Download Murine leukemia virus transmembrane protein R

Journal of General Virology (2006), 87, 1583–1588
Short
Communication
DOI 10.1099/vir.0.81527-0
Murine leukemia virus transmembrane protein
R-peptide is found in small virus core-like
complexes in cells
Klaus Bahl Andersen, Huong Ai Diep and Anne Zedeler3
Department of Pharmacology and Pharmacotherapy, The Danish University of Pharmaceutical
Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark
Correspondence
Klaus Bahl Andersen
[email protected]
Received 14 September 2005
Accepted 30 January 2006
The core of the retrovirus Murine leukemia virus (MLV) consists of the Gag precursor protein and
viral RNA. It assembles at the cytoplasmic face of the cell membrane where, by an unclear
mechanism, it collects viral envelope proteins embedded in the cell membrane and buds off.
The C-terminal half of the short cytoplasmic tail of the envelope transmembrane protein (TM) is
cleaved off to yield R-peptide and fusion-active TM. In Moloney MLV particles, R-peptide was
found to bind to core particles. In cells, R-peptide and low amounts of uncleaved TM were found
to be associated with small core-like complexes, i.e. mild detergent-insoluble, Gag-containing
complexes with a density of 1?23 g ml”1 and a size of 150–200 S. Our results suggest that TM
associates with the assembling core particle through the R-peptide before budding and that this
is the mechanism by which the budding virus acquires the envelope proteins.
During the formation of the retrovirus particle, the preproteins Gag and Env have different routes of formation
and co-localize at the plasma membrane before virus
budding. Gag [Pr65gag in Murine leukemia virus (MLV)] is
translated in the cytoplasm. The assembly of Gag and viral
RNA into the core particle differs among retroviruses. Betaand spumaretroviruses, as exemplified by Mason–Pfizer
monkey virus (MPMV), assemble their core in the cytoplasm, whereas most other retroviruses, including MLV and
human immunodeficiency virus (HIV), utilize the type C
pathway, i.e. assemble their core at the inner face of the
plasma membrane, although assembly and budding into
intracellular vesicles have been reported in some cell types
[see, for example, Sherer et al. (2003)]. Fully assembled cores
are not observed until just before budding. Env (gPr80env in
MLV) is translated into the rough endoplasmic reticulum,
trafficked to the Golgi, glycosylated and cleaved into the
surface protein (SU) and the transmembrane protein (TM)
by a cellular protease. SU and TM are linked by S–S bridges
and join to form trimer complexes, which are trafficked to
the cytoplasmic membrane (Einfeld & Hunter, 1988).
Gag plays a central role in the assembly of the retroviral
particle (reviewed by Cimarelli & Darlix, 2002; Demirov &
Freed, 2004). MLV Gag is transported rapidly to the membrane (Suomalainen et el., 1996), where it directs particle
formation. SU–TM co-localizes with Gag (HermidaMatsumoto & Resh, 2000) and, during budding, SU–TM
becomes concentrated in the viral membrane, whereas
3Present address: Department of Forensic Genetics, University of
Copenhagen, Frederik V’s Vej 11, DK-2100 Copenhagen, Denmark.
0008-1527 G 2006 SGM
cellular membrane proteins are excluded (Kuznetsov et al.,
2004). Gag particles can bud in the absence of SU–TM
(Kuznetsov et al., 2004), but SU–TM is necessary for
efficient budding (Fischer et al., 1998) and high infectivity
(Rein et al., 1994).
The mechanism ensuring that budding virions contain
SU–TM is unknown. In immature virions of HIV, a specific
association between the cytoplasmic TM tail and Gag has
been shown (Wyma et al., 2000), but whether this is important for the assembly process is not known. After budding,
the core matures by cleavage of Gag into the mature core
proteins, the matrix (MA), capsid (CA) and nucleocapsid
proteins (p15C, p30 and p10, and the additional p12 in
MLV). However, cleavage and budding do not occur
synchronously, as illustrated by the existence of both mature
core proteins in cells and uncleaved Gag in virions [see,
for example, Melamed et al. (2004)]. Analytically, immature
viral cores can be distinguished from mature cores by their
electron-dense structure and their ability to resist mild
detergents (Oshima et al., 2004). In this respect, cellular core
material behaves (at least in part) in the same way as the
immature core (Hansen et al., 1993). Thus, after treatment
with mild detergents, Gag is expected to sediment and CA to
be in solution. TM is then expected to be in low-density
material or in solution, depending on its raft location.
In some retroviruses (mammalian type C, MPMV and others;
Bobkova et al., 2002), TM matures further by cleavage by the
viral protease around the time of budding. The C-terminal
part of the cytoplasmic tail, called R-peptide (p2E in MLV),
is cleaved off. R-peptide per se was first observed in MLV
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1583
K. B. Andersen, H. A. Diep and A. Zedeler
virions by Henderson et al. (1984). In Moloney MLV
(MoMLV), the sequence is H-VLTQQYHQLKPIEYEP-OH.
The R-peptide tail apparently prevents premature fusion of
the immature TM (pre-TM) by keeping it in a fusioninactive state (Ragheb & Anderson, 1994; Rein et al., 1994).
The events following TM cleavage are intriguing, as the
majority of R-peptide is found in cells (Olsen & Andersen,
1999), whereas mature, cleaved TM is seen mainly in virions
(Green et al., 1981; Olsen & Andersen, 1999).
The sedimentation coefficient (sw,20) of the retrovirus
particle is reported to be in the range 580–750 S [MoMLV
(Sharma et al., 1997); Avian myeloblastosis virus and Rous
sarcoma virus (Bellamy et al., 1974)]. Despite the smaller size,
the core has almost the same sw,20 [750 S, as calculated for
intracellular, fully assembled Gag MPMV cores from data by
Parker & Hunter (2000) and Parker et al. (2001)]. This is due
to the higher density of 1?24 g ml21 of the core in sucrose
(Wyma et al., 2000) than of the whole virion (1?16 g ml21).
The s value generally follows d2Dr, where d is the particle
diameter and Dr is the density difference between particle
and centrifugation medium. Cellular core material of retroviruses using the type C pathway does not show a uniform
size. Hansen et al. (1993) estimated the core material to be
larger than 165 S and in vitro assembly studies have shown a
large profile for Gag material (Lingappa et al., 1997).
Previously, we found R-peptide as a 7 kDa band in Tricine
gels by immunoblotting with an antiserum raised against a
synthetic MoMLV R-peptide. Despite the fact that the
formula mass of the R-peptide is 1968 Da, we believe that
the 7 kDa band is R-peptide for the following reasons (Olsen
& Andersen, 1999): (i) the antiserum also recognized Env
and pre-TM, but not cleaved TM; (ii) the 7 kDa band was
observed only in infected cells; (iii) the band resolved at an
isoelectric point of 5?5, which was correct according to the
sequence; and (iv) it is known that small peptides can run
irregularly in Tricine gels (Schägger & von Jagow, 1987).
Furthermore, the band was shown to be associated with
palmitic acid, possibly changing its electrophoretic properties. Kubo & Amanuma (2003) observed R-peptide in a
haemagglutinin-tagged form, which changed its size.
NIH 3T3 cells infected chronically with a molecular clone of
MoMLV derived from pMov3 (Reik et al., 1985) were grown
as described previously (Olsen & Andersen, 1999). Cell
monolayers were washed twice with PBS, once with NTE
[100 mM NaCl, 10 mM Tris/HCl (pH 7?5), 1 mM EDTA]
and lysed with 1 % Triton X-100 (Rohm and Haas) in icecold NTE containing Protease Inhibitor Cocktail tablets
(Roche). Nuclei and large cellular debris were removed by
centrifugation at 1000 r.p.m. for 5 min.
The Svedberg unit of centrifugation is calculated as
S=1013 s21 ln(rfinal/rinitial)/v2t, where v is the angular
speed and r is the radius from the rotational axis. Values
were corrected to the corresponding value in water at 20 uC
(sw,20) by the factor gs,TDrw/gw,20Drs, where gs,T and gw,20
were, respectively, the viscosities of the centrifugation
1584
medium at the temperature used (5 uC) and that of water
at 20 uC [1?00461023 Pa s (1?004 centipoise)]. Drw and Drs
were density differences between the centrifuged particles
and water, respectively, and the centrifugation medium. In
sucrose gradients, the sw,20 values were integrated as:
ð
1013 s{1 rfinal gs,T Dow
sw,20 ~
dr
u2 t
rinitial gw,20 Dos r
The density of 1?23 g ml21 for the particles was used in
calculations unless otherwise noted. Fractions from sucrose
gradients were concentrated by fourfold dilution and centrifugation in a microfuge (22 000 r.p.m. for 7 h, yielding 13S
material). Material at equilibrium in 1?23 g sucrose ml21
was thus required to be larger than 37sw,20 to pellet fully.
Because low-S material was expected at the top of the
gradients, the top fractions were also analysed directly.
Proteins were separated by Tricine SDS-PAGE (Schägger &
von Jagow, 1987) and blotted on to Hybond-P membranes
(Amersham Biosciences) followed by immunostaining
using rabbit antiserum raised against synthetic R-peptide
(Olsen & Andersen, 1999), TM (from Alan Rein, National
Cancer Institute, MD, USA), CA (from Bjørn A. Nexø,
Department of Human Genetics, University of Aarhus,
Denmark) and actin (Sigma-Aldrich). The secondary antibody was horseradish peroxidase-conjugated swine antirabbit immunoglobulin (Dako). Bands were visualized by
ECL Plus (Amersham Biosciences).
The location of R-peptide in virions was analysed (Fig. 1).
Virions were density-purified, treated with Triton X-100 or
left untreated and rerun to equilibrium on new sucrose
gradients. When virions were untreated, they ran as expected
at 1?16 g ml21, as seen for the majority of each tested
protein. Small amounts of Gag, CA and cleaved TM ran at
approximately 1?06 g ml21, which probably represented
fragments of virions. After treatment with Triton X-100, Rpeptide shifted to 1?22 g ml21 together with large amounts
of Gag, a hallmark of the immature core. Minor amounts of
pre-TM, cleaved TM and CA were also observed in this
band. In conclusion, R-peptide appeared to be bound to
immature cores, but whether all R-peptide was bound was
difficult to judge due to the small amount of R-peptide
present in virions.
Previously, in infected cells, we observed R-peptide in membrane preparations (Olsen & Andersen, 1999). Membranes
were prepared from cells according to the method of Maeda
et al. (1983). Of the total cellular amounts, the majority
(63–88 %) of R-peptide, Gag and CA and all (97 %) pre-TM
were found in membranes.
To determine whether R-peptide was attached to cellular
core material, we carried out size and density separation
of cells lysed in Triton X-100, which dissolves membranes.
A post-nuclear lysate was pelleted stepwise to decreasing S
values. Gag and R-peptide co-sedimented in two ranges:
above 62 000 S and between 16 and 1000 S (results not
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Journal of General Virology 87
MLV R-peptide is found in complexes
No detergent
V T 1.03
a-CA
Gag
1.10
1.06
1.17
1.23
1.27
53
i
CA
a-TM
pre-TM
TM
34
23
17
13
12
a-R
R
7
Triton X-100
a-CA
V T 1.03
Gag
1.10
1.05
1.16
1.22
1.27
i
53
CA
a-TM
pre-TM
TM
a-R
R
34
23
17
13
12
7
shown). The low-S material could have been cores in the
process of assembly and the high-S material could have been
core material bound to cellular debris/cytoskeletal fragments. Gag is known to bind to actin in the cytoskeleton
(Chen et al., 2004; Wilk et al., 1999). Approximately onethird of the total R-peptide was present in each size range,
with the last third in a final 5S supernatant, which, as
expected, contained the majority of CA and pre-TM.
The density of the low-S complexes was examined by equilibrium centrifugation (Fig. 2). The majority of R-peptide
was clearly located in a band of 1?23 g ml21 together with a
considerable amount of Gag and a small amount of pre-TM,
Pelleted fractions
a-CA Gag
properties similar to those of the viral core seen in Fig. 1. As
also observed in the stepwise centrifugation, some R-peptide
and most of the CA were present in solution. Some Gag and
most of the pre-TM were also present as low-density pelletable material, possibly rafts.
The size of the core-like material was examined by velocity
centrifugation (Fig. 3a). R-peptide and Gag co-located,
both in the gradient pellet (larger than 460 S) and in a band
around 170 S. In similar experiments, R-peptide was not
observed in the range between 400 and 1200 S (not shown),
where whole cores are expected. Pre-TM and CA were
mainly present in solution, as expected; however, low
53
i
CA
34
a-R
pre-TM
17
13
12
7
R
T 1.05
Fractions directly
a-R + a-CA
Gag
i
CA
pre-TM
R
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Fig. 1. Equilibrium centrifugation of MoMLV
with or without Triton X-100 treatment. Two
0?5 ml portions of density-purified MoMLV
from a total of 250 ml medium were diluted
fivefold in NTE buffer with or without 1 %
Triton X-100 for 1 h on ice and rerun on
10 ml 13–65 % sucrose gradients at
36 000 r.p.m. for 20 h in an SW40 rotor.
Fractions (~1 ml) are indicated by density
(g ml”1; determined by refractometry).
Pelleted fractions (600 ml) were analysed
together with 20 ml of the loaded virus
dilutions (V) and 30 ml of the top fractions
(T) by immunoblotting using antisera against
R-peptide (a-R), CA (a-CA) and TM (a-TM).
Possible Gag cleavage intermediates are
indicated by ‘i’.
1.15
V
1.21
53
34
23
17
13
12
7
C
1.27
1.32
Fig. 2. Equilibrium centrifugation of Triton
X-100 lysate from infected NIH 3T3 cells. A
Triton X-100 lysate from ~66106 cells was
centrifuged to give a 25 000S supernatant
(3 ml), which was run on a 10 ml 13–65 %
sucrose gradient at 36 000 r.p.m. for 20 h in
an SW40 rotor. Fractions (~1 ml) are indicated by density (g ml”1). T, Top fraction.
Fractions were analysed either directly (30 ml)
or after pelleting of 300 ml by immunoblotting
using antisera against R-peptide, CA or
both (a-R + a-CA). In the CA immunoblot
of pelleted fractions, fractions were pooled as
indicated. MoMLV and MoMLV cores were
run in parallel and their densities are indicated
as V and C, respectively.
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1585
K. B. Andersen, H. A. Diep and A. Zedeler
(a)
a-CA
Gag
Fractions directly
T
<40 60 98 135 170 206
Pelleted fractions
P
T
>460 <40 60 98 135 170 206 245 282 320 360 400 445
Gag
53
i
CA
a-R
pre-TM
53
23
17
13
12
34 pre-TM
23
17
13
R
7
(b)
T
P
<50 120 180 240 300 370 440 520 600 670 760 870 1050 >1100
a-CA
Gag
L
D
53
CA
34
a-R
pre-TM
23
17
13
12
7
R
a-Actin
actin
Fig. 3. Velocity centrifugation of Triton X-100 lysates from infected NIH 3T3 cells. (a) A 0?5 ml Triton X-100 lysate from
approximately 2?56107 cells was loaded on to a 5–20 % sucrose gradient. (b) A 1?6 ml Triton X-100 lysate from
approximately 7?26107 cells was centrifuged to yield a 40 000S supernatant. This supernatant was equilibrium-centrifuged
on a 13–65 % sucrose gradient at 36 000 r.p.m. for 20 h in an SW40 rotor. Fractions with densities of 1?20–1?26 g ml”1
were pooled, diluted 3?6-fold to 1?2 ml and loaded onto a 20–35 % sucrose gradient. Both the 5–20 and 20–35 % sucrose
gradients were centrifuged in an SW40 rotor for 2 h at 30 000 r.p.m. The fractions (~1 ml) are indicated by their mid-sw,20
values. T, Top fractions; P, pellets in the velocity centrifugations; L, loaded lysate; D, pool from the equilibrium centrifugation.
Material analysed by immunoblotting was: pelleted fractions from 800 ml (a) and 700 ml (b), 30 ml of fractions directly (a), half
of P (a), all of P (b), 5 ml L (b) and 15 ml D (b). Antisera against R-peptide, CA and actin were used. The sw,20 of virions of
MoMLV was analysed in parallel and found to be approximately 750 S at 1?16 g ml”1.
amounts of pre-TM also appeared in the 170S band and in
the gradient pellet above 460 S.
To determine whether the 1?23 g ml21 and 170S material
were identical, a double separation was carried out. A Triton
X-100 cell lysate was first density-fractionated, and the 1?20–
1?26 g ml21 fractions were pooled, diluted and examined
on a velocity gradient (Fig. 3b). R-peptide was observed in
a band of almost the same size as that seen in Fig. 3(a) of
approximately 180 S, which also contained Gag and small
amounts of pre-TM. Most Gag, but only a small amount of
R-peptide, was present in the gradient pellet above 1100 S. It
was again noteworthy that no proteins were observed at
around 750 S, the size of viral cores. Gag is known to bind
to actin, so this was tested. Actin was present only in the
pellet. In conclusion, the experiments showed that cellular
R-peptide co-sedimented with membrane-linked, core-like
complexes at 150–200 S and that the large Gag complexes
were probably bound to the cytoskeleton.
The co-sedimentation of R-peptide and pre-TM with Gag
does not necessarily show a direct binding. A binding
was indicated by co-precipitation using a goat CA/MA
antiserum that did not recognize R-peptide or pre-TM in
immunoblots. However, it precipitated significant amounts
of R-peptide and pre-TM together with Gag, CA and MA
from Triton X-100 lysates (results not shown).
1586
The location of R-peptide and pre-TM in cellular core-like
material can explain directly how Env proteins become
associated with the assembling Gag core in the cell. The
extensive binding of R-peptide indicates that pre-TM is
associated by its R-tail. Mutational analysis supports the
importance of the TM–Gag association. In R-peptidecontaining retroviruses, R-cleavage-negative mutants do
produce virions, whereas R-truncated mutants give a low
yield; in both cases, the specific virus infectivity is low (Rein
et al., 1994; Kubo & Amanuma, 2003). Low infectivity of R
truncations is also observed for MPMV (Brody et el., 1994),
indicating that the R-peptide–core association is not restricted
to retroviruses with the type C assembly pathway. Furthermore, mutations in MPMV MA, to which the binding
presumably occurs, have been shown to suppress cleavage of
pre-TM (Brody et al., 1992). In HIV, mutations in MA
prevent incorporation of envelope proteins into virions (Yu
et al., 1992; Freed & Martin 1995, 1996; Cimarelli & Darlix,
2002) and incorporation can be restored by truncations in
the TM cytoplasmic tail (Mammano et al., 1995). Furthermore, the TM–core association in HIV has been shown to
have implications for fusion ability (Wyma et al., 2004).
Some cleaved TM also appeared to be bound to viral cores
(Fig. 1). This association cannot be explained directly by the
R-peptide association. It may occur indirectly through the
R-tail of pre-TM in mixed pre-TM/TM oligomers, as TM
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Journal of General Virology 87
MLV R-peptide is found in complexes
oligomers are stable in mild detergents (Einfeld & Hunter,
1988).
The cellular core-like complexes had the same density as
viral cores (1?23 g ml21), but were of a smaller size. According to the proportionality between S and d2, the core-like
complexes had approximately half the diameter of viral
cores. We do not know whether the complexes are involved
in the formation of virus particles or whether they represent
abortive material. The large amount of R-peptide associated
with the cellular core-like complexes compared with the
amount of R-peptide in virions suggests an accumulation in
cells. Further studies are required, therefore, to elucidate the
fate of R-peptide and the core-like complexes.
Henderson, L. E., Sowder, R., Copeland, T. D., Smythers, G. &
Oroszlan, S. (1984). Quantitative separation of murine leukemia
virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products. J Virol
52, 492–500.
Hermida-Matsumoto, L. & Resh, M. D. (2000). Localization of human
immunodeficiency virus type 1 Gag and Env at the plasma membrane by confocal imaging. J Virol 74, 8670–8679.
Kubo, Y. & Amanuma, H. (2003). Mutational analysis of the R
peptide cleavage site of Moloney murine leukaemia virus envelope
protein. J Gen Virol 84, 2253–2257.
Kuznetsov, Y. G., Low, A., Fan, H. & McPherson, A. (2004). Atomic
force microscopy investigation of wild-type Moloney murine
leukemia virus particles and virus particles lacking the envelope
protein. Virology 323, 189–196.
Lingappa, J. R., Hill, R. L., Wong, M. L. & Hegde, R. S. (1997). A
Acknowledgements
We thank Helle Dyhrfjeld Jensen for excellent technical assistance.
multistep, ATP-dependent pathway for assembly of human
immunodeficiency virus capsids in a cell-free system. J Cell Biol
136, 567–581.
Maeda, T., Balakrishnan, K. & Mehdi, S. Q. (1983). A simple and
rapid method for the preparation of plasma membranes. Biochim
Biophys Acta 731, 115–120.
References
Bellamy, A. R., Gillies, S. C. & Harvey, J. D. (1974). Molecular weight
of two oncornavirus genomes: derivation from particle molecular
weights and RNA content. J Virol 14, 1388–1393.
Bobkova, M., Stitz, J., Engelstädter, M., Cichutek, K. & Buchholz,
C. J. (2002). Identification of R-peptides in envelope proteins of C-
type retroviruses. J Gen Virol 83, 2241–2246.
Brody, B. A., Rhee, S. S., Sommerfelt, M. A. & Hunter, E. (1992). A
Mammano, F., Kondo, E., Sodroski, J., Bukovsky, A. & Göttlinger,
H. G. (1995). Rescue of human immunodeficiency virus type 1
matrix protein mutants by envelope glycoproteins with short
cytoplasmic domains. J Virol 69, 3824–3830.
Melamed, D., Mark-Danieli, M., Kenan-Eichler, M. & 7 other authors
(2004). The conserved carboxy terminus of the capsid domain of
human immunodeficiency virus type 1 Gag protein is important for
virion assembly and release. J Virol 78, 9675–9688.
viral protease-mediated cleavage of the transmembrane glycoprotein
of Mason–Pfizer monkey virus can be suppressed by mutations
within the matrix protein. Proc Natl Acad Sci U S A 89, 3443–3447.
Olsen, K. E. P. & Andersen, K. B. (1999). Palmitoylation of the
Brody, B. A., Rhee, S. S. & Hunter, E. (1994). Postassembly cleavage
Oshima, M., Muriaux, D., Mirro, J., Nagashima, K., Dryden, K.,
Yeager, M. & Rein, A. (2004). Effects of blocking individual maturation
of a retroviral glycoprotein cytoplasmic domain removes a necessary
incorporation signal and activates fusion activity. J Virol 68, 4620–4627.
Chen, C., Weisz, O. A., Stolz, D. B., Watkins, S. C. & Montelaro, R. C.
(2004). Differential effects of actin cytoskeleton dynamics on equine
intracytoplasmic R peptide of the transmembrane envelope protein
in Moloney murine leukemia virus. J Virol 73, 8975–8981.
cleavages in murine leukemia virus Gag. J Virol 78, 1411–1420.
Parker, S. D. & Hunter, E. (2000). A cell-line-specific defect in the
infectious anemia virus particle production. J Virol 78, 882–891.
intracellular transport and release of assembled retroviral capsids.
J Virol 74, 784–795.
Cimarelli, A. & Darlix, J. L. (2002). Assembling the human immuno-
Parker, S. D., Wall, J. S. & Hunter, E. (2001). Analysis of Mason-
deficiency virus type 1. Cell Mol Life Sci 59, 1166–1184.
Demirov, D. G. & Freed, E. O. (2004). Retrovirus budding. Virus Res
106, 87–102.
Ragheb, J. A. & Anderson, W. F. (1994). pH-independent murine
Einfeld, D. & Hunter, E. (1988). Oligomeric structure of a prototype
retrovirus glycoprotein. Proc Natl Acad Sci U S A 85, 8688–8692.
Fischer, N., Heinkelein, M., Lindemann, D., Enssle, J., Baum, C.,
Werder, E., Zentgraf, H., Müller, J. G. & Rethwilm, A. (1998). Foamy
virus particle formation. J Virol 72, 1610–1615.
Freed, E. O. & Martin, M. A. (1995). Virion incorporation of envelope
glycoproteins with long but not short cytoplasmic tails is blocked by
specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J Virol 69, 1984–1989.
Freed, E. O. & Martin, M. A. (1996). Domains of the human
immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail
required for envelope incorporation into virions. J Virol 70, 341–351.
Green, N., Shinnick, T. M., Witte, O., Ponticelli, A., Sutcliffe, J. G. &
Lerner, R. A. (1981). Sequence-specific antibodies show that maturation
of Moloney leukemia virus envelope polyprotein involves removal of a
COOH-terminal peptide. Proc Natl Acad Sci U S A 78, 6023–6027.
Hansen, M., Jelinek, L., Jones, R. S., Stegeman-Olsen, J. & Barklis,
E. (1993). Assembly and composition of intracellular particles
formed by Moloney murine leukemia virus. J Virol 67, 5163–5174.
http://vir.sgmjournals.org
Pfizer monkey virus Gag particles by scanning transmission electron
microscopy. J Virol 75, 9543–9548.
leukemia virus ecotropic envelope-mediated cell fusion: implications
for the role of the R peptide and p12E TM in viral entry. J Virol 68,
3220–3231.
Reik, W., Weiher, H. & Jaenisch, R. (1985). Replication-competent
Moloney murine leukemia virus carrying a bacterial suppressor
tRNA gene: selective cloning of proviral and flanking host sequences.
Proc Natl Acad Sci U S A 82, 1141–1145.
Rein, A., Mirro, J., Haynes, J. G., Ernst, S. M. & Nagashima, K.
(1994). Function of the cytoplasmic domain of a retroviral
transmembrane protein: p15E-p2E cleavage activates the membrane
fusion capability of the murine leukemia virus Env protein. J Virol
68, 1773–1781.
Schägger, H. & von Jagow, G. (1987). Tricine-sodium dodecyl
sulfate-polyacrylamide gel electrophoresis for the separation of
proteins in the range from 1 to 100 kDa. Anal Biochem 166,
368–379.
Sharma, S., Murai, F., Miyanohara, A. & Friedmann, T. (1997).
Noninfectious virus-like particles produced by Moloney murine
leukemia virus-based retrovirus packaging cells deficient in viral
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 15:52:57
1587
K. B. Andersen, H. A. Diep and A. Zedeler
envelope become infectious in the presence of lipofection reagents.
Proc Natl Acad Sci U S A 94, 10803–10808.
Sherer, N. M., Lehmann, M. J., Jimenez-Soto, L. F. & 7 other
authors (2003). Visualization of retroviral replication in living cells
reveals budding into multivesicular bodies. Traffic 4, 785–801.
Suomalainen, M., Hultenby, K. & Garoff, H. (1996). Targeting of
Moloney murine leukemia virus Gag precursor to the site of virus
budding. J Cell Biol 135, 1841–1852.
Wilk, T., Gowen, B. & Fuller, S. D. (1999). Actin associates with the
nucleocapsid domain of the human immunodeficiency virus Gag
polyprotein. J Virol 73, 1931–1940.
1588
Wyma, D. J., Kotov, A. & Aiken, C. (2000). Evidence for a stable
interaction of gp41 with Pr55Gag in immature human immunodeficiency virus type 1 particles. J Virol 74, 9381–9387.
Wyma, D. J., Jiang, J., Shi, J., Zhou, J., Lineberger, J. E., Miller, M. D.
& Aiken, C. (2004). Coupling of human immunodeficiency virus type
1 fusion to virion maturation: a novel role of the gp41 cytoplasmic
tail. J Virol 78, 3429–3435.
Yu, X., Yuan, X., Matsuda, Z., Lee, T.-H. & Essex, M. (1992). The
matrix protein of human immunodeficiency virus type 1 is required
for incorporation of viral envelope protein into mature virions.
J Virol 66, 4966–4971.
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