Determining the Frequency and Mechanisms of HIV-1 and HIV-2 RNA Copackaging by Single-Virion Analysis

Article (PDF Available)inJournal of Virology 85(20):10499-508 · August 2011with22 Reads
DOI: 10.1128/JVI.05147-11 · Source: PubMed
Abstract
HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity. Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, ∼10% of the viral particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimerization or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and HIV-2 RNA copackaging. These results define the cis- and trans-acting elements required for and affecting the heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.

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JOURNAL OF VIROLOGY, Oct. 2011, p. 10499–10508 Vol. 85, No. 20
0022-538X/11/$12.00 doi:10.1128/JVI.05147-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Determining the Frequency and Mechanisms of HIV-1 and HIV-2
RNA Copackaging by Single-Virion Analysis
Kari A. Dilley, Na Ni, Olga A. Nikolaitchik, Jianbo Chen, Andrea Galli, and Wei-Shau Hu*
HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland 21702
Received 18 May 2011/Accepted 8 August 2011
HIV-1 and HIV-2 are derived from two distinct primate viruses and share only limited sequence identity.
Despite this, HIV-1 and HIV-2 Gag polyproteins can coassemble into the same particle and their genomes can
undergo recombination, albeit at an extremely low frequency, implying that HIV-1 and HIV-2 RNA can be
copackaged into the same particle. To determine the frequency of HIV-1 and HIV-2 RNA copackaging and to
dissect the mechanisms that allow the heterologous RNA copackaging, we directly visualized the RNA content
of each particle by using RNA-binding proteins tagged with fluorescent proteins to label the viral genomes. We
found that when HIV-1 and HIV-2 RNA are present in viral particles at similar ratios, 10% of the viral
particles encapsidate both HIV-1 and HIV-2 RNAs. Furthermore, heterologous RNA copackaging can be
promoted by mutating the 6-nucleotide (6-nt) dimer initiation signal (DIS) to discourage RNA homodimeriza-
tion or to encourage RNA heterodimerization, indicating that HIV-1 and HIV-2 RNA can heterodimerize prior
to packaging using the DIS sequences. We also observed that the coassembly of HIV-1 and HIV-2 Gag proteins
is not required for the heterologous RNA copackaging; HIV-1 Gag proteins are capable of mediating HIV-1 and
HIV-2 RNA copackaging. These results define the cis- and trans-acting elements required for and affecting the
heterologous RNA copackaging, a prerequisite for the generation of chimeric viruses by recombination, and
also shed light on the mechanisms of RNA-Gag recognition essential for RNA encapsidation.
The two etiological agents of AIDS, human immunodefi-
ciency virus type 1 (HIV-1) and HIV-2, were introduced into
the human population by zoonotic transmission of simian im-
munodeficiency viruses (SIVs) that naturally infect chimpan-
zees or gorillas (SIV
cpz
or SIV
gor
) and sooty mangabeys
(SIV
sm
), respectively (12, 13, 17, 19, 26, 30, 32). Consequently,
these lentiviruses, which are the only two known to infect
humans, share only limited sequence identity (48% nucleo-
tide [nt] identity at the genome level and 60% amino acid
identity between their Gag polyproteins). Although both vi-
ruses can cause AIDS, HIV-1 is more pathogenic in humans
and is distributed worldwide, whereas HIV-2 infection is lim-
ited geographically and mostly located in West Africa (20).
However, infection with one of the HIVs does not provide
protection from infection with the other HIV. Indeed, there is
a significant population of people who are dually infected with
both HIV-1 and HIV-2 (11, 14). HIV-1 and HIV-2 target the
same cell types, using both the same receptor and the same
coreceptors for virus entry (1, 31, 34). The possible interplay
between these two viruses has been an intriguing topic of
research aimed at understanding the extent and the mecha-
nisms of such molecular interactions.
One unique feature of retrovirus replication is that two cop-
ies of the full-length RNA are packaged into a viral particle.
The specific packaging of the viral RNA, which makes up less
than 1% of the total cellular RNA, is mediated by at least two
determinants. One determinant is the viral structural protein
Gag and especially the nucleocapsid domain in the polypro-
tein. The second determinant is the packaging signal in the
viral RNA, most of which resides at the 5 end of the viral
genome, including part of the 5 untranslated region, and often
extends into part of gag. The interactions between Gag and
viral RNA ensure the selection and encapsidation of the
proper RNAs into the particles. Retroviral RNA packaging is
generally specific and limited to the RNAs of the same virus or
closely related viruses. However, cross-packaging does occur,
including two known examples of nonreciprocal RNA packag-
ing of distantly related viruses; one set is HIV-1 and HIV-2
(18), and the other set is murine leukemia virus (MLV) and
spleen necrosis virus (SNV) (3). SNV Gag polyprotein can
efficiently package both SNV and MLV RNA; however, MLV
Gag polyprotein does not package SNV RNA. Similarly, it has
been shown that HIV-1 Gag can package HIV-2 RNA,
whereas HIV-2 Gag does not package HIV-1 RNA (18). How-
ever, certain aspects of RNA packaging mechanisms used by
HIV-1 and HIV-2 are similar. HIV-1 RNA has been shown to
dimerize or select its copackaged RNA partner prior to encap-
sidation (4, 23, 24). A palindromic sequence located at the top
of stem-loop 1 (SL1), termed the dimerization initiation signal
(DIS), plays an important role during the RNA partner selec-
tion process (6, 8). HIV-1 RNAs that have complementary DIS
sequences can copackage together more efficiently than those
that do not (7, 23, 28). It is thought that the DIS sequences of
the two RNAs form intermolecular base pairings to initiate the
dimerization or the RNA partner selection process. Our recent
studies showed that the RNA packaging mechanisms of HIV-2
have many features similar to those of HIV-1, including RNA
partner selection prior to encapsidation and the involvement of
a palindromic sequence in this process (27).
It has been shown that HIV-1 and HIV-2 can interact mo-
lecularly in several ways. For example, despite sharing a low
* Corresponding author. Mailing address: HIV Drug Resistance
Program, NCI-Frederick, P.O. Box B, Building 535, Room 336, Fred-
erick, MD 21702. Phone: (301) 846-1250. Fax: (301) 846-6013. E-mail:
Wei-Shau.Hu@nih.gov.
Published ahead of print on 17 August 2011.
10499
level of amino acid sequence identity, the Gag polyproteins
from HIV-1 and HIV-2 can coassemble into the same particle
and complement each other’s functions to carry out infection
(2). It has also been shown in a cell culture system that HIV-1
and HIV-2 can recombine and generate hybrid genomes, albeit
at a rate far lower than that occurring between two HIV-1 or
between two HIV-2 genomes (25). Retroviral recombination is
the result of the reverse transcriptase (RT) switching between
the two copackaged RNA templates during DNA synthesis.
Therefore, the observation that HIV-1 and HIV-2 can recom-
bine indicates that the RNA genomes of these two different
viruses must be able to be packaged into the same particle
(heterologous RNA copackaging). However, it was unclear
how frequently these heterologous RNAs can be copackaged
and whether elements in the viral RNAs exist that constrain
such copackaging. Furthermore, it is unclear whether the coas-
sembly of the HIV-1 and HIV-2 Gag polyproteins is required
to promote copackaging of the heterologous RNAs by each
interacting with its own RNA.
In this study, we used the single-virion analysis for direct
measurement of the frequency of HIV-1 and HIV-2 RNA
copackaging. This system, based on labeling HIV-1 and HIV-2
genomes with fluorescent protein-tagged RNA-binding pro-
teins, offers a direct measurement of viral RNA content at
single-virion resolution. Unlike standard biochemical assays
that measure the efficiency with which a given RNA is pack-
aged, this microscopy-based method allows us to determine
whether two RNAs are copackaged into the same virion. We
found that when HIV-1 and HIV-2 RNAs are packaged at
similar levels, 10% of the particles contained both HIV-1 and
HIV-2 RNA. The heterologous RNA copackaging frequency
can be influenced by the identities of the DIS sequences, in-
dicating that HIV-1 and HIV-2 RNAs can heterodimerize us-
ing the DIS sequences prior to packaging. We also determined
that HIV-1 Gag alone is sufficient for heterologous RNA co-
packaging.
MATERIALS AND METHODS
Viral vectors and plasmid construction. The HIV-1 constructs GagCeFP-
MS2SL and Gag-MS2SL have been previously described (4); for clarity, in this
report these plasmids are referred to as HIV-1–GagCeFP-MS2SL and HIV-1–
Gag-MS2SL, respectively. Briefly, these two constructs were derived from the
molecular clone NL4-3; portions of the pol, vif, vpr, vpu, and env genes were
deleted, rendering the encoded proteins nonfunctional. Additionally, 24 copies
of the stem-loop recognized by the bacteriophage MS2 coat protein were in-
serted in the pol gene. All cis-acting elements required for HIV-1 replication,
including the packaging signal, are present in these genomes. The two plasmids
have similar structures, except the HIV-1–GagCeFP-MS2SL expresses Gag
tagged with cerulean fluorescent protein (CeFP), whereas HIV-1–Gag-MS2SL
expresses untagged Gag. In all experiments, tagged Gag-CeFP and untagged Gag
are coexpressed to avoid possible distortions of viral particle morphology. For
brevity, only GagCeFP-expressing constructs are mentioned.
The mutant with altered DIS sequences, HIV-1–DIS6C–GagCeFP-MS2SL,
was derived from HIV-1–GagCeFP-MS2SL by changing the GCGCGC sequence
in the DIS into CCCCCC. This was done by replacing the SphI-XhoI DNA
fragment in 6CDIS–GagCeFP-BglSL with the corresponding fragment from
HIV-1–GagCeFP-MSL; both the Gag-CeFP and the untagged Gag versions were
generated (4). A Gag mutant, HIV-1–noGag-MSL, which contains a stop codon
after amino acid 109 of CA, was generated by introducing a 4-bp frameshift
mutation in the gag gene of HIV-1–Gag-MS2SL. This was achieved by digesting
HIV-1–Gag-MS2SL with SpeI, followed by a fill-in reaction using the Klenow
fragment of Escherichia coli polymerase and DNA ligation.
The HIV-2 constructs 2-GagCeFP-BglSL, 2-Gag-BglSL, and 2-6G-GagCeFP-
BSL have been previously described (27); for clarity, these constructs are re-
ferred to as HIV-2–GagCeFP-BglSL, HIV-2–Gag-BglSL, and HIV-2–DIS6G-
GagCeFP-BglSL in this report. These two constructs were derived from the
ROD12 molecular clone and contained all cis-acting elements important for viral
replication and inactivating mutations in pol, vpr, and env. Additionally, both
constructs contained 18 copies of stem-loops (BglSL) in pol recognized by the E.
coli BglG protein. Construct HIV-2–GagCeFP-BglSL expresses Gag tagged with
CeFP, whereas HIV-2–Gag-BglSL expresses untagged Gag (27). As with HIV-1,
only the GagCeFP construct is mentioned, although both CeFP-tagged HIV-2
Gag and wild-type HIV-2 Gag were coexpressed in all experiments.
Construct HIV-2–GagCeFP-BglSL-noTatRev was generated by digesting
HIV-2–GagCeFP-BglSL plasmid DNA with BsmBI, followed by a fill-in reaction
using the Klenow fragment from E. coli DNA polymerase and DNA ligation.
This procedure generated an inactivating frameshift mutation in both tat and
rev. The HIV-2–DIS6G-GagCeFP-BglSL-noTatRev construct was generated us-
ing the same procedure by starting with HIV-2–DIS6G-GagCeFP-BglSL plas-
mid. The HIV-2–noGag-BglSL was derived from a previously described 2*T-17
mutant that contained two stop codons in gag, one at codon 17 of Gag in MA and
one at codon 171 of CA (27). DIS mutants in the HIV-2–noGag-BglSL context
were constructed using 2-step PCR. Primers harboring the DIS mutations were
used to amplify the HIV-2–noGag-BglSL template, yielding a 2.4-kb DNA frag-
ment containing DIS mutations and both stop codons in Gag that was cloned into
the HIV-2–GagCeFP-BglSL backbone by the use of the NgoMIV and SwaI
restriction sites. The general structures of the newly generated plasmids were
determined by restriction enzyme mapping, and regions generated by PCR were
characterized by DNA sequencing to avoid inadvertent mutations.
The MS2-yellow fluorescent protein (MS2-YFP) construct was a generous gift
from Robert Singer (Albert Einstein Medical College); the Bgl-mCherry plasmid
has been previously described (4).
Cell culture, transfection, and virus production. The 293T human embryonic
kidney cell line was grown in a humidified 37°C incubator with 5% CO
2
and
maintained in Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 g/ml). In all
experiments, plasmids expressing Gag and GagCeFP were cotransfected at a 1:1
molar ratio; DNA transfections were performed using FuGeneHD (Roche)
according to manufacturer’s recommendations. Supernatants were harvested
from 293T cells 19 to 20 h posttransfection, clarified through a 0.45-m-pore-size
filter, and either stored at 80°C or used immediately for image acquisition.
Single-virion analyses. To prepare particles for microscopy, clarified culture
supernatant was mixed with Polybrene and the mixture was plated on a 35-mm-
diameter glass-bottom plate (MatTek Corp.) and incubated at 37°C for 2 h. An
inverted Nikon Eclipse Ti microscope with a 100 oil objective and a numerical
aperture of 1.40 and an X-Cite 120 system (EXFO Photonic Solutions Inc.) were
used for image capture as previously described (27). The filter settings were
427/10 nm and 480/40 nm (CeFP), 504/12 nm and 542/27 nm (YFP), and 577/25
nm and 632/60 nm (mCherry) for excitation and emission, respectively. Digital
images were acquired by the use of an AndOr technology iXon camera and NIS
Element AR software (Nikon). Custom software developed with Matlab was
used to identify particles and their colocalization with RNA.
RESULTS
Determining HIV-1 and HIV-2 RNA genome copackaging
frequency. To measure the frequency of HIV-1 and HIV-2
RNA copackaging, we used two previously described con-
structs that express near-full-length HIV-1 and HIV-2 ge-
nomes containing stem-loop sequences recognized by RNA-
binding proteins (Fig. 1A) (4, 27). These viral genomes allowed
us to fluorescently tag and directly visualize encapsidated RNA
in individual viral particles. The modified HIV-1 genome HIV-
1–GagCeFP-MS2SL expresses HIV-1 Gag tagged with CeFP,
Tat, and Rev; additionally, this construct also contains 24 cop-
ies of MS2SL, the stem-loop sequence specifically recognized
by the bacteriophage MS2 coat proteins. The modified HIV-2
genome, HIV-2–GagCeFP-BglSL, expresses viral gene prod-
ucts, including HIV-2 Gag tagged with CeFP, Tat, and Rev;
additionally, it contains 18 copies of BglSL, the stem-loop
sequence specifically recognized by the E. coli BglG protein.
Two versions of each viral construct were generated; one ex-
10500 DILLEY ET AL. J. VIROL.
presses wild-type Gag and the other expresses Gag tagged with
CeFP. In all experiments, GagCeFP and Gag were coexpressed
to preserve normal particle morphology; for simplicity, only
the names of the GagCeFP constructs are mentioned.
To examine the RNA content in the HIV particles, viral
constructs were transfected into 293T cells along with two
plasmids that express fluorescently tagged RNA-binding pro-
teins, MS2-YFP and Bgl-mCherry (Fig. 1B). The supernatant
was harvested 19 to 20 h posttransfection and clarified, and
images of viral particles were obtained using fluorescence mi-
croscopy. Examples of images of viral particles obtained from
293T cells transfected with HIV-1–GagCeFP-MS2SL, MS2-
YFP, and Bgl-mCherry are shown in the upper panels of Fig.
1C. As a portion of the HIV-1 Gag polyproteins were tagged
with CeFP, viral particles can be identified by their signals in
the CeFP channel. The full-length RNA expressed by HIV-1–
GagCeFP-MS2SL contained sequences recognized by the MS2
coat protein; as a result, the majority (90%) of the CeFP
particles also contained YFP signals but not mCherry signals
(summarized in Table 1). In contrast, HIV-2 construct HIV-
2–GagCeFP-BglSL contains sequences recognized by Bgl pro-
teins; hence, most of the CeFP
particles generated from
cotransfection of HIV-2–GagCeFP-BglSL, MS2-YFP, and
Bgl-mCherry also contained mCherry signals but not YFP sig-
nals (Fig. 1C, middle panels; Table 1). These results showed
that most of the HIV particles contained viral RNA ge-
nomes; furthermore, the signals observed from MS2-YFP or
Bgl-mCherry were specific to the stem-loops in the viral
genomes and had little background and nonspecific labeling.
We then generated particles by coexpressing HIV-1–
GagCeFP-MS2SL, HIV-2–GagCeFP-BglSL, MS2-YFP, and
Bgl-mCherry (Fig. 1C, lower panels). Most of the CeFP
par
-
ticles also had either YFP or mCherry signals, indicating that
they contained either HIV-1 or HIV-2 RNA, respectively;
some of the CeFP
particles were positive for YFP and
mCherry signals, indicating that they contained both HIV-1
and HIV-2 RNA. As the relative levels of abundance of these
two viral RNAs affect the copackaging frequency, in our ex-
periments, we used only results from experiments that showed
similar levels of HIV-1 and HIV-2 RNA signals. In 13 inde-
pendent experiments, between 6% and 13% of the particles in
the viral population exhibited CeFP, YFP, and mCherry sig-
nals, with an average of 9.7% and standard deviation of 2.8%
(Table 1).
Examining the influence of DIS on copackaging of heterol-
ogous viral RNAs. In both HIV-1 and HIV-2, RNA partner
selection occurs prior to the encapsidation of the viral genome;
additionally, the DIS sequences play a major role in the selec-
tion process (4, 6, 8, 23, 24, 27). However, previous experi-
ments examined copackaging of RNAs derived from the same
viral species; it is unclear whether DIS can affect copackaging
of heterologous viral RNAs. We sought to determine whether
RNAs from genetically distinct viruses can interact through
DIS sequences and be copackaged. The sequences of the 5
untranslated regions of HIV-1 and HIV-2 have little homol-
ogy; the two molecular clones we used had 42% nucleotide
sequence identity, but the two sequences had similar predicted
RNA structures (Fig. 2A and B). The DIS sequences for
HIV-1 and HIV-2 are discordant; the subtype B HIV-1 used in
this study has a GCGCGC sequence whereas HIV-2 has a
gag-cefp
env
rev
tat
HIV-1-GagCeFP-MS2SL
pol
Bgl-mCherry
NLS
MS2-YFP
NLS
gag-cefp
env
rev
tat
vpx
vif
HIV-2-GagCeFP-BglSL
B.
pol
HIV-1
HIV-2
HIV-1 + HIV-2
CeFP
mCherry
YFP
merged
and
shifted
CeFP
mCherryYFP
CeFP
mCherryYFP
merged
and
shifted
merged
and
shifted
gag-cefp
env
rev
tat
HIV-1-GagCeFP-MS2SL
pol
Bgl-mCherry
NLS
Bgl-mCherry
NLS
MS2-YFP
NLS
NLS
gag-cefp
env
rev
tat
vpx
vif
HIV-2-GagCeFP-BglSL
pol
HIV-1
HIV-2
HIV-1 + HIV-2
CeFP
mCherry
YFP
merged
and
shifted
CeFP
mCherryYFP
CeFP
mCherryYFP
merged
and
shifted
merged
and
shifted
A.
C.
FIG. 1. System used to determine HIV-1 and HIV-2 RNA copack-
aging efficiency. (A) General structures of the HIV constructs. HIV-1
construct HIV-1–GagCeFP-MS2SL contains in its RNA stem-loops
recognized by the bacteriophage MS2 coat protein. HIV-2 construct
HIV-2–GagCeFP-BglSL contains in its RNA stem-loops recognized by
the E. coli BglG protein. Both constructs express Gag tagged with
CeFP. In all experiments, these constructs were coexpressed with sister
constructs that were identical except that they expressed wild-type Gag
proteins without CeFP. For simplicity, the constructs expressing wild-
type Gag are not shown. White boxes represent HIV-1 sequences,
whereas black boxes represent HIV-2 sequences. (B) General struc-
tures of RNA-binding proteins tagged with fluorescent proteins
containing nuclear localization signals (NLS) at the C terminus.
(C) Representative images of single-virion analyses. MS2-YFP and
Bgl-mCherry were cotransfected along with viral constructs in all sam-
ples. All three channels were merged and shifted (the YFP and
mCherry channels were shifted 5 and 10 pixels to the right, respec-
tively) as shown in the fourth column to illustrate the RNA signals
associated with each particle.
V
OL. 85, 2011 HIV-1 AND HIV-2 HETEROLOGOUS RNA COPACKAGING 10501
GGTACC sequence (Fig. 2). Using an in vitro assay, it was
previously shown that RNA dimerization occurs only when
HIV-1 and HIV-2 RNA contain the same DIS (9). To examine
whether the frequency of heterologous RNA copackaging can
be altered, we generated viral constructs containing various
DIS sequences and tested the abilities of their RNAs to be
copackaged with RNA from another virus. We first mutated
the HIV-2 DIS sequence to GCGCGC so that this mutant
(HIV-2–DIS3GC-GagCeFP-BglSL; Fig. 2D) had the same
DIS sequence as the HIV-1 construct HIV-1–GagCeFP-
MS2SL. We found that the presence of matching and comple-
mentary DIS sequences did not have a significant effect on the
frequency of heterologous RNA copackaging; approximately
12% of the particles contained both HIV-1 RNA and HIV-2
RNA with GCGCGC at the DIS (range in 7 experiments, 10%
to 14%) (Table 2).
Although mutating the HIV-2 DIS sequence to GCGCGC
may have allowed base pairing of the DIS sequences in the
HIV-1 and HIV-2 RNA, this mutation did not interfere with
the ability of two RNAs from the same virus to base pair at the
DIS sequences. To simultaneously promote heterodimeriza-
tion and discourage homodimerization of the viral RNAs, we
mutated the HIV-1 DIS sequence to CCCCCC (HIV-1–
DIS6C–GagCeFP-MS2SL; Fig. 2C) and the HIV-2 DIS se-
quence to GGGGGG (HIV-2–DIS6G-GagCeFP-BglSL; Fig.
2D), coexpressed these constructs with RNA-binding proteins,
and examined the resulting viruses. Indeed, the copackaging
frequency of these two RNAs increased to 31% (range in 9
TABLE 1. Single-virion analyses of HIV-1 and HIV-2 RNA copackaging
Coexpressed constructs
a
CeFP
particles
RNA labeling
efficiency
c
No.
analyzed
YFP
(%)
mCherry
(%)
YFP
mCherry
(%)
b
HIV-1–GagCeFP-MS2SL
Expt 1 1,005 93.2 0.0 0.0 93.2
Expt 2 1,772 93.7 0.1 0.3 94.1
Expt 3 1,786 92.9 0.0 0.1 93.0
Expt 4 2,369 92.0 0.0 0.0 92.0
Expt 5 5,032 92.8 0.0 0.0 92.8
Expt 6 5,060 91.8 0.0 0.0 91.8
Expt 7 4,568 91.6 0.0 0.0 91.6
Expt 8 5,578 91.5 0.0 0.2 91.7
Expt 9 7,505 91.3 0.0 0.3 91.6
Expt 10 3,655 92.3 0.0 0.1 92.4
Expt 11 8,138 93.0 0.0. 0.0 93.0
Expt 12 20,159 91.4 0.0 0.4 91.8
Expt 13 16,632 92.0 0.0 0.0 92.0
HIV-2–GagCeFP-BglSL
Expt 1 1,644 0.1 92.7 0.4 93.2
Expt 2 2,902 0.0 93.7 0.2 93.9
Expt 3 5,708 0.0 93.8 0.1 93.9
Expt 4 4,738 0.0 93.3 0.3 93.6
Expt 5 6,467 0.0 92.7 0.5 93.2
Expt 6 1,077 0.0 91.8 1.0 92.8
Expt 7 8,240 0.0 92.8 0.4 93.2
Expt 8 10,027 0.0 95.6 0.3 95.9
Expt 9 15,232 0.0 91.4 0.2 91.6
Expt 10 15,107 0.0 94.1 0.0 94.1
Expt 11 2,384 0.0 92.1 0.5 92.6
HIV-1–GagCeFP-MS2SL HIV-2–
GagCeFP-BglSL
Expt 1 1,025 43.8 44.4 6.1 94.3
Expt 2 1,251 45.2 42.0 5.7 92.9
Expt 3 1,651 53.0 34.0 5.6 92.6
Expt 4 3,072 32.8 50.7 9.5 93.0
Expt 5 5,983 50.4 33.6 8.0 92.0
Expt 6 12,462 36.6 46.0 10.2 92.8
Expt 7 15,366 44.2 37.6 10.9 92.7
Expt 8 13,505 49.9 32.1 10.9 92.9
Expt 9 7,259 52.1 33.0 8.0 93.1
Expt 10 12,956 37.4 41.1 13.1 91.6
Expt 11 12,245 36.9 42.6 12.9 92.4
Expt 12 10,749 35.4 44.3 12.1 91.8
Expt 13 10,431 36.0 42.4 12.7 91.1
a
Although not indicated, both Bgl-mCherry and MS2-YFP were coexpressed in all experiments.
b
Average plus standard deviation (SD) for HIV-1-GagCeFP-MS2SL HIV-2-GagCeFP-BglSL, 9.7 2.8.
c
Calculated by adding the values from the three columns under the CeFP
heading.
10502 DILLEY ET AL. J. VIROL.
independent experiments, 28% to 33%) (Table 2). These re-
sults suggested that the increased copackaging of HIV-1 and
HIV-2 RNAs is the result of heterodimerization that occurs
before RNA encapsidation and is not the result of the pack-
aging of one HIV-1 and one HIV-2 RNA each as a monomer.
The increased copackaging of HIV-1–DIS6C–GagCeFP-
MS2SL and HIV-2–DIS6G-GagCeFP-BglSL can occur through
two distinct mechanisms. The presence of CCCCCC in HIV-1
RNA and GGGGGG in HIV-2 RNA can encourage RNA
heterodimerization or discourage RNA homodimerization or
both. To distinguish among those possible mechanisms, we
measured the copackaging frequency of HIV-1 and HIV-2
RNAs when both genomes contained CCCCCC in their DIS
sequences, that is, HIV-1–DIS6C–GagCeFP-MS2SL and HIV-
2–DIS6C-GagCeFP-BglSL (Fig. 2D). Both RNAs derived
from these viruses were discouraged from homodimerization;
however, the lack of complementarity of the two DIS se-
quences also do not encourage heterodimerization of these
two RNAs. In 8 experiments using the particles generated by
coexpressing the two constructs containing CCCCCC in DIS
sequences, we observed a modest increase in the heterologous
RNA copackaging frequency to 16% (range, 14% to 18%)
(Table 2).
Taken together, these data indicate that the copackaging of
HIV-1 and HIV-2 RNAs can be influenced by the sequence at
the DIS. Simply allowing intermolecular interaction by chang-
ing the HIV-2 DIS to the same sequence as the HIV-1 DIS had
little effect on the copackaging frequency, indicating that other
elements are present that favor RNA homodimerization. The
level of heterologous RNA copackaging was increased slightly
when the DIS was mutated to discourage RNA homodimeriza-
tion. However, it was when the two DIS sequences were mu-
tated to simultaneously discourage RNA homodimerization
and encourage RNA heterodimerization that we observed the
largest increase of the copackaging frequency of HIV-1 and
HIV-2 RNAs.
Determining the roles of HIV-1 and HIV-2 Gag polyproteins
in heterologous RNA copackaging. We previously showed that
HIV-1 and HIV-2 Gag can coassemble into the same particle
(2). It is possible that the coassembly of these two Gag pro-
teins, by each interacting with its corresponding RNA, can
promote the copackaging of HIV-1 and HIV-2 RNAs. To
investigate the individual and collective contributions of HIV-1
and HIV-2 Gag proteins in heterologous RNA copackaging,
we examined the effects of eliminating one of the Gag proteins
in the system. We first examined the RNA encapsidation that
resulted when only HIV-2 Gag was present in the system. It
was previously shown that HIV-2 Gag does not package HIV-1
RNA (18); however, it is unclear whether HIV-1 RNA pack-
aging can be promoted by interactions with HIV-2 RNA. For
this purpose, we introduced an inactivating frameshift muta-
tion into the gag gene of the HIV-1 construct to generate
HIV-1–noGag-MS2SL (Fig. 3A), which can produce near-full-
length viral RNA with the packaging signal but does not ex-
press functional Gag proteins. As expected, HIV-1–noGag-
MS2SL did not generate CeFP
particles but its RNA was able
to be packaged into particles when coexpressed with another
HIV-1 that expresses Gag (data not shown). We then tested
whether heterologous RNA copackaging can be observed
when only HIV-2 Gag was present. HIV-1–noGag-MS2SL,
HIV-2–GagCeFP-BglSL, MS2-YFP, and Bgl-mCherry were
cotransfected into 293T cells, and the resulting viruses were
examined by single-virion analysis. We found that over 90% of
the CeFP
particles had mCherry signals, indicating that
HIV-2 RNA was packaged efficiently in these viruses; however,
we did not observe a significant level of YFP signals, indicating
FIG. 2. Examining the role of the DIS in copackaging of HIV-1 and
HIV-2 RNA. (A) Predicted secondary structure of a portion of the 5
leader sequences of HIV-1, containing stem-loop 1, stem-loop 2, and
stem-loop 3 (labeled SL1, SL2, and SL3) (33). (B) Predicted secondary
structure of a portion of the 5 leader of HIV-2 containing stem-loop
1, stem-loop 2, and stem-loop 3 (labeled SL1, SL2, and SL3) (9). DIS,
dimer initiation signal. Nucleotides that comprise the DIS are out-
lined. (C) General structures of HIV-1 constructs and their DIS se-
quences. The wild-type DIS sequence (GCGCGC) of HIV-1–
gagCeFP-MS2SL is shown for comparison. (D) General structures of
HIV-2 constructs and their DIS sequences. The wild-type HIV-2 DIS
sequence (GGUACC) is shown as a reference.
VOL. 85, 2011 HIV-1 AND HIV-2 HETEROLOGOUS RNA COPACKAGING 10503
that HIV-1 RNA was not packaged efficiently, and copackag-
ing was not observed (Table 3).
To address the possibility that HIV-1 RNA may not have
been expressed in the cells transfected with HIV-2 and there-
fore not packaged, we generated HIV-2–GagCeFP-BglSL-no-
TatRev, a construct that contains a frameshift mutation in the
overlapping region of the HIV-2 tat and rev genes, to abolish
the expression of functional Tat and Rev (Fig. 3B). Lacking
functional Tat and Rev, HIV-2–GagCeFP-BglSL-noTatRev
does not produce Gag and therefore does not generate viral
particles (data not shown). However, when cotransfected with
HIV-1–noGag-MS2SL that expressed functional Tat and Rev,
HIV-2–GagCeFP-BglSL-noTatRev was able to produce viral
particles. This strategy ensures the expression of HIV-1 RNA
in the virus-producing cells. Analyses of these particles re-
vealed abundant mCherry signals, indicating efficient packag-
ing of HIV-2 RNA but very little YFP signals, indicating the
lack of encapsidation of HIV-1 RNA (Table 3). To investigate
whether HIV-1 RNA packaging can be rescued by base pairing
in the DIS sequences with HIV-2 RNA, we generated HIV-1–
DIS6C-noGag-MS2SL and HIV-2–DIS6G-GagCeFP-BSL-no-
TatRev, two variants containing DIS sequences that, when
both HIV-1 and HIV-2 Gag proteins were expressed, pro-
moted heterologous RNA packaging. Single-virion analyses
revealed that the complementary DIS mutations were not suf-
ficient to promote HIV-1 RNA packaging to detectable levels
(Table 3).
Next, we examined the effects of eliminating HIV-2 Gag in
heterologous RNA copackaging experiments. We generated
HIV-2–noGag-MS2SL, which contains two inactivating muta-
tions in the gag gene: a substitution mutation in the MA-
encoding region that generated a stop codon and a frameshift
in the CA-encoding region that disrupts the Gag reading
frame. We then analyzed particles generated by coexpressing
HIV-1–GagCeFP-MS2SL, HIV-2–noGag-MS2SL, MS2-YFP,
and Bgl-mCherry and observed a heterologous RNA copack-
aging frequency of 10% (range, 9% to 11%) (Table 4), which
is similar to the frequency observed when both HIV-1 and
HIV-2 Gag were present (Table 1). We then generated HIV-2
constructs that contained mutations in DIS sequences (Fig.
3C) and examined whether these mutations had the same ef-
fects on RNA copackaging when only HIV-1 Gag was present
in the system. Along with MS2-YFP and Bgl-mCherry, coex-
pression of HIV-1–DIS6C–GagCeFP-MS2SL and HIV-2–
DIS6C-noGag-BglSL resulted in 16% heterologous RNA
copackaging (Table 4), whereas coexpression of HIV-1–
TABLE 2. The effects of DIS sequences on HIV-1 and HIV-2 RNA copackaging
Coexpressed constructs
a
CeFP
particles
RNA labeling
efficiency
c
Total no.
analyzed
YFP
(%)
mCherry
(%)
YFP
mCherry
(%)
b
HIV-1–GagCeFP-MS2SL HIV-2–
DIS3GC-GagCeFP-BglSL
Expt 1 10,420 35.0 45.9 12.4 93.3
Expt 2 7,558 42.6 38.6 12.2 93.4
Expt 3 5,866 47.9 32.5 11.3 91.7
Expt 4 8,644 47.6 33.1 12.5 93.2
Expt 5 9,474 49.7 33.1 9.6 92.4
Expt 6 11,034 39.2 37.5 13.6 90.3
Expt 7 11,664 33.4 45.6 12.6 91.6
HIV-1–DIS6C-GagCeFP-MS2SL
HIV-2–DIS6G-GagCeFP-BglSL
Expt 1 2,762 26.2 34.2 31.9 92.3
Expt 2 2,853 24.1 40.7 28.6 93.4
Expt 3 2,896 28.7 33.4 31.9 94.0
Expt 4 6,605 36.6 26.4 31.0 94.0
Expt 5 6,647 36.0 25.0 33.0 94.0
Expt 6 6,387 32.4 29.2 32.3 93.9
Expt 7 5,095 37.7 26.6 28.5 92.8
Expt 8 4,806 35.2 29.3 28.5 93.0
Expt 9 5,702 36.3 26.2 31.1 93.6
HIV-1–DIS6C-GagCeFP-MS2SL
HIV-2–DIS6C-GagCeFP-BglSL
Expt 1 3,085 35.4 38.7 16.9 91.0
Expt 2 3,374 36.7 37.3 17.7 91.7
Expt 3 3,259 35.0 40.7 15.1 90.8
Expt 4 6,100 35.7 40.6 14.4 90.7
Expt 5 4,209 39.8 35.2 16.3 91.3
Expt 6 4,609 41.0 33.7 15.3 90.0
Expt 7 8,688 34.1 42.2 14.8 91.1
Expt 8 9,626 33.2 42.5 16.6 92.3
a
Although not indicated, both Bgl-mCherry and MS2-YFP were coexpressed in all experiments.
b
Average SD for HIV-1–GagCeFP-MS2SL HIV-2–DIS3GC-GagCeFP-BglSL, 12.0 1.3; average SD for HIV-1–DIS6C-GagCeFP-MS2SL HIV-2–
DIS6G-GagCeFP-BglSL, 30.8 1.8; average SD for HIV-1–DIS6C-GagCeFP-MS2SL HIV-2–DIS6C-GagCeFP-BglSL, 15.9 1.2.
c
Calculated by adding the values from the three columns under the CeFP
heading.
10504 DILLEY ET AL. J. VIROL.
DIS6C–GagCeFP-MS2SL and HIV-2–DIS6G-noGag-BglSL
resulted in 39% RNA copackaging (Table 4). The introduc-
tion of DIS mutations in the absence of HIV-2 Gag influenced
copackaging in a manner parallel to that observed when both
HIV-1 and HIV-2 Gag were present. Therefore, HIV-1 Gag
alone is most likely responsible for HIV-1/HIV-2 RNA co-
packaging; although coassembly of HIV-1 and HIV-2 Gag
occurs frequently, this event is not driving heterologous RNA
copackaging.
DISCUSSION
During the assembly process of all known retroviruses, Gag
proteins select from a large pool of total cellular RNAs and
package two copies of full-length, single-stranded viral RNA
into viral particles. In some viruses, such as HIV-1 and HIV-2,
RNA selects its copackaged partner (or dimerizes) prior to
encapsidation (4, 23, 27). Considering the specificity of this
process, the RNA-RNA and RNA-protein interactions in-
volved are likely to be highly regulated and coordinated events.
The packaging of two distinct retroviral RNAs into the same
virus particle requires at least a partial overlap, spatially as well
as temporally, of the two RNAs in the producer cell, as well as
similar packaging determinants and mechanisms of the two
viruses. The work described in this report explored the factors
that influence the copackaging of two divergent human retro-
viruses, HIV-1 and HIV-2. The copackaging of the heterolo-
gous RNAs is a determinant for potential genetic interactions;
FIG. 3. General structures of constructs used to examine the roles
of HIV-1 and HIV-2 Gag proteins in heterologous RNA copackaging.
(A) HIV-1 constructs that do not express functional Gag. These con-
structs harbor a frameshift mutation in gag. (B) HIV-2 constructs that
do not express functional Tat and Rev. These constructs harbor a
frameshift mutation that simultaneously prevents expression of func-
tional HIV-2 Tat and that of functional Rev. (C) HIV-2 constructs that
do not express functional Gag and their DIS sequences. Each construct
contains two mutations in gag to prevent expression of functional Gag.
DIS sequences of the constructs are indicated. Frameshift mutations
are indicated by asterisks.
TABLE 3. Single-virion analyses of RNAs packaged by HIV-2 Gag
Coexpressed constructs
a
CeFP
particles
RNA labeling
efficiency
c
Total no.
analyzed
YFP
(%)
mCherry
(%)
YFP
mCherry
(%)
b
HIV-1–noGagCeFP-MS2SL
HIV-2–GagCeFP-BglSL
Expt 1 2,405 0.0 92.6 0.9 93.5
Expt 2 835 0.0 96.3 0.2 96.5
Expt 3 1,227 0.0 92.3 2.1 94.4
Expt 4 1,089 0.0 93.8 1.0 94.8
Expt 5 909 0.0 97.4 0.9 98.3
Expt 6 614 0.0 98.4 0.0 98.4
Expt 7 1,548 0.0 91.5 1.2 92.7
HIV-1–noGagCeFP-MS2SL
HIV-2–GagCeFP-BglSL-
noTatRev
Expt 1 495 0.0 97.0 0.6 97.6
Expt 2 428 0.0 93.9 1.4 95.3
Expt 3 391 0.0 98.0 0.3 98.3
Expt 4 1,176 0.0 90.3 0.7 91.0
HIV-1–DIS6C-noGagCeFP-MS2SL
HIV-2–DIS6G-GagCeFP-BglSL-
noTatRev
Expt 1 687 0.0 91.7 0.9 92.6
Expt 2 527 0.0 91.7 0.8 92.5
Expt 3 1,146 0.0 94.9 1.6 96.5
Expt 4 810 0.0 90.6 1.5 92.1
a
Although not indicated, both Bgl-mCherry and MS2-YFP were coexpressed in all experiments.
b
Average SD for HIV-1–noGagCeFP-MS2SL HIV-2–GagCeFP-BglSL, 0.9 0.7; average SD for HIV-1–noGagCeFP-MS2SL HIV-2–GagCeFP-BglSL-
noTatRev, 0.8 0.5; average SD for HIV-1–DIS6C-noGagCeFP-MS2SL HIV-2–DIS6G-GagCeFP-BglSL-noTatRev, 1.2 0.4.
c
Calculated by adding the values from the three columns under the CeFP
heading.
VOL. 85, 2011 HIV-1 AND HIV-2 HETEROLOGOUS RNA COPACKAGING 10505
furthermore, this system allowed us to study the mechanisms of
retroviral RNA dimerization and packaging.
In this study we found that, within a population of particles
containing similar proportions of HIV-1 and HIV-2 RNAs,
about 10% of the particles contained both HIV-1 and HIV-2
RNAs. Equal expression and random assortment of RNAs
from two proviruses predict that 50% of the virions would be
heterozygous particles, containing one RNA derived from each
provirus. The 10% copackaging frequency implies that HIV-1
and HIV-2 RNA packaging mechanisms have sufficient over-
lap to yield a modest but reproducible level of copackaging but
are divergent enough that HIV-1 and HIV-2 RNAs still prefer
to homodimerize. We have previously measured recombina-
tion betwen HIV-1 and HIV-2 using two modified genomes,
each carrying a mutated green fluorescent protein (gfp) gene;
recombination between the two mutated genes could reconsti-
tute a functional gfp gene, and its expression could be mea-
sured. We found that a very small percentage (0.5%) of
infected cells had a GFP
phenotype (25), which is much lower
than that generated between two HIV-1 or two HIV-2 viruses
(7%) (5, 29). In all three studies, the gfp gene was used as a
target sequence for reporting recombination; thus, regardless
of the sequence diversity elsewhere in the viral genomes, the
target regions between the mutations in the gfp genes contain
identical sequences. Results from this study indicate that
10% of the particles have copackaged HIV-1 and HIV-2
RNAs and that the low copackaging frequency was a contrib-
uting factor but not the only one that caused the lower recom-
bination rate. Together, these studies revealed that there is a
block or barrier in the reverse transcription step that limits the
completion of DNA synthesis or recombination in the target
gfp gene between these heterologous genomes. The details of
the mechanism for this block or barrier are currently unknown.
It is possible that the two copackaged RNAs have different
structures, thereby inhibiting DNA synthesis or preventing RT
from switching templates; alternatively, it is also possible that
the low sequence homology flanking the gfp genes reduces RT
template switching within the gfp gene. Further experimenta-
tion is needed to distinguish between these possibilities.
In this report, we studied factors that may restrict the het-
erodimerization of HIV-1 and HIV-2 RNA and thus restrict
copackaging. We found that changing the DIS to the same pal-
indrome (GCGCGC) to allow the DIS sequences of HIV-1 and
HIV-2 RNA to form base pairs did not increase the copackaging,
most likely because the two RNAs still homodimerize efficiently.
The largest improvement of heterologous RNA copackaging
was observed when we simultaneously encouraged RNA het-
erodimerization and discouraged homodimerization by changing
TABLE 4. Single-virion analyses of RNAs packaged by HIV-1 Gag
Coexpressed constructs
a
CeFP
particles
RNA labeling
efficiency
c
Total no.
analyzed
YFP
(%)
mCherry
(%)
YFP
mCherry
(%)
b
HIV-1–GagCeFP-MS2SL HIV-2–
noGagCeFP-BglSL
Expt 1 1,243 46.3 33.8 10.1 90.2
Expt 2 349 22.6 57.9 9.5 90.0
Expt 3 1,399 45.6 36.7 10.1 92.4
Expt 4 1,405 47.0 35.9 9.1 92.0
Expt 5 1,240 42.5 40.9 9.0 92.4
Expt 6 2,839 27.2 53.9 8.9 90.0
Expt 7 2,376 45.5 37.7 9.9 93.1
Expt 8 4,052 27.8 54.8 9.7 92.3
Expt 9 2,016 47.2 37.1 9.0 93.3
Expt 10 4,701 53.9 26.9 10.3 91.1
Expt 11 5,218 58.0 23.7 10.5 92.2
Expt 12 6,564 48.9 31.7 10.5 91.1
Expt 13 6,789 42.7 38.7 9.3 90.7
HIV-1–DIS6C-GagCeFP-MS2SL
HIV-2–DIS6C-noGagCeFP-BglSL
Expt 1 3,152 45.4 34.1 12.7 92.2
Expt 2 4,006 47.8 30.3 15.4 93.5
Expt 3 5,282 48.0 29.2 17.5 94.7
Expt 4 5,703 45.6 33.1 15.8 94.5
Expt 5 6,045 44.7 27.5 18.5 90.7
HIV-1–DIS6C-GagCeFP-MS2SLL
HIV-2–DIS6G-noGagCeFP-BglSL
Expt 1 3,881 29.4 28.7 36.7 94.8
Expt 2 4,589 33.7 21.5 39.8 95.0
Expt 3 5,334 34.4 22.7 38.3 95.4
Expt 4 5,020 29.6 26.1 38.8 94.5
Expt 5 4,862 24.8 28.5 41.1 94.4
a
Although not indicated, both Bgl-mCherry and MS2-YFP were coexpressed in all experiments.
b
Average SD for HIV-1–GagCeFP-MS2SL HIV-2–noGagCeFP-BglSL, 9.7 0.6; average SD for HIV-1–DIS6C-GagCeFP-MS2SL HIV-2–DIS6C-
noGagCeFP-BglSL, 16.0 2.2; average SD for HIV-1–DIS6C-GagCeFP-MS2SLL HIV-2–DIS6G-noGagCeFP-BglSL, 38.9 1.6.
c
Calculated by adding the values from the three columns under the CeFP
heading.
10506 DILLEY ET AL. J. VIROL.
the HIV-1 DIS to CCCCCC and the HIV-2 DIS to GGGGGG.
These results not only revealed that DIS sequences can mediate
dimerization of the heterologous RNAs but also shed light on the
mechanisms of Gag-RNA interactions.
Although RNA partner selection occurs prior to the encap-
sidation of HIV-1 or HIV-2 RNA, it is unclear how HIV Gag
proteins distinguish between monomeric and dimeric RNAs.
Studies of MLV packaging showed that the monomeric and
dimeric RNAs have very different secondary structures (10, 15,
21). Furthermore, dimerization of MLV RNAs exposes high-
affinity binding sites for nucleocapsids, which is critical for
RNA packaging (16, 22). It is thought that exposure of these
sites allows Gag binding, leading to the incorporation of RNA
into virus particles. Despite its attractiveness, this model has
been difficult to extend to the RNA packaging mechanisms of
HIV-1, mainly because structural analyses of HIV-1 RNA have
not revealed a drastic difference between monomeric RNA
and dimeric RNA (35); hence, the relationship between
switching the RNA states and RNA-Gag molecular recogni-
tion is not well defined. Our study revealed surprising insights
into this process. Despite the low homology of HIV-1 and
HIV-2 leader sequences, when DIS sequences were manipu-
lated to form base pairing between these two heterologous
RNAs but not between homologous RNAs, we observed in-
creased copackaging. If the HIV-1 and MLV Gag-RNA rec-
ognition mechanisms are similar, then our results suggest that
base pairing of the DIS between these two heterologous RNAs
can expose the hidden high-affinity binding sites recognized by
HIV-1 Gag. The limited homology between the 5 leader se-
quences of HIV-1 and HIV-2 makes it less likely that there are
many other conserved interactions between these two RNAs;
hence, this hypothesis implies that base pairing of DIS provides
a molecular switch that directly or indirectly exposes Gag-
binding sites.
A possible model for the mechanism of heterologous RNA
copackaging is that such events are driven by the coassembly of
Gag polyproteins from different viruses. However, our results
indicated that HIV-1 Gag, in the absence of HIV-2 Gag, can
mediate heterologous RNA copackaging at levels similar to
those observed with both HIV-1 and HIV-2 Gag proteins. This
observation defines the trans-acting element required for the
heterologous RNA copackaging, that is, the Gag protein that
recognizes its own RNA and the heterologous RNA.
Replication strategies used by retroviruses ensure frequent
generation of recombinants, as one viral particle has two full-
length viral genomes and RT switches templates during DNA
synthesis. Although copackaged RNAs are derived from the
same types of viruses most of the time, there are infrequent
occurrences of heterologous RNA copackaging, which pro-
vides the backdrop for the generation of chimeric viruses from
two distantly related viruses. Delineating the requirements and
factors that influence heterologous RNA copackaging allows
us to understand the potential for the genetic interactions
between distantly related viruses and also provide insights into
the general mechanisms of RNA-RNA and RNA-Gag inter-
actions that lead to encapsidation of viral genomes.
ACKNOWLEDGMENTS
We thank Vinay K. Pathak for discussions throughout the project
and critical reading of the manuscript.
This research was supported in part by the Intramural Research
Program of the NIH, National Cancer Institute, Center for Cancer
Research; IATAP funding, NIH.
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10508 DILLEY ET AL. J. VIROL.
    • "Of course, not all viral processes necessarily proceed from a wellmixed pool of viral proteins (e.g., certain poliovirus proteins required for genome synthesis are not efficiently complemented in trans [20]). Nevertheless, in viral processes where mass-action mixing readily occurs, if two viral variants are present in a cell, their genomes inevitably compete for shared viral products, such as capsid and envelope proteins [21,22]. This competition between viral genomes can generate a fitness cost, as illustrated by a phenomenon described in the 1940s by both Henle and von Magnus. "
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    Full-text · Article · May 2016
    • "HIV-2 genomic RNA is packaged as a dimer into the virions and, as in other retroviruses, Gag is involved in genome dimerization and packaging [35, 37, 38]. It was presented that, similarly to HIV-1, the intact NC domain within the uncleaved HIV-2 Gag confers specific binding of dimerization and Ψ signals via its two zinc finger motifs [37] . "
    Full-text · Article · Mar 2016
  • [Show abstract] [Hide abstract] ABSTRACT: Influenza A virus possesses a segmented genome of eight negative-sense, single-stranded RNAs. The eight segments have been shown to be represented in approximately equal molar ratios in a virus population; however, the exact copy number of each viral RNA segment per individual virus particles has not been determined. We have established an experimental approach based on multicolor single-molecule fluorescent in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Colocalization analysis showed that a high percentage of virus particles package all eight different segments of viral RNAs. To determine the copy number of each RNA segment within individual virus particles, we measured the photobleaching steps of individual virus particles hybridized with fluorescent probes targeting a specific viral RNA. By comparing the photobleaching profiles of probes against the HA RNA segment for the wild-type influenza A/Puerto Rico/8/34 (PR8) and a recombinant PR8 virus carrying two copies of the HA segment, we concluded that only one copy of HA segment is packaged into a wild type virus particle. Our results showed similar photobleaching behaviors for other RNA segments, suggesting that for the majority of the virus particles, only one copy of each RNA segment is packaged into one virus particle. Together, our results support that the packaging of influenza viral genome is a selective process.
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