Selective and nonselective packaging of cellular RNAs in retrovirus particles

Abstract

Assembly of retrovirus particles normally entails the selective encapsidation of viral genomic RNA. However, in the absence of packageable viral RNA, assembly is still efficient, and the released virus-like particles (termed "Psi-" particles) still contain roughly normal amounts of RNA. We have proposed that cellular mRNAs replace the genome in Psi- particles. We have now analyzed the mRNA content of Psi- and Psi+ murine leukemia virus (MLV) particles using both microarray analysis and real-time reverse transcription-PCR. The majority of mRNA species present in the virus-producing cells were also detected in Psi- particles. Remarkably, nearly all of them were packaged nonselectively; that is, their representation in the particles was simply proportional to their representation in the cells. However, a small number of low-abundance mRNAs were greatly enriched in the particles. In fact, one mRNA species was enriched to the same degree as Psi+ genomic RNA. Similar results were obtained with particles formed from the human immunodeficiency virus type 1 (HIV-1) Gag protein, and the same mRNAs were enriched in MLV and HIV-1 particles. The levels of individual cellular mRNAs were approximately 5- to 10-fold higher in Psi- than in Psi+ MLV particles, in agreement with the idea that they are replacing viral RNA in the former. In contrast, signal recognition particle RNA was present at the same level in Psi- and Psi+ particles; a minor fraction of this RNA was weakly associated with genomic RNA in Psi+ MLV particles.

FIG. 1.

Linkage of SRP RNA to MLV dimeric RNA in MLV virions. RNA was extracted from wild-type virions and cleaved with RNase H in the presence of an oligodeoxynucleotide complementary to nt 754 to 783 of the viral genome. The digest was then heated to the temperatures shown above the image and analyzed (A) by nondenaturing Northern blotting using a probe against nt 215 to 739 of viral RNA. The membrane was then stripped and reprobed (B) with a probe complementary to SRP RNA. D, dimeric viral RNA; 5′D, dimer of nt 1 to 754 of viral RNA; 5′F, nt 1 to 754 of viral RNA; S, SRP RNA; Un, unheated.

FIG. 2.

Frequency distribution of fold change values for individual probe sets.

FIG. 3.

Copy numbers of individual RNA species were divided by total amounts of RNA in the respective VLP and cellular RNA preparations.

FIG. 4.

Copy numbers of individual RNA species in MLV VLP preparations were divided by amounts of Gag in the preparation.

FIG. 5.

Encapsidation efficiencies of individual RNAs. Encapsidation efficiency is defined as copy numbers per ng RNA in VLPs divided by copy numbers per ng RNA in the virus-producing cells.

FIG. 6.

Encapsidation of an MLV-derived retroviral vector by MLV and HIV-1 Gag proteins. 293T cells were stably transfected with pLXSH and then transiently transfected with plasmids expressing either Ψ⁻ MLV or HIV-1 Gag. Copies of pLXSH RNA were enumerated using the MLV psi primer/probe set. (A) Copy numbers of individual RNAs per ng of RNA in VLPs or in cells. (B) Encapsidation efficiencies of RNA species.