JOURNAL OF VIROLOGY, June 2007, p. 6623–6631
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 12
Selective and Nonselective Packaging of Cellular RNAs in
Samuel J. Rulli, Jr.,1Catherine S. Hibbert,1Jane Mirro,1Thoru Pederson,2
Shyam Biswal,3and Alan Rein1*
HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland 217021; Department of Biochemistry and
Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 016052; and Department of
Environmental Health Sciences, Bloomberg School of Public Health, and Department of Oncology,
School of Medicine, Johns Hopkins University, Baltimore, Maryland 212053
Received 21 December 2006/Accepted 20 March 2007
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
“??” particles) still contain roughly normal amounts of RNA. We have proposed that cellular mRNAs replace
the genome in ??particles. We have now analyzed the mRNA content of ??and ??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 ??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 ??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 ?5-
to 10-fold higher in ??than in ??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 ??and ??
particles; a minor fraction of this RNA was weakly associated with genomic RNA in ??MLV particles.
During the assembly of infectious, wild-type retrovirus par-
ticles, the viral Gag protein selectively encapsidates the viral
genomic RNA. The selection of this RNA for incorporation
into the nascent virion is presumably due to the recognition by
Gag of a cis-acting “packaging signal,” termed “?,” in the viral
RNA. However, the packaging of this RNA is completely dis-
pensable for efficient particle assembly (22, 26). We previously
reported that murine leukemia virus (MLV) particles formed
in the absence of packageable genomic RNA (“??” particles)
still contain measurable amounts of RNA. Moreover, RNA
appears to play a structural role in MLV particles, as RNase
digestion of detergent-stripped immature particles solubilizes a
substantial fraction of the Gag protein. We observed this effect
both with particles containing genomic RNA (“??” particles)
and with ??particles (29).
What is the nature of the RNA present in virions? In addi-
tion to two copies of genomic RNA, a wild-type particle is
known to contain between 10 and 50 tRNA molecules (two of
which are annealed to the genomic RNA and will serve as
primers for reverse transcription) and at least one molecule of
signal recognition particle (SRP) RNA (initially known as 7SL
RNA), the RNA component of the signal recognition particle
(4, 5, 10, 25, 39). Several cellular mRNAs have also been
detected in purified virus particles (1, 3, 13, 20, 29). It is
significant that retroviral genomic RNA is also the mRNA for
the Gag and Gag-Pol proteins and that, like most cellular
mRNAs, it is capped at its 5? end and polyadenylated at its 3?
Little is known about the mechanism by which Gag selects
??RNA for incorporation into the nascent virion. It seems
possible that the selection of cellular RNAs for incorporation
into retrovirus particles could provide clues regarding this
mechanism. We now present a quantitative analysis of the
cellular RNAs present in both ??and ??MLV-derived virus-
like particles (VLPs). Our analysis indicates that cellular
mRNAs are packaged to replace the genomic RNA in ??
particles. There seems to be very little selectivity in this pro-
cess, as the majority of the thousands of mRNA species
present in VLP-producing cells are incorporated into the VLPs
simply in proportion to their representation in the cells. How-
ever, mitochondrial mRNAs are not packaged in the VLPs. In
addition, a small number of cellular mRNAs are very signifi-
cantly enriched in the VLPs: at least one species is encapsi-
dated at the same efficiency as ??RNA. Despite this enrich-
ment, these species do not constitute a major fraction of the
RNA in the VLPs, because they are at such low levels in the
cells. We also expressed the human immunodeficiency virus
type 1 (HIV-1) Gag protein in mammalian cells and analyzed
the cellular RNAs packaged in the VLPs assembled in these
cells; the results were quite similar to those obtained with
MATERIALS AND METHODS
Production of VLPs. This study used two Moloney MLV proviral clones. The
“??” clone is the protease-negative (PR?) active-site mutant D32L (12), while
the ??clone is a chimera in which the 5? region of the genome, up to the XhoI
site at nucleotide (nt) 1560, is from pPAM3 (27), and the remainder is from the
* Corresponding author. Mailing address: National Cancer Insti-
tute—Frederick, P.O. Box B, Frederick, MD 21702-1201. Phone: (301)
846-1361. Fax: (301) 846-6013. E-mail: firstname.lastname@example.org.
?Published ahead of print on 28 March 2007.
PR?D32L clone. pPAM3 (and thus our ??clone) contains a deletion in the
leader RNA sequence from nt 215 to 535 of the MLV genome (27). All MLV
genomes are in the plasmid vector pGCcos3neo, a derivative of pSV2neo (36).
The HIV-1 Gag protein was expressed from plasmid pCMV55M1-10 (a kind
gift from B. Felber, National Cancer Institute). This plasmid encodes a Rev-
independent HIV-1 gag gene, containing a number of silent mutations eliminat-
ing the Rev requirement, under the control of the cytomegalovirus major late
VLPs were produced by transient transfection of HEK 293T cells using Tran-
sit-293 (Mirus) as recommended by the manufacturer. Twenty-four-hour har-
vests were collected 48 and 72 h after transfection. Supernatants were filtered
through 0.45-?m filters, and VLPs were isolated by pelleting through 20%
sucrose in TNE (10 mM Tris-HCl [pH 7.4]–0.1 M NaCl–1 mM EDTA). The
pellets were redissolved in TNE before further analysis. In some experiments, the
293T cells had previously been stably transfected with the MLV-derived vector
Isolation of RNA from VLPs and Cells. RNA was isolated from VLPs in PK
lysis buffer (50 mM Tris-Cl [pH 7.4], 100 mM NaCl, 10 mM EDTA, 1% sodium
dodecyl sulfate, 100 ?g/?l proteinase K [Ambion]), followed by phenol-chloro-
form extraction, exactly as described previously (12), except that GlycoBlue
(Ambion), rather than tRNA, was used as a carrier in ethanol precipitations.
Extracted RNA was treated with 100 U/ml RQ1 DNase (Promega) for 60 min at
37°C. DNase was then inactivated by the addition of guanidine HCl to 2 M, and
the RNA was reprecipitated before further analysis.
Yields of RNA from VLPs were determined by the Ribogreen (Invitrogen)
assay, which was performed according to the manufacturer’s instructions. Esch-
erichia coli 16S and 23S rRNA provided by the manufacturer were used as a
For the isolation of cellular RNA, transfected cells were first collected in
phosphate-buffered saline and washed by centrifugation. The cellular pellet was
resuspended in 0.5 ml phosphate-buffered saline, and RNA was then extracted
using TRIzol (Invitrogen) according to the manufacturer’s protocol. Cell extracts
were digested with RQ1 DNase, and the RNA was reprecipitated before further
analysis. Cellular RNA was quantitated by measuring the A260. When these RNA
concentrations were checked by Ribogreen assay, the results were in excellent
Isolation of RNA from VLPs for use in Affymetrix microarrays. Each HIV-1 or
MLV (??) VLP RNA sample was analyzed on a separate Affymetrix Human
Genome U133 2.0 Plus array. In each case, the mRNA composition of a VLP
sample was compared with that of the transfected cells that produced the VLPs.
Several protocols were used to prepare the VLP RNA for the microarray
analyses: in one, the extracted RNA was purified on RNeasy spin columns
(QIAGEN) and treated with DNase I; in another, the particles were treated with
DNase I prior to RNA extraction; in a third protocol, the VLPs were digested
with subtilisin (32) and pelleted through 20% sucrose before RNA extraction.
These variations in the experimental procedures appeared to have no effect on
the overall results.
Affymetrix Gene Chip and data analysis. RNAs were first processed using the
Invitrogen SuperScript Double-Stranded cDNA synthesis kit and T7-oligo (dT)24
primers to make double-stranded cDNA. The samples were then labeled using the
Enzo BioArray High Yield RNA Transcript Labeling kit to generate biotinylated
cRNA. After purification by the Affymetrix GeneChip Sample Module Cleanup kit,
12.5 ?g of cRNA was fragmented at 94°C for 35 min. The fragmented cRNA was
placed in a hybridization cocktail and applied to Affymetrix Human U133 Plus 2.0
arrays (over 47,000 transcripts) for 18 h in the GeneChip Hybridization Oven 640
(Affymetrix). Probe signals were amplified using streptavidin-phycoerythrin and bi-
otinylated antibody stains in the GeneChip Fluidics Station 400. Arrays were then
scanned with the GeneChip Scanner 3000. Chip output files were created using
GeneChip Operating software v1.4 and normalized to one before comparison as
previously described (33, 38). In each case, VLP RNA was compared with RNA
from the cells that produced the VLPs. To avoid possible inaccuracies associated
with measurements of extremely rare RNAs, we excluded species with signals in the
cell RNA preparations that were less than 450 from this analysis. Each probe signal
comparison was assigned a call (increase, decrease, or no change) based on the
Wilcoxon signed-rank test P values. Comparisons were further analyzed using the
Affymetrix Data Mining Tool to calculate signal log ratios. These ratios were con-
verted to “fold change” values with the following formula:
fold change ??
2signal log ratio
??1? ? 2??signal log ratio?
signal log ratio ? 0
signal log ratio ? 0
Results from this analysis were virtually identical in replicate experiments in
which the HIV-1 Gag protein was expressed in 293T cells.
Real-time RT-PCR measurements. Typically, each real-time reverse transcrip-
tion-PCR (RT-PCR) reaction mixture contained, in a total of 30 ?l, about the
equivalent of 100 ?l of supernatant or 100 ng of cellular RNA. RNA was reverse
transcribed into DNA using random primers (Invitrogen) and Superscript II
(Invitrogen) in 1? PCR buffer II supplemented with 2.5 mM MgCl2(ABI) and
0.5 mM deoxynucleotide triphosphates (Invitrogen). The DNA was then quan-
titated using real-time PCR. Standard curves were generated by using RNA from
in vitro transcription (Promega) of linearized plasmids containing the target
sequence of interest. For PGK1 (Mammalian Gene Collection clone 3917985),
ASB1 (clone 3842384), and PLEKHB2 (clone 3639839), clones containing the
cDNA were obtained from the NIH Mammalian Gene Collection through In-
vitrogen or the American Type Culture Collection. MLV standards were gener-
ated by in vitro transcription of pMXH linearized with HindIII (11) or from a
small PCR fragment containing the target sites of both the Mo2421 and SRP
(7SL) primer/probe sets. Unincorporated nucleoside triphosphates were re-
moved from RNA standards using G-50 Sephadex spin columns (Roche), and
the RNA transcripts were quantitated by measuring the A260. In some experi-
ments, the transcript was further purified by electrophoresis in 6% Tris-borate-
EDTA–urea polyacrylamide gels, quantitated by Ribogreen, and stored at
?20°C in aliquots. The results were independent of how the standards were
prepared and quantitated. Primers/probes were ordered from Applied Bio-
systems for PGK1 (4310885E), ASB1 (Hs00211548_m1), and PLEKHB2
(Hs00215820_m1) as 20? ready-made primers/probes. Other primer/probe sets
were synthesized by Biosource. Primers and probes are detailed in Table 1.
Real-time RT-PCR results from multiple experiments were pooled to generate
the data presented in this report. Data are presented as means ? standard errors
of the means.
Western blots and quantitation of MLV and HIV-1 Gag. Proteins were re-
solved on 4% to 12% sodium dodecyl sulfate-polyacrylamide gels (Invitrogen),
and MLV Pr65Gagwas detected with rabbit anti-p30CA, while HIV-1 Gag was
detected by using goat anti-p24CAantibody (David Ott, AIDS Vaccine Program,
SAIC–Frederick), followed by the appropriate horseradish peroxidase-labeled
secondary antibody (BioChain Institute, Inc.). The protein of interest was quan-
titated using Supersignal West Dura extended-duration substrate (Pierce) on an
Alpha Innotech Corp. ChemiImager 5500. Highly purified recombinant MLV or
HIV-1 Gag proteins (?p6) (7) (a kind gift of S. A. K. Datta, National Cancer
Institute) were used as absolute standards. Multiple dilutions of samples that fell
within the standard curve were averaged and reported.
Northern analysis of SRP RNA. The association of SRP RNA with the dimeric
genomic RNA extracted from virions was analyzed by nondenaturing Northern
analysis in 1% agarose as described previously (17). In some experiments, the
dimeric RNA was extracted from a gel slice using the Zymoclean gel RNA
recovery kit (Zymo Research). Specific cleavage of the genomic RNA by RNase
H in the presence of an oligodeoxynucleotide complementary to nt 754 to 783
was described previously (18). Viral RNA was detected with a32P-labeled ribo-
probe complementary to nt 215 to 739 of the viral RNA, while SRP RNA was
detected with a32P-labeled riboprobe complementary to human SRP RNA. The
probes were transcribed from a PCR product that included a T7 polymerase
promoter for the transcription of the SRP RNA sequences.
Microarray data accession number. Microarray results have been deposited in
the GEO database (http://www.ncbi.nlm.nih.gov/geo) under the accession num-
Presence of SRP RNA in VLPs. It has long been known that
retrovirus particles contain significant amounts of SRP RNA,
the ?300-nt RNA component of the signal recognition particle
(5, 10). However, the mechanism of its encapsidation is still not
understood. We have quantitated the level of SRP RNA in
both MLV and HIV-1 immature VLPs by real-time RT-PCR.
In agreement with the findings of Onafuwa-Nuga and col-
leagues (30, 31), we found approximately 1 copy of SRP RNA
per copy of genomic RNA in ??MLV particles (see below).
While this roughly 1:1 stoichiometry might suggest that the
association with genomic RNA is responsible for SRP encap-
sidation, we also found, in further agreement with data re-
ported previously by Onafuwa-Nuga et al. (31), that the level of
SRP per ng of Gag protein in ??particles, which have much
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