that most RNA viruses tolerate few nonviral bases beyond their natural termini. The use of a baculovirus to express a small RNA virus opens avenues for investigating replication of dicistroviruses and may allow large-scale production of these viruses for use as biopesticides.

FIG. 1.

Cloning strategy, map of the full-length RhPV cDNA clone in the baculovirus expression vector pFastBac1, and sequences of genome termini. (Top) Genome organization of RhPV RNA. UTR, untranslated region; IGR, intergenic region; hel, helicase domain; prot, proteinase domain; pol, RNA-dependent RNA polymerase active site; VP, virion protein. The positions of the PCR primers (P1 to P6) used for the amplification of cDNA clones in relation to the RhPV RNA genome are indicated by the solid arrows below the primer designations and above the amplified cDNA fragments (open rectangles). EcoRI and KpnI restriction sites were introduced artificially (indicated by parentheses) into the PCR primers at the 5′ and 3′ ends, respectively. The three cDNA fragments were sequentially introduced into pFastBac1 so that the viral genome rests between the EcoRI and KpnI sites (positions −11 and 10,011, respectively), as described in Materials and Methods. The sequence below the map shows the RhPV sequence insert in bold and the flanking vector sequence, which includes the polh core promoter sequences (underlined) and SV40 polyadenylation/termination signal (bottom line). The open arrows and singly underlined bases indicate the 5′ and 3′ ends detected by RACE following the passaging of baculovirus-derived virions in aphids for 32 days.

FIG. 2.

Accumulation of RhPV particles and proteins in baculovirus-infected cells. (A) Electron micrograph of negatively stained R. padi-derived virus particles of wild-type RhPV. (B) RhPV particles recovered from Sf21 cells infected with AcRhPV6. Images of RhPV particles (at right) and larger particles of unknown identity (at left) from the same micrograph are shown for direct comparison. (C and D) Immunogold labeling of baculovirus-produced RhPV proteins. Sf21 cell infected with AcRP23LacZ (C) or AcRhPV6 (D). RhPV-specific antibody binding in the nucleus was detected by goat anti-rabbit serum conjugated with 10-nm colloidal gold. Baculovirus nucleocapsids (N), which accumulate in the nucleus prior to occlusion, are indicated. All bars represent 200 nm.

FIG. 3.

Detection of RhPV RNA in (A) recombinant baculovirus infected Sf21 cells and (B) baculovirus-produced virus particles. (A) Northern blot hybridization of total cellular RNA from Sf21 cells infected with AcRhPV6 at 4 and 5 dpi or AcRP23lacZ at 5 dpi. Lane V contains 100 ng of viral RNA (10 kb). ³²P-labeled RNA complementary to the 3′ end of the RhPV genome was used as a probe. The bottom panel represents ethidium bromide-stained rRNA (used as a loading control). (B) RT-PCR of RNA extracted from purified virus particles. Lane M, 1-kb double-stranded DNA ladder; RhPV, RhPV RNA from aphid-derived virions; AcRP23lacZ, RNA extracted from AcRP23LacZ-infected Sf21 cells; AcRhPV6, RNA extracted from purified RhPV particles from AcRhPV6-infected Sf21 cells.

FIG. 4.

Detection of (A) positive- and (B) negative-sense RhPV RNAs in AcRhPV6-infected Sf21 cells and in aphids fed on baculovirus-derived virus particles by RT-PCR. Lane M, 1-kb double-stranded DNA ladder. (A) Templates: C, control with no reverse transcriptase; +, positive-sense T7 in vitro transcript of RhPV RNA; AcRhPV6, AcRhPV6-infected Sf21 cells; 1 through 3, total RNA from three replicates of aphids 17 days after feeding on baculovirus-derived RhPV6 virus particles; A, virus-free aphids; RhPV, RhPV-infected aphids. (B) Templates: AcRhPV6, total RNA from AcRhPV6-infected Sf21 cells; A1, total RNA from aphids fed on baculovirus-derived RhPV6 virus particles; −, positive control, negative-sense transcript (SP6); +, negative control, positive-sense transcript (T7); AcRP23lacZ, RNA from AcRP23lacZ-infected Sf21 cells; C, control with no reverse transcriptase.

FIG. 5.

Transmission electron micrographs of RhPV6 particles derived from aphids. A, virus-free aphids (negative control); B, RhPV-infected aphids (positive control); C and D, baculovirus-derived RhPV6 virus particles obtained 47 dpi of aphids. Representative stained virus particles approximately 27 nm in diameter in the virus minipreps are indicated by arrowheads. All scale bars represent 200 nm.

FIG. 6.

Stability and transmission of RhPV in aphids. (A) Western immunoblot analysis of virus preparations obtained from aphids at 47 dpi with antibody against RhPV virions. Protein was extracted from virus-free aphids (A−), RhPV-infected aphids (A+), and aphids fed on baculovirus-derived virus particles (A1 and A2). (B) RT-PCR showing aphid-to-aphid transmission of RhPV6 at 17 dpi. Lane M, 1-kb ladder. Templates: total RNA from lanes 1 to 5, progeny aphids from five separate plants harboring aphids fed on baculovirus-derived virus particles; A−, virus-free aphids; A+, known RhPV-infected aphids; +, positive-sense T7 in vitro transcript or RhPV RNA.