An Mpsi-containing heterologous RNA, but not env mRNA, is efficiently packaged into avian retroviral particles

Abstract

Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MPsi, is located upstream of the 5' splice site and therefore is present on both genomic and spliced RNAs. We now show that an MPsi-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MPsi. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MPsi is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3' end of MPsi was found to reduce the packaging efficiency of heterologous RNAs.

FIG. 1

(A) Predicted secondary structure of MΨ, obtained from the mfold computer program (32, 33, 63). The O3 and L3 stem-loop structures, as well as the 5′ and 3′ ends of the RNA, are indicated. Solid lines are shown next to sequences deleted in the MΨ3′Δ40 construct; dashed lines are shown next to sequences deleted in the MΨ3′Δ20 construct. (B) RPA of virions released from G418-resistant mass cultures of Q2bn cells transfected with plasmids expressing the heterologous RNA indicated above each lane. RNAs were protected with an antisense neo probe. The expected locations of free probe and of the protected neo bands are indicated. MWM, molecular weight markers. (C) RIPA of viral particles described for panel B. Proteins were precipitated with an α-PrB-specific antibody. The expected size of the CA band is indicated. (D) Average packaging efficiencies of the RNAs from three replications of the experiment relative to CMVneo-MΨ RNA. Error bars represent standard deviations. Packaging efficiencies were calculated as the ratio of neo RNA packaged into particles, as measured by RPA, to the number of viral particles, as measured by RIPA.

FIG. 2

Schematic of the assay to determine the packaging efficiency of genomic RSV RNA relative to CMVneo-MΨ RNA. Q2bn packaging cells were transfected with both pASY194, which expressed CMVneo-MΨ, and pRCASBPpuro, which expresses a full-length infectious clone of RSV. Control cells were transfected with pASY194 only or pRCASBPpuro only. RPAs were performed on viral and whole-cell or cytoplasmic lysates. RIPA was performed on viral particles. g, gag gene encoding viral structural proteins; p, pol gene encoding reverse transcriptase and integrase; e, env gene encoding glycoproteins; Pu, puro gene encoding puromycin acetyltransferase; NEO, neo gene encoding neomycin phosphotransferase.

FIG. 3

(A) RPA of whole-cell lysates from cells described in the legend to Fig. 2. The RNA expressed in the cells is indicated above each lane. Lane 4 contains lysates from untransfected Q2bn cells. The sample in lane 5 had no RNase added; the sample in lane 6 had no cell lysate added. CMVneo-MΨ was detected with an antisense neo probe. RCASBPpuro RNA was detected with an antisense puro probe. A cellular message, gapdh, was detected with antisense gapdh probe. The bands corresponding to free neo, puro, and gapdh probes, as well as the protected bands, are indicated. Free puro and gapdh probes are of similar size and appear as one band in this gel. MWM, molecular weight markers. (B) RPA of viral lysates. The RNA expressed in the cells from which the virions were released is indicated above each lane. The puro and neo probes described for panel A were also used in this assay. (C) RIPA of viral particles released from the cell lines, using PrB-specific antibody. The band corresponding to CA protein is indicated. (D) Formula used for calculating packaging efficiency of each RNA. The level of each RNA in virions was measured by RPA of viral lysates, shown in panel B. The level of the RNA relative to gapdh in cells was measured by RPA of cell lysates, shown in panel A. The number of viral particles was determined by RIPA, shown in panel C.

FIG. 4

(A) The RCASBPneo genome showing the location of the minimal packaging signal, MΨ, relative to the viral splice sites. Unspliced and spliced viral mRNAs were detected in RCASBPneo-infected cells and viral particles by RPA, using a probe antisense to the env 3′ss. The regions of the two RNAs protected by the probe, as well as the predicted size of the protected region, are shown. gag, gene encoding viral structural proteins; pol, gene encoding reverse transcriptase and integrase; env, gene encoding glycoproteins; neo, gene encoding neomycin phosphotransferase. (B) RPA of viral, cytoplasmic, and nuclear lysates, using the antisense 3′ss probe described for panel A. The sample in lane 4 was not treated with RNase; the sample in lane 5 had no lysate added. The expected locations of free probe and of protected unspliced and spliced RNA bands are indicated. MWM, molecular weight markers.