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Retrovirology

BioMed Central

Open Access

Research

Cross-packaging of genetically distinct mouse and primate
retroviral RNAs
Noura Salem Al Dhaheri, Pretty Susan Phillip, Akela Ghazawi, Jahabar Ali,
Elizabeth Beebi, Soumeya Ali Jaballah and Tahir A Rizvi*
Address: Department of Microbiology & Immunology, Faculty of Medicine and Health Sciences (FMHS), United Arab Emirates University (UAEU),
Al Ain, UAE
Email: Noura Salem Al Dhaheri - 200301922@uaeu.ac.ae; Pretty Susan Phillip - prettys@uaeu.ac.ae; Akela Ghazawi - akelag@uaeu.ac.ae;
Jahabar Ali - jahabar@uaeu.ac.ae; Elizabeth Beebi - elizabithb@uaeu.ac.ae; Soumeya Ali Jaballah - 200770003@uaeu.ac.ae;
Tahir A Rizvi* - tarizvi@uaeu.ac.ae
* Corresponding author

Published: 14 July 2009
Retrovirology 2009, 6:66

doi:10.1186/1742-4690-6-66

Received: 22 March 2009
Accepted: 14 July 2009

This article is available from: http://www.retrovirology.com/content/6/1/66
© 2009 Al Dhaheri et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: The mouse mammary tumor virus (MMTV) is unique from other retroviruses in
having multiple viral promoters, which can be regulated by hormones in a tissue specific manner.
This unique property has lead to increased interest in studying MMTV replication with the hope of
developing MMTV based vectors for human gene therapy. However, it has recently been reported
that related as well as unrelated retroviruses can cross-package each other's genome raising safety
concerns towards the use of candidate retroviral vectors for human gene therapy. Therefore, using
a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and
propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV) that has intracellular
assembly process similar to that of MMTV.
Results: Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the
heterologous virus particles reciprocally suggesting that pseudotyping between two genetically
distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could
not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To
further confirm that the specificity of cross-packaging was conferred by the packaging sequences
(ψ), we cloned the packaging sequences of these viruses on expression plasmids that generated
non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was
primarily due to the recognition of ψ by the heterologous virus proteins.
Conclusion: The results presented in this study strongly argue that MPMV and MMTV are
promiscuous in their ability to cross-package each other's genome suggesting potential RNAprotein interactions among divergent retroviral RNAs proposing that these interactions are more
complicated than originally thought. Furthermore, these observations raise the possibility that
MMTV and MPMV genomes could also co-package providing substrates for exchanging genetic
information.

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Retrovirology 2009, 6:66

Background
The mouse mammary tumor virus (MMTV) is a betaretrovirus that has been primarily implicated in causing breast
cancer and, to some extent, T-cell lymphomas in mice,
reviewed in Mustafa et al., [1,2]. Classically, MMTV has
been categorized as a simple retrovirus containing only
the structural and regulatory genes needed to complete
the virus life cycle. However, recently, it has been proposed that MMTV be reclassified as a more complex
murine retrovirus because of the presence of accessory/
regulatory factors such as sag [3,4], Naf [5], and the
recently identified Rem/RmRE regulatory pathway of
MMTV [6-8]. In addition, it has recently been shown that
Rev and Rex proteins of human complex retroviruses can
interact with MMTV Rem responsive element [9]. Furthermore, MMTV is unique from other simple and complex
retroviruses in harboring several promoters for the expression of its various gene products [10-12]. Of these several
promoters, two have been identified in the long terminal
repeats (LTR) and two in the envelope region, reviewed in
Mustafa et al., [1]. The LTR promoters are under the influence of steroid hormones in a tissue-specific manner due
to the presence of hormone responsive elements (HREs),
making them inducible [13]. Therefore, the tissue-specific
and inducible MMTV promoters have lead to increased
interest in studying MMTV replication with the ultimate
goal of developing MMTV based vectors for their potential
use in targeted gene therapy. In one recent study, MMTV
promoters have been utilized in murine leukemia virus
(MLV) based vectors used for targeted enzyme prodrug
therapy both in vivo and in vitro [14]. The use of MMTV
based vectors would not only provide tissue-specific and
inducible expression of the therapeutic gene, but also its
non-primate nature may circumvent potential safety concerns. Such concerns include cross-packaging and copackaging of the transfer vector RNA genome by related
primate retroviruses or retrovirus-like elements resulting
in the generation of recombinant variants with unknown
pathogenic potential.
Packaging of retroviral genomic RNA by the assembling
virus particles is a crucial step in the virus life cycle. RNA
packaging among retroviruses is unique and a highly specific phenomenon during which two copies of full length
"unspliced" genomic RNA are preferentially packaged
from amongst a wide pool of cellular and other spliced
viral RNAs, reviewed by D'Souza and Summers [15] and
Lever [16]. The specificity towards RNA packaging by the
newly assembling viral particles is conferred by the recognition of specific cis-acting sequences, the packaging signal (ψ), present at the 5'end of the viral genome, and the
nucleocapsid (NC) protein is responsible for discriminating between spliced and unspliced viral RNA, reviewed by
D'Souza and Summers [15] and Lever [16]. Despite this
specificity, in some cases, it has been shown that evolu-

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tionary related, however molecularly different, retroviruses can cross- and co-package each other's genome
suggesting that phylogenetically related retroviruses are
capable of using similar protein-and RNA-packaging elements, reviewed by D'Souza and Summers [15] and Lever
[16]. Such cross- and co-packaging among retroviruses
have been shown to exchange genetic information resulting in recombinant variants [17,18] undermining many
advantages of using retroviral vectors in human gene therapy studies. The use of phylogenetically distant nonhuman retroviral vectors, such as those based on MMTV,
should minimize the chances of recombination with
unrelated primate exo-and/or endogenous retroviruses.
In spite of the advantages of using non-human retroviral
vectors with inducible tissue-specific promoters as in the
case of MMTV, so far, no detailed studies have been conducted to investigate the ability of MMTV RNA to be crosspackaged in human cells by heterologous primate retroviral proteins. However, in one earlier report, Günzburg and
Salmons have reported that MMTV RNA could not be
packaged by Moloney murine leukemia virus (MoMLV)
packaging cell lines [19]. Such limited information
regarding MMTV RNA packaging and cross-packaging
studies in the literature can be attributed to 1) a trans complementation assay for MMTV was not developed until
recently, and 2) MMTV is not expressed very efficiently in
human cells due to the promoter's low transcriptional
activity. In order to overcome these drawbacks, we have
successfully replaced the U3 region of MMTV 5'LTR with
the human cytomegalovirus (hCMV) promoter in MMTV
based vectors to allow for its efficient expression in
human cells; and we have also developed a three-plasmid
trans complementation assay for MMTV to study its RNA
packaging and propagation [20]. Using this in vivo packaging and transduction assay, we investigated the ability
of MMTV RNA to be cross-packaged by a primate retrovirus, the Mason-Pfizer monkey virus (MPMV) that, like
MMTV, also preassembles in the cytoplasm before budding. Our results showed that both of these viruses could
cross-package each other's RNAs. However, the crosspackaged RNA could not be propagated further and therefore failed to transduce the target cells suggesting a block
at post RNA packaging events of the retroviral life cycle
such as reverse transcription and/or integration. Our
results further demonstrated that this cross-packaging specificity was conferred specifically by the packaging
sequences, which were in turn recognized by the heterologous proteins; since cloning of these sequences in plasmids, which generate non-viral RNAs, resulted in the
encapsidation of these non-viral RNAs by MMTV and
MPMV proteins reciprocally. The results presented in this
study strongly suggest that MPMV and MMTV are promiscuous in their ability to cross-package each other's
genome and that interactions between retroviral RNAs

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and Gag polyprecursors are more complicated than originally thought.

Results and discussion
In vivo packaging and transduction assay for MMTV and
MPMV
To study cross-packaging between MMTV and MPMV, we
used three-plasmid trans complementation assays developed earlier by our laboratory [20,21]. These MMTV and
MPMV trans complementation assays consist of a packaging construct, pJA10 or pTR301, which expresses either
MMTV or MPMV gag/pol genes, respectively, which results
in the production of viral particles, which are capable of
encapsidating viral RNA containing ψ. The source of the
packageable RNA is provided by MMTV (pDA024,
pSS013) and MPMV (pKAL11, pSS015) transfer vectors
(Figure 1A and 1B). These transfer vectors contain the
sequences responsible for RNA packaging, in addition to
the cis-acting sequences needed for viral replication,
which include Primer Binding Site (PBS), Poly Purine
Tract (PPT) (needed for reverse transcription), and U3 and
U5 attachment (att) sites (required for integration). In
addition, these transfer vectors express hygromycin resistance and/or enhanced green fluorescence protein (EGFP)
gene from an internal simian virus (SV40) promoter (SVhygr/SV-EGFP), which allows for the monitoring of the
successful propagation of the transfer vector RNAs by

A

rem

MMTV
Genome
pDA024

gag

U3 R U5

U3 R U5

env

pol

sag

sag

CMV

R U5

400 bp gag

hygr

SV

U3 R U5

transducing the target cells with these marker genes. An
envelope expression plasmid (MD.G) based on vesicular
stomatitis virus envelope G (VSV-G) was also used to enable the study of steps involved in both packaging and
propagation of the transfer vector RNAs [22].
Briefly, in these assays, the three plasmids are co-transfected into 293T producer cells, which will generate virus
particles containing the encapsidated RNA, the replication
of which is limited to a single round because reinfection
of the target cells cannot take place. These virus particles
can be used to: 1) directly examine the viral RNA content
in the virus particles using reverse transcriptase polymerase chain reaction (RT-PCR) and 2) to infect target cells
resulting in the transduction of these cells with the marker
gene, thus allowing for monitoring the propagation of the
transfer vector RNA. The number of Hygromycin-resistant
(Hygr) colonies and/or EGFP positive cells obtained
should be directly proportional to the amount of RNA
that is packaged into the virus particles providing an indirect estimate of RNA packaging.
MMTV RNA can be cross-packaged but cannot be
propagated by MPMV proteins
To determine whether MMTV RNA can be cross-packaged
by the heterologous primate retrovirus (MPMV) proteins,
we co-transfected MMTV transfer vectors (pDA024 and

B
MPMV
Genome

gag

U3 R U5

env

pol

pKAL011

U3 R U5

282 bp gag

SV

hygr

U3

CMV

R U5

400 bp gag

SV

U3 R U5

EGFP

pSS015

U3 R U5

282 bp gag

SV

U3

EGFP

5’ UTR

CMV

pNF008

CMV

R U5

pND015

CMV

R U5

pND016

CMV

pND001

BGHPoly A

EGFP

400 bp gag

NotI

NotI

R U5

CMV

R U5

CMV

R U5

hygr

SV

CMV

5’ UTR

pND011

U3 R U5

SV

U3 R U5

hygr

U3 R U5

hygr

U3 R U5

pND013

pND014

U3 R U5

CTE

hygr

SV

hygr

MPMV PBS &

U3 R U5
CTE

NotI

SV

MPMV

U3 R U5
CTE

NotI

MMTV PBS &

U3 R U5

BGHPoly A

EGFP
SV

MMTV

NotI

U3 R U5
CTE

BGHPoly A

EGFP

NotI

NotI

CTE

SV

282 bp gag

282 bp gag

NotI

CTE

NotI
MMTV PBS &

5’ UTR

R U5

NotI

pND012

hygr

NotI
MMTV

U3 R U5

pND002

CTE

NotI

NotI

pND018

SV

MPMV PBS &
NotI

pND017

CTE

NotI
MPMV

BGHPoly A

EGFP

400 bp gag

CMV

CTE

CTE
5’ UTR

R U5

CTE

CTE

pNF007

R U5

CTE

CTE

pSS013

U3 R U5
CTE

hygr

U3 R U5
CTE

NotI

SV

hygr

U3 R U5
CTE

Figure 1
Schematic representation of MMTV and MPMV based vectors
Schematic representation of MMTV and MPMV based vectors. (A) MMTV genome, MMTV transfer vectors, and
expression plasmids containing MMTV or MPMV packaging signal. (B) MPMV genome, MPMV transfer vectors, and expression
plasmids containing MPMV or MMTV packaging signal. The design and construction of these vectors is described in materials
and methods and can be further obtained from authors upon request. CMV, human cytomegalovirus promoter; SV, Simian
virus 40 promoter; hygr, hygromycin resistance gene; CTE, constitutive transport element from Mason-Pfizer monkey virus
(MPMV); EGFP, enhanced green fluorescence protein gene; BGH, bovine growth hormone.

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Retrovirology 2009, 6:66

pSS013) with MPMV packaging construct (pTR301) along
with the envelope expression plasmid (MD.G) into 293T
cells. As a control, MMTV transfer vectors were also cotransfected with the homologous packaging construct
(pJA10) along with the envelope expression plasmid.
Supernatants from the transfected cultures were used to
isolate the viral RNA to determine vector RNA packaging
and to infect human HeLa CD4+ cells in order to study
vector RNA propagation.
To ensure that the transfer vector RNAs are efficiently and
stably expressed and properly transported from the
nucleus to the cytoplasm, RNAs from the transfected cells
were fractionated into cytoplasmic and nuclear fractions.
To verify that there was no contaminating plasmid DNA
in our cytoplasmic RNA preparations, which may confound the interpretation of our results, cytoplasmic RNAs
were treated with RNase free DNase and were PCR amplified. The lack of a positive PCR signal, following 30 cycles
of amplification, indicated that the contamination in our
cytoplasmic RNA preparations is below the detection level
(Figure 2A). After making the cDNA, we confirmed that
the transfer vector RNAs were properly transported from
the nucleus to the cytoplasm by ensuring that no compromise was made on the integrity of the nuclear membrane
during the fractionation process based on the absence of
unspliced β-actin mRNA in the cytoplasmic RNA fraction
as detected by RT-PCR. Unspliced β-actin mRNA is found
exclusively in the nucleus, while the spliced form is found
in both the nucleus and the cytoplasm [23]. To ensure that
each cytoplasmic sample in the unspliced β-actin PCRs
contained amplifiable cDNA, therefore, as an ancillary
control, PCRs were conducted for 25 cycles in the presence of primers/competimers for 18S ribosomal RNA. Figure 2B shows that there was a total lack of unspliced βactin message in the cytoplasmic fraction (upper panel)
suggesting that there was no leakage of RNA from the
nucleus. The presence of spliced β-actin mRNA observed
in the cytoplasmic fraction (lower panel) confirmed that
the transfer vector RNAs were properly transported to the
cytoplasm. To exclude the possibility of poor expression
and/or instability of the transfer vector RNAs, cDNAs prepared from the cytoplasmic fractions were amplified using
viral specific primers and were found to be stably
expressed (Figure 2C).
Having confirmed that all transfer vector RNAs were stably expressed and efficiently transported to the cytoplasm,
we examined the ability of MPMV proteins to cross-package MMTV transfer vector RNAs. Like cytoplasmic RNA
fractions, viral RNAs were treated with DNase, reverse
transcribed, and amplified using viral specific primers for
varying number of cycles. In addition, Southern blotting
was performed on the PCR products using transfer vector
RNA specific probe as described previously [24]. Test of

http://www.retrovirology.com/content/6/1/66

the transfer vector RNAs packaged into the viral particles
by RT-PCR revealed that MPMV proteins (pTR301) were
able to cross-package MMTV transfer vector (pDA024 and
pSS013) RNAs. The cross-packaged RNAs, following RTPCR, could be visualized by ethidium bromide staining
within 20 cycles of PCR, but Southern blotting was
needed to appreciate cross-packaging after 15 PCR cycles
(Figure 2E). However, the level of cross-packaging efficiency was lower when compared to MMTV vectors
(pDA024 and pSS013) packaged by the homologous
MMTV proteins (pJA10) (Figure 2E). Taken together,
these results demonstrated that MMTV transfer vector
RNAs could be cross-packaged by MPMV proteins within
the detectable range.
Since our vectors contained a hygromycin resistance marker
or an EGFP gene, we examined the propagation of these
vectors by infecting the target cells with supernatants produced by the transfected cells. Following infection, if the
vectors were properly propagated, these marker genes
(hygromycin resistance gene in the case of pDA024 and
EGFP gene in the case of pSS013) will transduce the target
cells resulting in Hygr colonies or EGFP positive cells suggesting the successful completion of crucial steps of the
viral life cycle such as reverse transcription and integration
following RNA packaging. As expected, the propagation of
the packaged MMTV transfer vector RNAs by homologous
MMTV proteins was readily observed as evidenced by the
presence of Hygr colonies in the case of pDA024 and EFGP
positive cells in the case of pSS013 (Table 1) indicating
that the MMTV transfer vectors are capable of efficiently
expressing the marker genes. On the other hand, the lack
of Hygr colonies or EGFP positive cells when MMTV vector
(pDA024 and pSS013) RNAs were cross-packaged by
MPMV proteins (pTR301) suggested that the cross-packaged RNAs could not be propagated (Table 1).
MPMV RNA can be cross-packaged but cannot be
propagated by MMTV proteins
To determine whether MMTV proteins can cross-package
MPMV RNA or not, MPMV transfer vectors (pKAL011 and
pSS015) were co-transfected along with MMTV packaging
construct (pJA10) and the envelope expression plasmid
MD.G. In parallel, as a control, MPMV transfer vectors
were also co-transfected with their homologous packaging
construct (pTR301) and the envelope expression plasmid.

After confirming the absence of any contaminating plasmid DNA in our cytoplasmic and viral RNA preparations
(Figure 3A and 3D), we confirmed that all MPMV transfer
vector RNAs were efficiently transported to the cytoplasm
and were stably expressed (Figure 3B and 3C). Next, we
investigated the ability of MPMV RNA to be cross-packaged by MMTV proteins by directly examining the viral
RNA content in MMTV particles. RT-PCR results in figure

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Spliced Actin

pDA024

pSS013

pTR301

pSS013

pJA10

-ve Control

C

pDA024

RT-PCR for Fr actionation Contr ol

MMTV Cytoplasmic cDNAs

+ve Control

Mock

pSS013

pSS013

pDA024

Mock

pSS013

MPMV
Proteins
(pTR301)

pDA024

Unspliced Actin

pSS013

18S rRNA

MMTV
Proteins
(pJA10)

pDA024

E

-ve Control

Nuclear
cDNA

pSS013

pDA024

DNase-Tr eated Vir al RNAs

pTR301

pSS013

-ve Control

B

pJA10

pDA024

DNase-Tr eated Cytoplasmic RNAs

pTR301

pJA10

pDA024

D

-ve Control

pSS013

pTR301

pDA024

pDA024

pSS013

pJA10

-ve Control

A

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+ ve Control

Retrovirology 2009, 6:66

Agarose
15X
Southern
Agarose
20X
Southern
25X

Agarose
MMTV Vir al cDNAs

Figure 2
MMTV transfer vectors RNA can be cross-packaged by MPMV proteins
MMTV transfer vectors RNA can be cross-packaged by MPMV proteins. (A) PCR amplification of cytoplasmic RNAs
treated with DNase to ensure the absence of any contaminating DNA in the RNA preparations using primers OTR537 and
OTR538. (B) Control for nucleocytoplasmic RNA fractionation technique to ensure that the transfer vector RNAs were
properly transported to the cytoplasm. Upper panel represents the multiplex RT-PCR for unspliced β-actin mRNA (found
exclusively in the nucleus) and 18S ribosomal RNA as a control for the presence of amplifiable cDNA in the PCR reactions as
described in the materials and methods and results sections. Unspliced β-actin was not detected in the cytoplasmic RNA fraction, ensuring that the transfer vector RNAs were properly transported to the cytoplasm without any compromise on the
integrity of the nuclear membrane. The lower panel represents RT-PCR on cytoplasmic RNA for spliced β-actin mRNA and
should be present in both nuclear and cytoplasmic fractions. (C) RT-PCR of cytoplasmic cDNA amplified using MMTV specific
primers to ensure that the transfer vector RNAs were stably expressed. (D) PCR amplification of DNase treated viral RNAs
to confirm the absence of any contaminating plasmid DNA carried over from the transfected cultures. (E) RT-PCR of viral
cDNAs amplified using virus specific primers and probed with the PCR product amplified using the same set of primers and
HYB MTV as a template. Amplifications were conducted for 15, 20, and 25 cycles and, in addition to agarose gel, Southern
blots are also shown. For this set of experiment, while amplifying DNase treated viral RNAs, cytoplasmic and viral cDNAs,
primers OTR643 and OTR676 were used and should amplify 585 bp fragment.
3E demonstrated that MPMV transfer vector (pKAL011
and pSS015) RNAs were cross-packaged by MMTV proteins (pJA10). Consistent with the results obtained for
MMTV RNA cross-packaging, the efficiency of the crosspackaged MPMV vector RNAs was lower when compared
to the homologous MPMV vector (pKAL011 and pSS015)
RNAs being packaged by its own proteins (pTR301) (Figure 3E). The cross-packaging efficiency of MPMV RNA by
MMTV proteins appeared to be lower when compared to
MMTV RNAs being cross-packaged by MPMV proteins
since Southern blotting was needed to appreciate crosspackaging after 20 cycles of PCR instead of the 15 cycles

needed to demonstrate the cross-packaging of MMTV
RNA by MPMV proteins (Figures 2E and 3E).
Similar to the results we obtained for MMTV cross-packaged RNA, MPMV cross-packaged transfer vector
(pKAL011 and pSS015) RNAs could not be propagated
further as evidenced by the lack of Hygr colonies or EGFP
positive cells in the infected cultures (Table 1). The presence of Hygr colonies or EGFP positive cells in the infected
cultures when MPMV transfer vector (pKAL011 and
pSS015) RNAs were packaged by its own proteins
(pTR301) assured that the marker genes were efficiently
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Table 1: Propagation of MMTV and MPMV transfer vectors RNA by homologous and heterologous proteins.

Titers (CFU/ml)a, b

Transfer Vector Description of the Transfer
Vector

% EGFP
Positive Cellsc

MMTV Protein
(JA10)

MPMV Protein
(TR301)

MMTV Protein
(JA10)

MPMV Protein
(TR301)

pDA024

Chimeric LTR, 5' region upto
400 bp of MMTV Gag, and
SV-hygr

3,676 ± 196

<1

-

-

pSS013

Chimeric LTR, 5' region upto
400 bp of MMTV Gag, and
SV-EGFP

-

-

19

<1

pND015

Same as DA024 but the putative
MMTV ψ has been replaced
with that of MPMV
(5' UTR + 282 bp of Gag)

2

8

-

-

pND016

Same as DA024 but the putative
MMTV ψ and PBS have been
replaced with that of MPMV

<1

7

-

-

pND017

Control MMTV vector in which
putative MMTV ψ has been
cloned back after creating NotI
site at PBS/UTR junction

187 ± 33

ND

-

-

pND018

Control MMTV vector in which
putative MMTV ψ and PBS have
been cloned back after creating
NotI site at U5/PBS junction

30 ± 4

ND

-

-

pKAL011

MPMV 5' region upto 282 bp
Gag and SV-hygr

<1

36,373 ± 3,972

-

-

pSS015

Same as KAL011 but has
SV-EGFP instead of SV-hygr

-

-

<1

41

pND011

Same as KAL011 but MPMV ψ
has been replaced with that of
MMTV
(5' UTR + 400 bp of Gag)

<1

3

-

-

pND012

Same as KAL011 but MPMV ψ
and PBS have been replaced
with that of MMTV

<1

23

-

-

pND013

Control MPMV vector in which
MPMV ψ has been cloned back
after creating NotI site at PBS/
UTR junction

ND

2813 ± 99

-

-

pND014

Control MPMV vector in which
MPMV ψ and PBS have been
cloned back after creating NotI
site at U5/PBS junction

ND

1660 ± 111

-

-

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Table 1: Propagation of MMTV and MPMV transfer vectors RNA by homologous and heterologous proteins. (Continued)

pTR174

Control vector containing
SV-hygr cassette and SIV 3'LTR
as poly(A)

<1

<1

-

-

pAG001

Control vector containing SVhygr cassette and FIV 3'LTR as
poly(A)

<1

<1

-

-

Mock

No DNA (control)

<1

<1

<1

<1

a CFU/ml, colony forming units per milliliter of non-concentrated supernatant from the transfected cultures. b Each value represents a mean of three
experiments performed in duplicates. c Percentage of EGFP positive cells represent the phenotypic analysis of the transduced target cells. Single cell
suspensions of the infected HeLa CD4+ cells were prepared, and 10,000 events (cells) per group were counted and analyzed using Becton Dickinson
FACS. < 1 indicates < 1 Hygr colonies in the target cells or < 1 EGFP positive cells out of 10,000 events (cells) analyzed.

expressed and that there was no compromise on the integrity of our transduction assay. Therefore, it is reasonable
to conclude that the absence of Hygr or EGFP positive cells
reflected a true lack of propagation of MPMV cross-packaged vector RNAs. A similar situation has been reported
earlier when MPMV RNA could be cross-packaged by heterologous feline immunodeficiency virus (FIV) proteins
but these proteins could not further propagate the crosspackaged MPMV RNA [21]. Although not quantitative,
the detection levels of the RT-PCR were consistent with
the titers obtained in the transduction assay. Using
homologous proteins, MPMV transfer vectors (pKAL011
and pSS015) were propagated at much higher efficiency
(36,373 CFU/ml and 41% EGFP positive cells) when
compared to MMTV transfer vectors (pDA024 and
pSS013) (3,676 CFU/ml and 19% EGFP positive cells).
Corroborating this observation, MPMV transfer vector
RNAs were packaged more efficiently when compared to
the same cycle of PCR amplification for MMTV transfer
vector RNAs being packaged by its own proteins (Table 1,
Figure 2E and 3E).
Over expressed non-specific RNAs cannot be packaged by
MMTV or MPMV proteins
As an additional control to rule out the possibility of nonspecific packaging and possible propagation of random
RNAs, we transfected control vectors pTR174 and pAG001
(lacking all the viral sequences at the 5'end) (Figure 4A)
separately with MPMV and MMTV packaging constructs
(pTR301 and pJA10) along with the envelope expression
plasmid MD.G. After taking into consideration all the necessary controls for plasmid DNA contamination, RNA
fractionation, and stability of the control transfer vector
RNAs (data not shown), we investigated the possibility of
packaging and propagation of these non-specific RNAs by
both MMTV and MPMV proteins using RT-PCR. Amplification was conducted using control vector specific primers
for 30 cycles to ensure the amplification of any control
vector RNAs that may have been non-specifically packaged. As shown in figure 4B, neither MPMV nor MMTV
proteins could package the control transfer vector RNA
(or, if packaged, the level of packaging was below the

threshold level of detection), and consequently the vector
RNA could not be propagated (Table 1). The lack of packaging of these non-specific RNAs further confirmed our
results that MMTV and MPMV proteins show relative specificity in recognizing the heterologous ψ and can efficiently encapsidate the heterologous RNA.
Comparison between MMTV and MPMV cis-acting
nucleotide sequences and amino acid sequences important
for reverse transcription and integration
The reciprocal cross-packaging of MPMV and MMTV
RNAs suggests that the ψ of these viruses are readily recognized by each other's NC domain of Gag protein facilitating their efficient cross-encapsidation. However, the
absence of Hygr colonies or EGFP positive cells in the
infected cultures suggests a block in post-packaging steps
of the viral life cycle such as reverse transcription and/or
integration. The successful propagation of MMTV and
MPMV transfer vector RNAs would require the recognition of a number of cis-acting sequences present on their
RNAs by the heterologous proteins following cross-packaging. Of these, reverse transcription and integration
would require specific enzymes namely reverse transcriptase (RT) and integrase to work on targets such as
PBS/PPT and att sites, respectively. Therefore, it is conceivable that MPMV and MMTV enzymes were not able to
function on the respective targets of the cross-packaged
RNAs resulting in the lack of propagation of these crosspackaged RNAs. With this rationale in mind, we compared the cis-acting sequences present on MMTV and
MPMV transfer vector RNAs. Our sequence analysis
shown in figure 5A revealed that PPT sequences between
the two viruses have 90% sequence homology, while PBS
showed a sequence homology of approximately 72%.
Comparison of att sites between the two viruses indicated
less than 50% sequence homology (U3 att 40% whereas
the U5 att 45%).

Regulatory enzymes working on the above-mentioned cisacting sequences may also have played an important role
in the inability of the cross-packaged RNAs to propagate
because of the sequence heterogeneity between these two
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+ve Control

Mock

pSS015

pSS015

pKAL011

pTR301 pJA10
pKAL011

D

-ve Control

pKAL011

pSS015

pKAL011

-ve Control

+ ve Control

pJA10

pTR301

pSS015

A

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RT-PCR for Fr actionation Contr ol

Mock

Agarose
20X

pSS015

pJA10
pKAL011

pSS015

pKAL011

-ve Control

C

pTR301

MMTV
Proteins
(pJA10)

pSS015

Unspliced Actin
Spliced Actin

pSS015

E

pKAL011

18S rRNA

-ve Control

MPMV
Proteins
(pTR301)

pKAL011

Nuclear
cDNA

pSS015

pKAL011

DNase-Tr eated Vir al RNA

pJA10

pSS015

pTR301
-ve Control

B

pKAL011

DNase-Tr eated Cytoplasmic RNAs

Southern
Agarose

25X

Agarose

30X
MPMV Vir al cDNAs

MPMV Cytoplasmic cDNAs

Figure 3
MPMV transfer vectors RNA can be cross-packaged by MMTV proteins
MPMV transfer vectors RNA can be cross-packaged by MMTV proteins. (A) PCR amplification of DNase treated
cytoplasmic RNAs using primers OTR537 and OTR538. (B) Control for nucleocytoplasmic RNA fractionation technique as
described for figure 2B. (C) RT-PCR of cytoplasmic cDNA amplified using MPMV specific primers confirming that the transfer
vector RNAs were stably expressed. (D) PCR amplification of DNase treated viral RNAs using viral specific primers. (E) RTPCR of viral cDNAs amplified (for 20, 25, and 30 cycles) using virus specific primers and probed with the PCR product amplified using the same set of primers and pKAL011 as template. For this set of experiments, while amplifying DNase treated viral
RNAs, cytoplasmic and viral cDNAs, primers OTR216 and OTR263 were used and should amplify 271 bp fragment.
viruses. Therefore, RNaseH and integrase were good candidates to be examined carefully since RNaseH plays a pivotal role in hydrolyzing the RNA-DNA hybrid [25] and its
inactivation leads to production of non-infectious virus
[26]. Similarly, integrase is essential for the integration of
the linear retroviral DNA, a crucial step for the completion
of the virus life cycle and has also been shown to promote
reverse transcription through interactions with the nucleoprotein reverse transcription complex [27]. Thus, using
the sequence alignment program, CLUSTAL W [28], we
compared the amino acid sequences of MPMV and MMTV
RNaseH and integrase. Because of the lack of any published sequences of MMTV integrase and RNaseH, we
took the amino acid sequences of MPMV integrase [29]
and RNaseH [30] and aligned them with the entire GagPro-Pol polyprotein amino acid sequence of exogenous
MMTV(C3H) (accession number AF228552) [31]. As
expected, the MPMV RNaseH and integrase sequences
aligned with those of the 3'end of Gag-Pro-Pol polyprotein of MMTV revealing varying degrees of heterogeneity.

The sequence homology between MPMV RNaseH and
integrase with the corresponding sequences of MMTV was
found to be 32% and 49%, respectively (Figure 5B).
Based on the sequence analyses of MPMV and MMTV cisacting sequences and those of the regulatory enzymes acting on them (and the fact that both of these viruses utilize
Lys tRNA primers; Lys-1, 2 for MPMV and Lys-3 for
MMTV), it is plausible to propose that the cross-packaged
RNAs have the potential to successfully initiate the process of reverse transcription; however, they may not have
been able to efficiently complete this event. In addition to
this, the possibility of a fully reverse transcribed unintegrated provirus cannot be ruled out as has been reported
earlier [32]. The inability of the cross-packaged RNAs to
reverse transcribe and/or to integrate should result in a
failed transduction of the target cells because our read out
assay is based on the expression of the marker genes from
an integrated provirus. This postulation stems from the
fact that inadequate integration has been implicated in
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A
hygr

pTR174 SV

OTR 637
SIV 3’LTR
U3
OTR 841
OTR 641

hygr

FIV 3’LTR
U3

Mock

pAG001

pTR174

pAG001

MMTV MPMV
Proteins Proteins
(pJA10) (pTR301)

pTR174

-ve Control

B

OTR 517

+ ve Control (pAG001)

CTE

+ ve Control (pTR174)

pAG001 SV

heterologous viral particles. Therefore, the region encompassing sequences containing MPMV [35] and the putative MMTV ψ (unpublished observations) were cloned
into expression plasmids that can generate non-viral
RNAs, which could act as a substrate for packaging into
the assembling virus particles. In a two-plasmid transcomplementation assay, the expression plasmids containing the putative MMTV ψ (5'UTR + 400 bp of Gag in the
case of pNF007 and R/U5 + 5'UTR + 400 bp of Gag in the
case of pNF008; Figure 1A) were transfected along with
MMTV (pJA10) as well as MPMV (pTR301) packaging
constructs into the 293T producer cells. Similarly, the
expression plasmids containing MPMV ψ (R/U5 + 5'UTR
+ 282 bp of Gag in pND001 and 5'UTR + 282 bp of Gag
in pND002; Figure 1B) were also transfected with the
homologous MPMV (pTR301) or the heterologous MMTV
(pJA10) packaging constructs.

earlier studies including those involving retroviral crosspackaged RNAs [33,34,21]. Therefore, it is reasonable to
propose that due to the great degree of amino acid
sequence divergence (in RNaseH and integrase) and the
heterogeneity in att sequences of these viruses, the crosspackaged RNAs could not be propagated.

Following trans-complementation, both cytoplasmic and
viral RNAs were isolated and after consideration of the
necessary controls (data not shown), the RNAs were
reverse transcribed and the RT-PCR was conducted to
determine the encapsidation of non-viral RNAs that contain either the MPMV or the putative MMTV ψ by the
homologous or the heterologous proteins. RT-PCR analysis conducted on the RNAs demonstrated that non-viral
(pNF007 and pNF008) RNAs were packaged by the
homologous (MMTV; pJA10) and the heterologous
(MPMV; pTR301) proteins (Figure 6A). These results further confirmed our findings of the ability of MPMV proteins to cross-package MMTV transfer vector RNAs in both
viral (pDA024 and pSS013) and non-viral (pNF007 and
pNF008) context (Figures 2E and 6A). In a reciprocal fashion, MMTV proteins (pJA10) cross-packaged non-viral
(pND001 and pND002) RNAs containing MPMV ψ (Figure 6B) confirming our initial observation of the crosspackaging of MPMV transfer vector (pKAL011 and
pSS015) RNAs by MMTV proteins in the viral context (Figure 3E). The cross-packaging of non-viral RNA containing
ψ further confirmed that the specificity towards RNA
packaging was conferred by ψ. Packaging of non-viral
RNAs containing the packaging sequences has also been
observed for other retroviruses such as bovine leukemia
virus (BLV) [36], FIV [24], and MoMLV [37].

Non-viral RNAs containing MMTV and MPMV packaging
sequences can be reciprocally cross-packaged by the
heterologous proteins
The results presented so far clearly indicate that MMTV
and MPMV proteins were able to recognize the ψ
sequence on each other's RNAs. If the recognition of the
packaging sequences by the heterologous proteins was
sufficient to encapsidate the RNAs, we would argue that
any RNA containing these sequences should also be able
to be cross-packaged into the homologous as well as the

Substitution of the packaging signal in MMTV and MPMV
transfer vectors resulted in efficient packaging but
drastically reduced vector RNA propagation
The results of the cross-packaging experiments between
MMTV and MPMV suggested that the reciprocal crosspackaging was due to the cross-recognition of packaging
sequences by the heterologous proteins. The lack of propagation of these cross-packaged transfer vector RNAs, on
the other hand, suggested that following packaging some
of the events imperative for the transduction of the target

Cytoplasmic
cDNAs
Vir al cDNAs

Figure
RNAs 4
MMTV and MPMV proteins cannot package non-specific
MMTV and MPMV proteins cannot package non-specific RNAs. (A) Schematic representation of control vectors for non-specific RNA packaging of an over expressed
RNA. (B) RT-PCR of cytoplasmic (upper panel) and viral
(lower panel) cDNA amplified using control vector specific
primers. Amplification was conducted for 30 cycles to ensure
the amplification of any control vector RNA that may have
been non-specifically packaged. While amplifying cytoplasmic
and viral cDNAs, primers OTR637 and OTR841 were used
for pTR174 and should amplify 368 bp fragment. For
pAG001, primers OTR641 and OTR517 were used and
should amplify 301 bp fragment.

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A

% Homology

PBS
MPMV TGG CGC CCA ACG TGG GGC
MMTV - - - - - - - -G - AC A- - - A-

72.2%

PPT
MPMV AAA AAG GGT GA
MMTV - - - - - - - -G - -

90.9 %

U3 att
MPMV CAT GCT CGG AGC CGT GCT GC
MMTV A- - - -C GC- CCT GCA - -A -A

40%

U5 att
MPMV ATC CCG CGG GTC GGG ACA GT
MMTV GGT -G - -C - ACT - C - G - - - C

45%

B
RNaseH

10

20

30

40

50

MPMV
MMTV

LNNALLVFTDGSSTG-MAAYTLTDTTIKFQTNLNSAQLVELQALIAVLSAFPNQPLNIYT
LEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAEIVAVITAFEEVS-QSFNLYT

MPMV
MMTV

DSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPFYIGHVRAHSGLPGPIAQG
DSKYVTGLFPEIET-ATLSPRTKIYTELRHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQG
Integr ase
150
160
170
120
130

MPMV
MMTV

NQRADLATKIVASNINTNLESAQNAHTLHHLNAQTLRLMFNIPREQARQIVKQCPICVTY
NAYADSLTRILTA-----LESAQESHALHHQNAAALRFQFHITREQAREIVKLCPNCPDW

60

180

70

190

80

200

90

210

100

220

110

230

MPMV
MMTV

LPVPHLGVNPRGLFPNMIWQMDVTHYSEFGNLKYIHVSIDTFSGFLLATLQTGETTKHVI
GHAPQLGVNPRGLKPRVLWQMDVTHVSEFGKLKYVHVTVDTYSHFTFATARTGEATKDVL

MPMV
MMTV

THLLHCFSIIGLPKQIKTDNGPGYTSKNFQEFCSTLQIKHITGIPYNPQGQGIVERAHLS
QHLAQSFAYMGFPQKIKTDNAPAYVSRSIQEFLARWKISHVTGIPYNPQGQAIVERTHQN

MPMV
MMTV

LKTTIEKIKKGEWYPRKGTPRNILNHALFILNFLNLDDQNKSAADRFWHNNPKKQFAMVK
IKAQLNKLQKAGKYY---TPHHLLAHALFVLNHVNMDNQGHTAAERHWGPISADPKPMVM

240

300

360

250

310

370

260

320

380

270

330

390

280

340

290

350

400

MPMV
MMTV

WKDPLDNTWHGPDPVLIWGRGSVCVYSQTYDAARWLPERLVR-----------------WKDLLAGSWKGPDVLITAGRGYACVFPQDAETPIWVPDRFIRPFTERKEATPTPGTAEKT

MPMV
MMTV

--------QVSNNNQS----RE----------------PPRDEKDQQKSPEDESSPHQREDGLATSAGVNLRSGGGS

410

Figure 5
Nucleotides and amino acid sequences comparison between MMTV and MPMV
Nucleotides and amino acid sequences comparison between MMTV and MPMV. (A) Comparison between MMTV
and MPMV cis-acting sequences needed for successful reverse transcription and integration. PBS, primer binding site; PPT poly
purine tract; U3 att, attachment site at 3'LTR; U5 att, attachment site at 5'LTR. The boxed areas represent the canonical TG
and CA dinucleotides in the U3 and U5 att sequences. The "-" represents homologous sequences, the differences are represented by the actual nucleotides. (B) Amino acid sequence alignment of MPMV RNaseH and integrase with the corresponding
region of MMTV using the sequence alignment program, CLUSTAL W. Identical amino acids are boxed.

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Retrovirology 2009, 6:66

cells were not carried out. Therefore, we rationalized that
since MPMV proteins are able to recognize the putative
MMTV ψ, we should be able to exchange successfully the
putative MMTV ψ with that of MPMV in the presence of
either MMTV or MPMV PBS (pND015 and pND016
respectively) or vice versa (pND011 and pND012) (Figure
1A and 1B). The chimeric transfer vectors (pND015 and
pND016) when co-transfected with MMTV packaging
constructs, in addition to being cross-packaged, should
also be able to propagate its RNA. Similar strategy has
been used to propagate chimeric lentiviral transfer vector
RNAs; for example, exchanging the packaging signal in
FIV transfer vectors with those of human and simian
immunodeficiency virus (HIV and SIV, respectively)
resulted not only in packaging but also in the propagation
of the chimeric FIV transfer vector RNA by FIV, HIV, and
SIV proteins, albeit at a much reduced efficiency [21].
A test of MMTV (pND015 & pND016) and MPMV
(pND011 & pND012) chimeric transfer vectors in our in
vivo packaging and transduction assay using either MMTV
(pJA10) or MPMV (pTR301) packaging constructs
revealed that the chimeric transfer vector RNAs were successfully packaged and cross-packaged by the respective
homologous and heterologous proteins (Figure 6C).
However, contrary to our expectations, the packaged
RNAs by the homologous or by the heterologous proteins
either could not be propagated at all or there was a drastic
drop in propagation as evidenced by very few Hygr colonies (< 25 CFU/ml compared to 3,676 CFU/ml when
pDA024 was packaged by pJA10 or 36,373 CFU/ml when
pKAL011 was packaged by pTR301; Table 1). Taken
together, these rather unexpected results pointed in the
direction that we may have disrupted the region around
U5/PBS/UTR during the course of exchanging sequences
encompassing ψ, and that this disruption proved to be
detrimental for vector RNA propagation and not RNA
packaging. During the course of cloning these chimeric
transfer vectors, an artificial NotI site either at PBS/UTR
(pND015 and pND011) or at U5/PBS junctions (pND016
and pND012) was created (Figure 1A and 1B). Thus, it is
conceivable that such non-natural sequences at these crucial junctions may have affected essential steps in the viral
life cycle such as reverse transcription thereby hindering
the propagation of these RNAs.
To confirm whether the introduction of such artificial
sequences could limit the ability of the transfer vector
RNAs to propagate, we created control vectors for both
MMTV (pND017 and pND018) and MPMV (pND013
and pND014). In these control transfer vectors, regions
encompassing MMTV and MPMV ψ were amplified and
cloned back at the same location after artificially creating
a NotI site as in the case of MMTV (pND015 and pND016)
and MPMV (pND011 and pND012) chimeric transfer vectors (Figures 1A and 1B). Test of these control transfer vec-

http://www.retrovirology.com/content/6/1/66

tors (pND013 and pND017) in which the NotI site was
created at the PBS/UTR junction revealed that their RNAs
were propagated to a much reduced level (20 fold reduction in the case of pND017 when compared to pDA024
and 13 folds reduction in the case of pND013 when compared to pKAL011; Table 1). On the other hand, when
NotI site was created at the U5/PBS junction (pND014
and pND018), the propagation of these control transfer
vectors was further hindered (120 fold reduction in
pND018 when compared to pDA024 and 22 fold reduction in pND014 when compared to pKAL011; Table 1).
We did not directly investigate the encapsidation of these
control transfer vector RNAs, because when similar
sequences were exchanged by creating artificial NotI site
on MMTV and MPMV chimeric transfer vectors (pND015,
pND016, pND011, and pND012), their RNAs were
shown to be efficiently packaged by both homologous
and heterologous proteins (Figure 6C).
The drastic drop in the propagation of the chimeric and
the control transfer vectors can be explained on the basis
that the introduction of NotI site in these vectors may have
disrupted RNA structural elements encompassing
sequences such as PBS (needed for reverse transcription)
and/or sequences encompassing or overlapping ψ
(needed for RNA dimerization) and these manipulations
in turn may have diminished the chances of viral RNA
propagation. Alterations/mutations in dimerization
region have been shown to affect multiple steps in the
viral life cycle, excluding packaging [38]. For example, in
the case of HIV-1, a stem loop structure downstream of
the PBS called dimerization initiation site (DIS) is able to
dimerize via a "kissing loop" model [[39], and further
reviewed in [16]]. In addition, the apical loop of the "kissing loop" hairpin contains a GC-rich autocomplementary
sequence called palindrome (pal) [40,41]. Mutations in
DIS region and in the palindrome have shown pronounced effects on viral infectivity, minor or no effects on
RNA packaging, and variable effects on RNA dimerization
[[41,42], and further reviewed in [43]]. Although the
existence of a "kissing loop" model and its potential role
in the life cycle of MPMV and MMTV has not been established yet, we have observed in close vicinity of the PBS a
GC-rich pal sequence in both MPMV (5' UCGCCGGCCGGCGA 3') and MMTV (5' GUCGGCCGAC 3') with 100%
autocomplementarity (unpublished observations). In
addition, using RNA structure prediction program, we
observed putative interaction(s) between NotI site (5'
GCGGCCGC 3') and PBS (MMTV PBS: 5'
UGGCGCCCGAACAGGGA
3';
MPMV
PBS:
5'
UGGCGCCCAACGUGGGGC 3') or pal in some of the
control transfer vectors showing no or much reduced vector RNA propagation (data not shown). The complementarity between these sequences and their GC-rich nature
might have disrupted the RNA structural elements in these
regions, which compromised the role of these biologically
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Mock

pND002

pND001

-ve Control

MMTV Cytoplasmic cDNAs

pND002

MPMV MMTV
Proteins Proteins
(pTR301) (pJA10)

B
Mock

pNF008

pNF007

pNF008

MMTV MPMV
Proteins Proteins
(pJA10) (pTR301)

pNF007

-ve Control

A

http://www.retrovirology.com/content/6/1/66

pND001

Retrovirology 2009, 6:66

MPMV Cytoplasmic cDNAs

Agarose

Agarose
20X

20X

Southern

Southern

Agarose

Agarose
25X

25X
Southern

Southern
MMTV Vir al cDNAs

MPMV Vir al cDNAs

Mock

pND012

pND011

pND016

pND015

MPMV
Proteins
(pTR301)

pND012

pND011

pND015

-ve Control

C

pND016

MMTV
Proteins
(pJA10)

Cytoplasmic cDNAs

Vir al cDNAs

Heterologous and chimeric transfer vector RNAs cross-packaging
Figure 6
Heterologous and chimeric transfer vector RNAs cross-packaging. (A) Reciprocal cross-packaging of heterologous
RNAs containing MMTV packaging signal. RT-PCR of cytoplasmic (upper panel) and viral (lower panel) cDNAs amplified using
MMTV specific primers and probed with the PCR product amplified using the same set of primers and HYB MTV as a template.
For this set of experiment, primers OTR567 and OTR560 were used and should amplify 149 bp fragment. (B) Reciprocal
cross-packaging of heterologous RNAs containing MPMV packaging signals. RT-PCR of cytoplasmic (upper panel) and viral
(lower panel) cDNAs amplified using MPMV specific primers and probed with the PCR product amplified using the same set of
primers and pKAL011 as template. For this set of experiments, primers OTR112 and OTR197 were used and should amplify
154 bp fragment. (C) MMTV and MPMV chimeric transfer vectors RNA containing each other's packaging sequences can be
packaged in a reciprocal fashion. Upper panel represents RT-PCR of cytoplasmic cDNAs using vector RNA specific primers to
ensure their stable expression. Lower panel represents RT-PCR of viral cDNAs amplified using chimeric vector specific primers. For this set of experiment, primers OTR567 and OTR560 were used for pND011 and pND012 and should amplify 149 bp.
For pND015 and pND016, primers OTR730 and OTR197 were used and should amplify 268 bp. However, for the sake of
clarity and to follow cross-packaging, the PCR products are shown next to each others in this figure.

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important sequences. Furthermore, It has earlier been
reported that the interaction between the PBS and the
tRNA 3' terminus is crucial for primer selection in MLV
reverse transcription and that mutations in PBS of both
MLV and HIV-1 involving aberrant reverse transcription
have been reported to hinder viral replication [44]; consequently, the interaction between the NotI site and PBS
may have hindered the accessibility of tRNA to PBS in our
control and chimeric transfer vectors. Therefore it is reasonable to propose that the substitution and/or introduction of the heterologous sequences, in addition to the
creation of the NotI site, may have disrupted the yet to be
identified "kissing loop" model in the case of MPMV and
MMTV, which thwarted post RNA packaging steps of retroviral life cycle, resulting in the abrogation and/or much
reduced propagation of the packaged RNAs in the control
as well as chimeric transfer vectors.
Owing to the wide use of retroviral vectors in human gene
therapy and the safety issues related with cross- and copackaging among retroviruses, these areas have been
extensively investigated and have revealed that reciprocal
as well as non-reciprocal cross-packaging among genetically distinct, simple and complex, retroviruses can take
place [17,21,45-54]. Not only cross-packaging but also copackaging, resulting in the exchange of genetic information, has been reported in genetically distant retroviruses
such as SNV and MLV [17] and HIV-1 and HIV-2 [18].
One of the most important consequences of exchanging
genetic information is the generation of viral variants with
unknown pathogenic potential. This has brought the vector safety concerns to the forefront especially in the light
of the mobilization of HIV-1 based vectors from the transduced cells following infection with the wild type virus
[55,56]. In addition, the generation of a replication competent retrovirus through the process of recombination
between the vector, the packaging construct, and endogenous retrovirus-like elements has also been reported
[57]. In light of these safety concerns, we tested whether
another non-primate retrovirus candidate for human gene
therapy, MMTV, would be prone to cross-packaging with
a primate retrovirus as has previously been shown that a
phylogenetically distant non-primate lentivirus (FIV)
could be packaged by primate lentiviruses [21]. Our study
showed that MMTV and MPMV packaging sequences are
promiscuous and could be recognized by each other's proteins suggesting potential RNA-protein interactions
among divergent retroviruses. It would be interesting to
determine how frequently MMTV RNA can be cross-packaged by other retroviral proteins and whether there are
obstacles to these RNA-protein interactions. These observations raise the possibility that MMTV and MPMV RNAs
could also co-package and exchange genetic information.
Therefore, it would be interesting to investigate whether
co-packaging between MMTV and MPMV could occur and

http://www.retrovirology.com/content/6/1/66

if so, at what frequency and what are the properties of the
resulting recombinant variants, if at all.

Conclusion
The results presented in this study raise important questions about the design and use of candidate vectors for
human gene therapy and suggest that sequences, which
could contribute to such cross-packaging, should be eliminated from the transfer vectors to allow the establishment
of safer and more effective vectors for human gene transfer
studies. The retrovirus life cycle may be exploited to
render the cis-acting sequences of an integrated transfer
vector inactive through a process called self-inactivation
(SIN) [45,55,58-61]. In addition to SIN vectors, a novel
super-split packaging system has also been recently developed, which incorporates many new safety features. In
such a system, gag was separated from pol and the protease
was provided in trans by a less cytotoxic mutant, while
maintaining high titers [62]. Surprisingly, despite taking
into consideration several safety aspects in modified packaging systems and transfer vectors, vector mobilization as
well as leaky transcriptional termination have been
reported [63-65]. Therefore, in order to construct efficient
and safe vectors, a thorough understanding of RNA packaging and cross packaging among retroviruses is imperative, and the understanding we will gain from such studies
should allow employing multiple strategies to prevent
viral replication through packaging and/or recombination
after delivery of the gene of interest into the target cells.

Methods
Numbering system
Nucleotide designation for MMTV is based on HYB MTV
molecular clone sequence [66]. Nucleotide designation
for MPMV is based on GenBank accession number
M12349[67].
Plasmid Constructions
MMTV packaging constructs and transfer vectors
The MMTV packaging construct, pJA10, has been
described previously [20]. Briefly, pJA10 expresses MMTV
gag/pol gene from hCMV promoter and contains constitutive transport element (CTE) from MPMV downstream of
MMTV gag/pol. pDA024 and pSS013 are MMTV based
transfer vectors that have been described previously [20]
and contain 5' chimeric LTR in which MMTV promoter
sequences have been replaced with that of hCMV (Figure
1A).

To asses whether the presence of regions encompassing
the putative MMTV ψ on non-viral RNAs would facilitate
the packaging of these RNAs, sequences containing the
putative MMTV ψ (5'UTR and 400 bp of Gag) and (R/U5/
5'UTR and 400 bp of Gag) were amplified using sense
primers OTR680 and OTR617 and an anti-sense primer

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Retrovirology 2009, 6:66

OTR552 using HYB MTV as a template. The PCR products
were cleaved with the artificially created HindIII and SpeI
sites and cloned into HindIII and XbaI sites of pNF003 (a
derivative of pcDNA3) generating pNF007 and pNF008,
respectively (Figure 1A).
pND015 and pND016 are pDA024 derivatives containing
MPMV ψ either in the presence (pND015) or absence
(pND016) of MMTV PBS and were generated through
series of cloning steps. Briefly, the 3'end of MMTV genome
containing SV-hygr cassette and MPMV CTE along with
3'LTR was obtained by digesting pDA024 with SpeI and
KpnI. The 5'region of MMTV genome containing 5'LTR
with and without PBS was amplified using sense primer
OTR617 and anti-sense primers OTR727 and OTR728
and pDA024 as a template. The resulting PCR products
were digested with the artificially created SacI and SpeI
sites. The resulting SacI-SpeI PCR fragment containing
5'end of the genome (without the packaging signal) and
SpeI-KpnI fragment containing the 3'end of the genome
were ligated in a three way ligation to a pIC based cloning
vector [68], which had already been cleaved with SacI and
KpnI resulting in pND005 and pND006, respectively.
Such a cloning strategy created an artificial NotI site at the
junction of PBS/UTR in the case of pND005 and at the
U5/PBS junction in the case of pND006. Next, sequences
encompassing MPMV packaging determinants (UTR and
282 bp of Gag) with and without MPMV PBS were amplified using sense primer OTR730 and OTR729 and
OTR731 as anti-sense primers. The resulting PCR products
were cleaved by the flanking artificially created NotI sites
and were cloned into the NotI site of pND005 and
pND006 resulting in the final clones pND015 and
pND016, respectively (Figure 1A).
To ensure that the creation of an artificial NotI site in the
region encompassing MMTV packaging sequences will
not affect RNA packaging or propagation adversely, we
created pND017 and pND018 as control transfer vectors.
Putative sequences that are involved in MMTV RNA packaging with and without the MMTV PBS were amplified
using sense primers OTR724 and OTR725 and anti-sense
primer OTR726 and pDA024 as a template. The combination of these primers created flanking artificial NotI sites in
the PCR products facilitating their cloning into NotI site of
pND005 and pND006 to generate pND017 and pND018,
respectively (Figure 1A).
MPMV packaging constructs and transfer vectors
The MPMV packaging construct, pTR301, has already
been described [21]. Briefly, it expresses MPMV gag/pol
genes from hCMV intron A promoter/enhancer and contains MPMV CTE between Pol termination codon and
bovine growth hormone (BGH) Poly (A) sequences. The
MPMV transfer vector, pKAL011, has also been described

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previously [21] and contains all cis-acting sequences necessary for RNA packaging and propagation. pSS015 is similar to pKAL011 except it contains SV-EGFP cassette
instead of SV-hygr cassette (Figure 1B).
To determine if the presence of regions encompassing
MPMV packaging sequences on non-viral RNAs would
facilitate the packaging of these RNAs, two different
regions encompassing MPMV ψ were cloned into
pNF003. A region encompassing MPMV ψ (R/U5, 5'UTR,
and 282 bp of Gag) was amplified using a sense primer
OTR615, an anti-sense primer OTR616, and pKAL011 as
a template. The PCR product was cleaved with the artificially created HindIII and NheI sites and was cloned into
HindIII and XbaI sites of pNF003 generating pND001.
pND002 is another version of pcDNA3 based clone containing MPMV ψ and was created by isolating SfoI-HpaI
fragment containing MPMV PBS, 5'UTR and 282 bp of
Gag from pKAL011 and was cloned into the blunted HindIII and XbaI sites of NF003 (Figure 1B).
pND011 and pND012 are MPMV based vectors in which
MPMV ψ has been replaced with that of MMTV either in
the presence (pND011) or absence (pND012) of MPMV
PBS and were created through series of cloning steps. As a
first step, the 3'end of MPMV genome containing 3'LTR
was obtained by digesting pKAL011 with NheI and
BamHI. The MPMV 5'LTR along with or without PBS were
amplified using sense primer OTR721 and anti-sense
primers OTR722 and OTR723 using pKAL011 as a template. The resulting XhoI-NheI PCR fragments containing
5'end of the genome (without the packaging signal) and
NheI and BamHI fragment containing the 3'end of the
genome were ligated in a three way ligation at the XhoI
and BamHI sites of a pIC based cloning vector resulting in
pND003 and pND004, respectively. Similar to the cloning
scheme of MMTV chimeric vectors, such a cloning strategy
created an artificial NotI site at the PBS/UTR and U5/PBS
junctions in the case of pND003 and pND004, respectively. Next, the putative MMTV ψ (5'UTR and 400 bp of
Gag) with and without MMTV PBS were amplified from
pDA024 using sense primers OTR725 and OTR724 and
OTR726 as the anti-sense primer. The resulting PCR products were cleaved by the flanking artificially created NotI
sites and were cloned into the NotI site of pND003 and
pND004 resulting in pND007 and pND008. Finally, SVhygr cassette containing flanking NheI sites was cloned
into the NheI site of pND007 and pND008 generating
pND011 and pND012 (Figure 1B).
pND013 and pND014 were created as controls to see the
effects of an artificially created NotI site within MPMV ψ.
Regions containing MPMV ψ (with and without the PBS)
were amplified using sense primers OTR729 and OTR730
and anti-sense primer OTR731 and pKAL011 as a tem-

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Retrovirology 2009, 6:66

plate. The PCR products were digested with the flanking
NotI site and cloned into the NotI site of pND003 and
pND004, respectively generating pND009 and pND010.
Finally, SV-hygr cassette containing flanking NheI sites was
cloned into NheI site of pND009 and pND010 generating
the final clones pND013 and pND014 (Figure 1B).
Plasmids for nonspecific RNA packaging
pTR174 has been described previously [49] and was created to test the possibility of non-specific packaging of an
over expressed RNA containing the SV-hygr cassette
through "retrofection" [69,70] and uses SIV 3'LTR for
transcript termination. pAG001 is similar to pTR174
except that at the 3'end it contains FIV 3'LTR for transcript
termination. Both of these control vectors lack all of the
viral sequences at the 5'end (Figure 4A).
Envelope expression construct
All these transfer vectors were psuedotyped in a three plasmid trans-complementation assay using vesicular stomatitis virus envelope G (VSV-G) expression plasmid (MD.G)
that has been previously described [22].
Transfection and infection of cells
The producer 293T cells were maintained and transfected
by the calcium phosphate method as described earlier
[71]. Seventy two hours post-transfection, viral supernatants were harvested for pelleting virus particles to isolate
viral RNA and some portion was used to infect HeLa CD4+
cells as described previously [21]. Forty-eight hours following infection, cells that were infected with transfer vectors containing hygromycin resistance gene were selected
with media containing hygromycin B phosphotranferase.
Hygr colonies that appeared were stained and counted
after 9–11 days as described previously [21]. Target cells
that were infected with transfer vectors containing EGFP
gene were trypsinized 48 hours post-infection, washed
with PBS, and analyzed on a Becton Dickinson Fluorescent Activating Cell Sorting (FACS) using the CellQuest
software.
Ultracentrifugation of virus particles
Viral supernatants collected seventy-two hours post-transfection were subjected to a low-speed centrifugation
(4000 rpm for 10 minutes) for removal of cellular debris.
Following which, viral particles were filtered using 0.2micron syringe filters and ultracentrifuged using 20%
sucrose cushion (using SW41 rotor at 26,000 rpm for 2
hours at 4°C) as described previously [24].
RNA isolation and Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR)
The transfected cells were taken off from the culture plates
without trypsinization and fractionated into cytoplasmic
and nuclear RNA fractions as described earlier [71]. For

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viral RNA isolation, pelleted virions resuspended in TNE
buffer were lysed in 500 μl of Trizol LS reagent containing
8 μl of polyacryl (used as a carrier) as described earlier
[24]. Following RNA isolation, both cytoplasmic and viral
RNAs were DNase treated and tested by PCR to confirm
the absence of any contaminating plasmid DNA that may
have been carried over from the transfected cultures. After
confirming that RNA preparations were devoid of any
contaminating DNA, cytoplasmic and viral RNAs were
reverse transcribed and amplified. cDNA prepared from
the cytoplasmic RNA fractions were amplified using vector specific primers to monitor the expression of the transfer vector RNA in the cells. In addition, cytoplasmic
fractions were also amplified to confirm the integrity of
the fractionation process in a multiplex PCR in the presence of primers/competimers for 18S ribosomal RNA as a
control for the presence of amplifiable cDNA in the PCR
reactions (18S Quantum competimer control, Ambion,
Austin, TX). Viral cDNAs were amplified using vector specific primer to determine the packaging of the transfer vector RNA. Amplified PCR products were analyzed on 2–4%
agarose gels and, in some cases, the gels were further processed for Southern blot analysis as described previously
[24]. Details of the primers used in cloning, RT-PCR, and
Southern hybridization can be obtained from authors
upon request.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
NSD, PSP, AG, JA, EB, and TAR performed the experiments (cloning, trans complementation assay, RNA
extraction, RT-PCR, Southern blotting). SAJ performed
RNase H and Integrase amino acid sequences alignment.
NSD and TAR conceived the study and participated in its
design. All authors read and approved the final manuscript.

Acknowledgements
This work was supported in part by grants from Terry Fox Foundation for
Cancer Research (2001/03) and a medical research grant from Sheikh Hamdan Award for Medical Sciences (MRG-8/2003-2004). We express our
thanks to Dr. Didier Trono (Ecole Polytechnique Fédérale de Lausanne,
Switzerland) for providing MD.G., Dr. Jaquelin Dudley (University of Texas
at Austin, Austin, TX) for providing HYB MTV molecular clone and Mr.
Allen Shahin (United Arab Emirates University, Al Ain, UAE) for helping in
performing the FACS analysis. We would also like to thank Ms. Suraiya
Jahan Aktar (United Arab Emirates University, Al Ain, UAE) for stimulating
discussions and critical reading of the manuscript. The authors also wish to
thank Ms. Noura Al Shamsi and Ms. Fatima Al Awadi for their assistance in
cloning some of the vectors while volunteering in the laboratory.

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