Preferential translation of vesicular stomatitis virus mRNAs is conferred by transcription from the viral genome

Abstract

Host protein synthesis is inhibited in cells infected with vesicular stomatitis virus (VSV). It has been proposed that viral mRNAs are subjected to the same inhibition but are predominantly translated because of their abundance. To compare translation efficiencies of viral and host mRNAs during infection, we used an enhanced green fluorescent protein (EGFP) reporter expressed from a recombinant virus or from the host nucleus in stably transfected cells. Translation efficiency of host-derived EGFP mRNA was reduced more than threefold at eight hours postinfection, while viral-derived mRNA was translated around sevenfold more efficiently than host-derived EGFP mRNA in VSV-infected cells. To test whether mRNAs transcribed in the cytoplasm are resistant to shutoff of translation during VSV infection, HeLa cells were infected with a recombinant simian virus 5 (rSV5) that expressed GFP. Cells were then superinfected with VSV or mock superinfected. GFP mRNA transcribed by rSV5 was not resistant to translation inhibition during superinfection with VSV, indicating that transcription in the cytoplasm is not sufficient for preventing translation inhibition. To determine if cis-acting sequences in untranslated regions (UTRs) were involved in preferential translation of VSV mRNAs, we constructed EGFP reporters with VSV or control UTRs and measured the translation efficiency in mock-infected and VSV-infected cells. The presence of VSV UTRs did not affect mRNA translation efficiency in mock- or VSV-infected cells, indicating that VSV mRNAs do not contain cis-acting sequences that influence translation. However, we found that when EGFP mRNAs transcribed by VSV or by the host were translated in vitro, VSV-derived EGFP mRNA was translated 22 times more efficiently than host-derived EGFP mRNA. This indicated that VSV mRNAs do contain cis-acting structural elements (that are not sequence based), which enhance translation efficiency of viral mRNAs.

FIG. 1.

(A) Genome of rVSV-EGFP recombinant virus that expresses EGFP as a foreign gene. Viral genes and leader (le) and trailer (tr) sequences are indicated. (B) EGFP-N1 plasmid DNA. EGFP mRNA synthesis is directed by a cytomegalovirus (CMV) promoter in stably transfected HeLa-EGFP cells. (C) Analysis of EGFP synthesis. HeLa cells were infected with rVSV-EGFP, and HeLa-EGFP cells were mock infected or infected with rwt virus. Cells were pulse-labeled with [³⁵S]methionine at 7.5 h postinfection and harvested at 8.5 h postinfection. EGFP was immunoprecipitated and analyzed by SDS-PAGE and phosphorimaging. Duplicate immunoprecipitates are shown. (D) Analysis of EGFP mRNA levels. HeLa cells were infected with rVSV-EGFP, and HeLa-EGFP cells were mock infected or infected with rwt virus. RNA was harvested at 8 h postinfection, and EGFP mRNA levels were analyzed by Northern blotting using a [³²P]dCTP-labeled EGFP probe and phosphorimaging. Samples were run in duplicate. (E) Translation efficiencies (rates of protein synthesis divided by mRNA levels) of EGFP mRNAs shown relative to HeLa-EGFP cells that were mock infected. Data are shown as means ± SEs for four or five experiments.

FIG. 2.

(A) Polysome profiles of mock-infected HeLa-EGFP cells, HeLa-EGFP cells infected with rwt virus, or HeLa cells infected with rVSV-EGFP. The bracket in the polysome profile from mock-infected cells shows the region of the gradient, containing monosomes and polysomes, that we are focused on. Abs, absorbance. (B) Distributions of EGFP mRNAs within sucrose gradients. Eight hours postinfection, cells were harvested for analysis of polysome profiles. Sucrose gradients were collected in 16 fractions, and EGFP mRNA levels in each fraction were analyzed by Northern blotting using a [³²P]dCTP-labeled EGFP probe and phosphorimaging. Fractions from the tops of the gradients are on the left. (C) Quantitations of EGFP mRNA distributions from multiple experiments were analyzed to show the distributions, as a percentage of the total EGFP mRNA in each fraction (± SE), for HeLa cells infected with rVSV-EGFP (○) or for HeLa-EGFP cells that were mock infected (▪) or infected with rwt virus (▴).

FIG. 3.

(A) Polysome profiles of HeLa-EGFP cells that were mock infected, infected with rwt virus, or mock infected and treated with puromycin. Abs, absorbance. (B to D) EGFP mRNA distributions in HeLa-EGFP cells that were mock infected, HeLa-EGFP cells infected with rwt virus, or HeLa cells infected with rVSV-EGFP, respectively, that were untreated (⧫) or treated with puromycin (⋄). Sixteen fractions were collected from sucrose gradients, and EGFP mRNA levels were analyzed by Northern blotting. EGFP mRNA in each fraction is shown as an average of multiple experiments ± SE. (E) Distributions of EGFP mRNAs in large polysomes (± SE); determined by subtracting EGFP mRNA signals in each fraction in puromycin-treated cells from the corresponding fraction in untreated cells, for HeLa-EGFP cells that were mock infected (▪), HeLa-EGFP cells infected with rwt virus (▴), or HeLa cells infected with rVSV-EGFP (○).

FIG. 4.

(A) Experimental design for infecting HeLa cells with rSV5-GFP followed by superinfection with rwt virus or mock superinfection. Cells were starved for methionine for 30 min, pulse-labeled with [³⁵S]methionine for 1 h, and harvested at 6.25 h postsuperinfection—or RNA was harvested at 6 h postsuperinfection. (B) Labeled GFP analyzed by immunoprecipitation, SDS-PAGE, and phosphorimaging. In rwt virus-superinfected cells, VSV M protein is the prominent band. Duplicate samples are shown. (C) Phosphorimage of Northern blot for GFP mRNA; samples were run in duplicate. (D) Translation efficiencies of GFP in rwt virus-superinfected cells relative to mock-superinfected cells; efficiencies were determined by dividing translation rates by mRNA levels (± SEs for three experiments).

FIG. 5.

(A) Recombinant viruses rVSV-P and rVSV-MCS that stably express EGFP from mRNA containing either the VSV P gene 5′ UTR or the 5′ UTR from the EGFP-N1 vector, respectively. (B) Analysis of EGFP translated from viral-derived mRNA. HeLa cells were infected with recombinant viruses and pulse-labeled at 7.5 h postinfection with [³⁵S]methionine and harvested 8.5 h postinfection. EGFP was immunoprecipitated and analyzed by SDS-PAGE and phosphorimaging. (C) EGFP synthesis in HeLa cells infected with recombinant viruses. Synthesis is shown as an average of two experiments ± SE, relative to synthesis in HeLa cells infected with rVSV-P virus. (D) Plasmid DNA that directs synthesis of EGFP-N1 and EGFP-P-G mRNAs in stably transfected HeLa cells. EGFP-N1 mRNA contains negative control UTRs from the pEGFP-N1 plasmid. EGFP-P-G mRNA contains the VSV P gene 5′ UTR and the VSV G gene 3′ UTR. (E) EGFP synthesis in stably transfected cells mock-infected or infected with rwt virus. At 7.5 h postinfection the cells were pulse-labeled with [³⁵S]methionine, and they were harvested at 8.5 h postinfection. EGFP was analyzed by immunoprecipitation, SDS-PAGE, and phosphorimaging. Duplicate immunoprecipitates are shown. (F) EGFP translation efficiencies in rwt virus-infected cells relative to mock-infected cells, determined by normalizing EGFP synthesis to mRNA levels (± SEs for three experiments).

FIG. 6.

Analysis of in vitro translation of RNA from infected cells. RNA isolated from infected cells at 8 h postinfection was translated in vitro in reticulocyte lysates in the presence of [³⁵S]methionine. EGFP synthesis was analyzed by immunoprecipitation, SDS-PAGE, and phosphorimaging (lanes 1 to 5). All translation products (3% of total) were also analyzed (lanes 6 to 10). VSV M protein and an unknown host protein (*) were nonspecifically immunoprecipitated along with EGFP. Translation was directed by 27 μg of total RNA from the following: HeLa-EGFP cells that were mock infected (lanes 1 and 6), HeLa-EGFP cells infected with rwt virus for 8 h (lanes 2 and 7), HeLa cells infected with rVSV-EGFP (1.4 μg) and mock-infected HeLa cells (25.6 μg) (lanes 3 and 8), HeLa cells infected with rVSV-EGFP (1.4 μg) and HeLa cells infected with rwt virus for 8 h (25.6 μg) (lanes 4 and 9), or mock-infected HeLa cells (lanes 5 and 10).