Evidence for preferential copackaging of Moloney murine leukemia virus genomic RNAs transcribed in the same chromosomal site

Abstract

Background: Retroviruses have a diploid genome and recombine at high frequency. Recombinant proviruses can be generated when two genetically different RNA genomes are packaged into the same retroviral particle. It was shown in several studies that recombinant proviruses could be generated in each round of HIV-1 replication, whereas the recombination rates of SNV and Mo-MuLV are 5 to 10-fold lower. The reason for these differences is not clear. One possibility is that these retroviruses may differ in their ability to copackage genomic RNAs produced at different chromosomal loci.

Results: To investigate whether there is a difference in the efficiency of heterodimer formation when two proviruses have the same or different chromosomal locations, we introduced two different Mo-MuLV-based retroviral vectors into the packaging cell line using either the cotransfection or sequential transfection procedure. The comparative study has shown that the frequency of recombination increased about four-fold when the cotransfection procedure was used. This difference was not associated with possible recombination of retroviral vectors during or after cotransfection and the ratios of retroviral virion RNAs were the same for two variants of transfection.

Conclusions: The results of this study indicate that a mechanism exists to enable the preferential copackaging of Mo-MuLV genomic RNA molecules that are transcribed on the same DNA template. The properties of Mo-MuLV genomic RNAs transport, processing or dimerization might be responsible for this preference. The data presented in this report can be useful when designing methods to study different aspects of replication and recombination of a diploid retroviral genome.

Figure 1

Structures of Mo-MuLV-based retroviral vectors used in this study. U3, R, U5, regions of long terminal repeat; SV, simian virus 40 early promoter region; ψ+, extended packaging signal; Neo, neomycin phosphotransferase gene; Puro, puromycin N-acetyltransferase gene. Δpbs and ΔEP indicate that the entire primer binding site and enhancer-promoter sequences from the U3 region are deleted.

Figure 2

Experimental scheme to study recombination frequencies for retroviral vectors located in the same or different chromosomal sites.

Figure 3

PCR analysis of plasmid DNA transfected into the packaging cell line GPenv-AM12. A. Analysis of tandemly integrated plasmid DNA. Amplification was performed with a 5' primer specific to neo sequences (T1, unique for pDHEneo) and a 3' primer specific to SV40 early promoter region (T2, unique for pDΔpbsSVpuro). Membrane was hybridized with 3' neo specific probe generated from a 150 bp SalI-ClaI fragment of pDHEneo. ST is GPenv-AM12 virus-producing cell clone ST2-1 generated by sequential transfection of pDHEneo and pDΔpbsSVpuro, and CT is cell clone CT2 generated by cotransfection of the same vectors. Molecular size markers are indicated on the right of the Southern blot. Similar results were obtained when four cell clones were analyzed. B. Control of amplification. Primers specific to the 5'- and 3'-end of neo gene (CND and CNR, respectively) were used to generate PCR products (1.63 kb) from ST and CT DNA samples. Membrane was hybridized with the same probe as in A. PCR products obtained from 200 and 40 ng of ST DNA sample (line 1 and 2); PCR products obtained from 200 and 40 ng of CT DNA sample (line 3 and 4). The result shows that specific PCR products could be amplified both from ST and CT DNA samples with this set of primers.

Figure 4

RT-PCR analysis of virion RNAs. A. Plasmid structures of retroviral leader regions. L1 and L2, primers used for PCR amplification; sizes of DNA fragments and positions of EcoRI sites are indicated. B. Leader sequences in virion RNAs were PCR amplified and analyzed by restriction digestion. PCR products obtained from virion RNAs of ST2-1 and ST2-2 packaging cell clones (lines 1 and 3); PCR products obtained from virion RNAs of CT1 and CT2 cell clones (lines 2 and 4); M, molecular weight markers. The ratios of puro/neo retroviral RNAs for ST2-1, ST2-2, CT1, and CT2 cell clones were 1.8, 2.0, 1.6, and 2.5, respectively.