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Journal of Virology, December 2001, p. 12081-12087, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12081-12087.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Restoration of Wild-Type Infectivity to Human
Immunodeficiency Virus Type 1 Strains Lacking nef by
Intravirion Reverse Transcription
Mahfuz
Khan,
Minerva
Garcia-Barrio, and
Michael D.
Powell*
Department of
Microbiology/Biochemistry/Immunology, Morehouse School of Medicine,
Atlanta, Georgia 30310
Received 9 April 2001/Accepted 5 September 2001
 |
ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) Nef protein exerts
several effects, both on infected cells and as a virion protein, which
work together to enhance viral replication. One of these activities is
the ability to enhance infectivity and the formation of proviral DNA.
The mechanism of this enhancement remains incompletely understood. We
show that virions with nef deleted can be restored to
wild-type infectivity by stimulating intravirion reverse transcription. Particle composition and measures of reverse transcriptase activity remain the same for Nef+ and Nef
virions both
before and after natural endogenous reverse transcription (NERT)
treatment. The effect of NERT treatment on virions pseudotyped with
murine leukemia virus envelope protein was similar to that on particles
pseudotyped with HIV-1 envelope protein. However, virions pseudotyped
with vesicular stomatitis virus G envelope protein showed no influence
of Nef on NERT enhancement of infectivity. These observations suggest
that Nef may function at a level prior to reverse transcription. Since
NERT treatment results in partial disassembly of the viral core, we
speculate that Nef may function at the level of core particle disassembly.
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INTRODUCTION |
The nef gene of human
immunodeficiency virus type 1 (HIV-1) modulates the viral life cycle in
several distinct ways (reviewed in references 10, 17, 22,
38, and 40). The absence of a functional
nef gene in virus infecting both humans and rhesus monkeys
diminishes viral loads and significantly increases the time of clinical
progression of disease (11, 28). Expression of
nef as a transgene in mice produces many of the pathological effects seen in AIDS (12, 21). In vitro studies
demonstrate that Nef can affect multiple cellular functions that help
explain how it modulates pathogenicity. Nef promotes the removal of CD4 molecules from the surface of infected cells (4, 16, 35) and downregulates the surface expression of major histocompatibility complex class I molecules (33, 34). These properties are
structurally separate features of the Nef molecule (3,
19). Nef also exerts a profound effect on infected cells by
altering cell signaling pathways (9, 23, 49) and inducing
chemokine release from infected macrophages (47).
One key feature of Nef's influence on pathogenesis is that it can
enhance the intrinsic infectivity of viral particles (7, 18, 26,
36, 37, 45). Viruses which have nef deleted are from
4- to 40-fold less infectious than wild-type (WT) HIV-1 in tissue
culture systems (7, 37). Enhancement of viral infectivity appears to be linked to an increase in the initiation of proviral DNA
(2, 43). However, the exact nature of how Nef enhances infectivity and proviral DNA synthesis remains elusive.
Several pieces of evidence from prior studies may be relevant in
explaining Nef's ability to enhance proviral DNA synthesis and
infectivity. Nef is a virion protein (39) and is present in the viral core (30). Although Nef can be cleaved by
viral protease to liberate the C-terminal core domain, this cleavage does not appear to correlate with the ability to stimulate virion infectivity (6). Viruses with nef deleted can
be restored to WT infectivity by coexpression of Nef in the virus
producer cells but not target cells (2, 7). Pseudotyping
of HIV-1 virions by vesicular stomatitis virus G (VSV-G) envelope
glycoprotein targets viral entry to the endocytic pathway and
suppresses the requirement for Nef as well as sensitivity to
cyclosporine (2). Recently, it has been shown that Nef may
enhance proviral DNA formation by increasing delivery of virions to the
cytoplasm of infected cells (42). This study suggests that
Nef may function as an entry factor, which may involve interactions
with viral envelope protein. In another recent study
Nef+ and Nef
virions
were allowed to undergo intravirion fusion by pseudotyping a donor
particle with gp160 and a target particle with CD4 (56). Nef+ donor virus could enhance the infectivity of
a Nef
target particle after fusion. An
interesting observation from this study was that the ability to
complement nef was dependent on envelope glycoprotein but
did not appear to be acting solely at the level of enhancing membrane
fusion. One possible explanation for this observation is that Nef may
act by altering the composition of the lipid rafts from which HIV-1
particles bud. Other studies have also implicated Nef in altering the
composition of membrane rafts as a possible mechanism for enhancement
of infectivity (8, 31). It has been shown that cell
surface expression of CD4 during virus production can lead to a
reduction in infectivity by blocking Env incorporation
(31). Since one of Nef's functions is to downregulate CD4
expression, it can also alter infectivity through such a mechanism. It
has also been noted that there is significant overlap in mutations known to affect cell sorting of CD4 and enhancement of infectivity (8, 42). Both functions appear to be highly dependent on a
functional dileucine motif of Nef. All of these studies suggest that
Nef may function at some level prior to reverse transcription to allow
an increase in proviral DNA formation.
Disassembly of HIV-1 normally occurs as a part of the entry process
and, in contrast to some other enveloped viruses, does not appear to
require a pH-dependent step (25). It has been shown that
HIV-1 particles pseudotyped with envelope proteins that fuse at low pH
do not require Nef for enhanced infectivity (5). One
method that can be used to uncouple disassembly from the entry process
is treating virions by natural endogenous reverse transcription (NERT)
(14, 52-55). In this procedure HIV-1 virions are exposed
to buffer containing high concentrations of deoxynucleoside triphosphates (dNTPs) and spermidine. The dNTPs enter the virion via
the amphipathic domains of the gp41 on the virion (51). Detailed electron microscopy studies have shown that treatment of HIV-1
virions by NERT results in partial disassembly of the viral core
(52). This disassembly also results in disruption of a
structure known as the core-envelope linkage (CEL), which is an
attachment between the smaller end of the core and the envelope (24). NERT treatment can restore vif deletion
viruses to WT infectivity (13), which is important since
the Vif protein is also thought to be involved in the formation and
stabilization of the early reverse transcription complex. Since NERT
treatment can rescue the activity of vif deletion virions
and it appears that NERT can induce partial disassembly and initiation
of reverse transcription, we were interested in how NERT might affect
nef deletion virions. In this study, we have treated
Nef+ and Nef
virions to
induce NERT. We show that pretreatment by NERT can restore infectivity
of nef deletion virions to WT levels.
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MATERIALS AND METHODS |
Plasmid and viral constructs.
A single-round infectivity
assay was developed from the viral clone pNL4-3. An env
deletion variant of this clone designated pNL4-3KFS was produced by
insertion of KpnI linkers into the env reading
frame, introducing a frameshift mutation (gift of Eric Freed, National
Institutes of Health [NIH]) (15). A further mutation of
pNL4-3KFS to delete nef was produced by insertion of tandem
stop codons at the beginning of the nef reading frame, and
this construct was designated pNL4-3KFS
Nef (gift of Judith Levin,
NIH). To produce infectious NL4-3KFS or NL4-3KFS
Nef virus stocks,
HeLa cells were cotransfected with either pNL4-3KFS or pNL4-3KFS
Nef
plasmid and the envelope plasmid pIIIenv3-1 (gift of Eric Freed)
(44). In some cases the HIV-1 Env plasmid was replaced
with pHCMV-G (50) (kindly provided by J. Burns,
University of California, San Diego) to pseudotype particles with the
VSV-G envelope glycoprotein or with pSVAMLVenv (32) to
pseudotype particles with the amphotrophic murine leukemia virus
(MLV) envelope glycoprotein. The transfections were done using
Effectene (Qiagen) according to the manufacturer's protocol.
Transfections were carried out in six-well plates using 1 µg of viral
plasmid and 83 ng of envelope plasmid per well. Cells were incubated
for 16 h at 37°C and then refed to remove the Effectene and any
residual plasmid. Inoculated cells were then incubated for an
additional 36 h before supernatants containing the pseudotyped
viral particles were collected. Typically transfections produced from 5 to 20 ng of viral p24 antigen per ml as determined by enzyme-linked
immunosorbent assay (National Cancer Institute AIDS Vaccine Program).
Viral particles were treated with 20 µg of DNase I (Roche) (2,000 U/mg) per ml for 30 min at 37°C to remove any residual plasmid DNA
prior to storage. Using this system, the virus produced will infect
HeLa CD4+ cells and undergo a single round of
replication, producing progeny that lack envelope proteins.
Single-round infection assay.
The relative infectivity of
viral particles was determined by the multinuclear activation of a
galactosidase indicator (MAGI) assay (29).
HeLa-CD4+-LTR-
gal cells (NIH AIDS Reagent
Program, catalog no. 1470) were maintained in Dulbecco modified Eagle
medium supplemented with 5% fetal bovine serum, 0.1 mg of G418
per ml, 0.05 mg of hygromycin B per ml, L-glutamine, 100 U
of penicillin per ml, and 100 µg of streptomycin per ml. Assays were
done in six-well plates seeded with 2 × 105
cells per well the day before the assay. Each well was infected with
pseudotyped virus at a concentration of 1 ng of p24 of either NL4-3KFS
or NL4-3KFS
Nef per ml and incubated for 48 h at 37°C. Cells
were then fixed with 0.2% glutaraldehyde and 1% formaldehyde for 5 min at room temperature. The cells were then washed twice with
phosphate-buffered saline (PBS) and stained in X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) solution (0.4 mg of X-Gal per ml dissolved in dimethylformamide-4 mM
potassium ferricyanide-4 mM potassium ferrocyanide-2 mM
MgCl2 in PBS). The staining was allowed to
continue for 50 min at 37°C, and then cells were washed twice with
PBS. Results were scored as the total number of blue cells per nanogram
of p24 equivalent of virus added.
Immunoblot analysis of viral particles.
Particle composition
was determined by Western blotting of disrupted whole virions. Ten
milliliters of supernatant from cells transfected as described above
was pelleted by centrifugation at 26,000 × g for
1 h and contained 200 ng of p24. Fifty nanograms of p24 equivalent
of virus was added to sodium dodecyl sulfate (SDS) loading buffer and
heated to 100°C before being loaded onto an SDS-7.5% polyacrylamide
gel. The separated proteins were detected by ECL (Amersham) using human
HIV immunoglobulin (AIDS Reagent Program, catalog no. 3957) and protein
A conjugated to horseradish peroxidase.
Detection of proviral DNA by PCR.
MAGI cells were infected
as described above. After 48 h at 37°C cells, were harvested by
trypsinization. The cells were pelleted and then resuspended in PBS
supplemented with 5 mM magnesium. The resuspended pellet was then
treated with DNase I (Roche; 20 µg/ml) for 30 min at 37°C. The
treated cells were then washed twice in PBS and left as a cell pellet.
The cell pellets were lysed and DNA was isolated using DNAeasy reagents
from Qiagen according to the manufacturer's protocol. The final DNA
extracts were adjusted to 150 µl in volume. Uninfected control cells
were also processed as described above.
Proviral DNA was detected by traditional PCR using the following primer
set to detect strong-stop DNA: forward primer 5'-GGC TAA CTA GGG AAC
CCA CTG CTT and reverse primer 5'-CTG CTA GAG ATT TTC CAC ACT GAC,
which amplify region 496 to 635 of NL4-3 (GenBank accession no.
AF070521). The amount of DNA from each cell was normalized by detection
of
-globin (forward primer 5'-TCT ACC CTT GGA CCC AGA GG and reverse
primer 5'-CTG AAG TTC TCA GGA TCC ACG). Quantitative results were
obtained using I-cycler real-time PCR (Bio-Rad) and SYBR green
(Perkin-Elmer) core reagents. In this case, the amount of cellular DNA
present in the preparations adversely affected measurement using the
-globin primer set, so the amount of DNA in each preparation was
confirmed using the
-actin Taqman control kit from Perkin-Elmer
instead. Plasmid DNAs from molecular clones of NL4-3 were used as
standards for quantitation of proviral DNA, and the human genomic DNA
from the Perkin-Elmer kit was used for standards for
-actin.
To detect the progress of proviral DNA during endogenous reverse
transcription (ERT) and NERT reactions, we also included
the following
primer set which would amplify late viral products:
forward primer
5'-GGC TAA CTA GGG AAC CCA CTG CTT and reverse
primer 5'-ATA CCG ACG
CTC TCG CAC CCA T, which amplify region
496 to 811 of NL4-3 (GenBank
accession no.
AF070521). In these
cases, the ERT and NERT reactions
were allowed to proceed for
4 h. At this time point the amount of
early NERT product had already
reached saturation, so additional time
points at 45, 120, and
240 min were included for the early primer
set.
RT assays.
Three types of reverse transcriptase (RT) assays
were employed in this study to test different aspects of reverse
transcription. The first assay is an exogenous RT assay using
detergent-treated virions and a poly(rA)-oligo(dT) template.
This assay measures the intrinsic enzymatic activity of the RT in
particles. The assay was done essentially as previously described
(27). The RT assay mixture contained 50 mM Tris-HCl (pH
7.8), 75 mM KCl, 2 mM dithiothreitol, 5 mM MgCl2,
5 µg of poly(rA)-oligo(dT) (Calbiochem) per ml, 0.05% NP-40, 1 mM
EDTA, and 10 µCi of [
-32P]dTTP (Amersham)
per ml. For each assay, 5 µl of supernatant from each transfection
was removed and mixed with 25 µl of RT assay mixture. Each sample was
then incubated at 37°C, and 6-µl aliquots were removed at 1, 10, 30, and 60 min and spotted on DE81 paper (Whatman). The filters were
dried and washed four times in 2× SSC (1× SSC is 0.15 M NaCl plus
0.015 M sodium citrate) for 5 to 10 min each. The filters were then
washed twice in 95% ethanol for 1 min each. Filters were then dried,
and radioactivity was counted by liquid scintillation. Counts were
normalized by p24 content prior to plotting.
The second assay is a classical ERT assay (
48) using
detergent-treated virions and the endogenous tRNA primer and viral
template. This assay measures the ability of each virus to initiate
reverse transcription on a viral template. Virus-containing
supernatants
from transfection of KFS or KFS

Nef plasmids were
normalized by
p24 content. Particles (5 ng) were pretreated with 30 U
of micrococcal
nuclease (Roche) for 1 h at 37°C in 50 µl of MN
buffer (50 mM
Tris HCl [pH 7.8], 5 mM NaCl, 2.5 mM
CaCl
2). To inactivate the
micrococcal nuclease
but not viral RT, EGTA was added to a final
concentration of 2 mM and
then 50 µl of endogenous buffer (50
mM Tris HCl [pH 7.5], 60 mM
KCl, 5 mM MgCl
2, 10 mM dithiothreitol,
10 µCi
of [

-
32P] dATP, and 0.05% NP-40) was added
to each reaction mixture.
The mixture was incubated at 37°C
overnight. The products were
analyzed by PCR using early and late
primers as described
above.
The third assay is the NERT reaction. Viral particles were normalized
by p24 content, and 4 ng of each stock was treated with
NERT cocktail
(1 mM dNTPs [Roche], 30 µM spermidine [pH 7.2] [Sigma],
2.5 mM
MgCl
2) for 4 h at 37°C as previously
described (
13).
One nanogram of treated virions was used
to infect MAGI cells
for infectivity measurement or was directly tested
by PCR using
early and late PCR primers to determine the progress of
the reaction.
In some cases the products of the NERT reaction were used
to test
intrinsic RT activity after NERT treatment. In these cases,
samples
were removed after 4 h of NERT treatment and diluted 1:50
to reduce
the relatively high concentration of cold dNTPs. The samples
were
then treated as described above for the exogenous RT
assay.
 |
RESULTS |
Deletion of Nef does not grossly affect particle composition.
Western blot analysis of whole viral particles did not reveal any
change in protein composition from Nef+ and
Nef
virions (Fig.
1). One way to explain the increase in
proviral DNA synthesis from virions that contain Nef could be that
there is a change in p24 relative to RT. Since particle numbers are generally normalized by p24 content, the increase in proviral DNA could
simply reflect a larger number of particles containing less total p24.
However, a comparison of overall content shows almost identical levels
of detectable proteins in each type of virion. Significantly, the
amounts of p24 were essentially the same in each virus preparation, and
the amounts of p66 and p51 of RT were the same (Fig. 1). This is also
confirmed by the observation that the RT activity of the same amount of
virions as judged by p24 content is essentially the same (Fig.
2A). This suggests that Nef is not
influencing proviral DNA synthesis through an artifactual change in
viral composition.

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FIG. 1.
Deletion of nef does not affect virion
composition. Equal amounts of virions as determined by p24 content were
loaded onto an SDS-7.5% polyacrylamide gel. The gel was blotted, and
HIV-1 proteins were detected using human antiglobulin to HIV-1. The
relative amounts of each protein detected in virions containing Nef
(KFS) and in virions in which Nef was absent [( )Nef] appear to be
identical. Numbers on the right are molecular weights in
thousands.
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FIG. 2.
Deletion of nef does not affect intrinsic
RT activity or residual RT activity after NERT treatment. (A) The
intrinsic RT activity of Nef-containing virions (KFS) and virions
lacking Nef [( )NEF] were tested using detergent-treated virions and
an exogenously added poly(rA)-oligo(dT) template. In both cases the RT
activity was essentially the same. (B) Intrinsic RT activity after NERT
treatment was also determined for Nef-containing virions and virions
lacking Nef. The samples were diluted 1:50 to lower the relatively high
concentration of dNTPs carried over from the NERT reaction, and this
may be responsible for the lower total counts. The error bars represent
the standard deviations of at least triplicate measurements.
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Intrinsic RT activities of Nef+ and Nef
virions are the same.
If Nef were somehow acting in the particle
to directly augment the catalytic activity of RT, one would expect that
the intrinsic activities of RT from the two types of particles could be
different. Therefore, we tested the intrinsic RT activities from
Nef+ and Nef
virions
using a poly(rA)-oligo(dT) template (Fig. 2A). In this assay the
particles are disrupted by treatment with NP-40 and tested using an
exogenously added template. As can be seen in Fig. 2A, the activities
of identical amounts of virus were essentially the same. This suggests
that Nef does not change the intrinsic catalytic activity of the RT in
the virions.
RT activities after NERT treatment of Nef+ and
Nef
virions are the same.
To ensure that NERT
treatment was not somehow altering the activities of the RT present in
Nef+ and Nef
virions in a
differential way, we tested the RT activities of NERT-treated virions.
Both Nef+ and Nef
virions
were allowed to undergo the NERT reaction. After NERT treatment, the
virions were diluted 1:50 to lower the high concentration of cold dNTPs
in the NERT cocktail. The virions were then tested for exogenous RT
activity using the standard poly(rA)-oligo(dT) substrate. The results
(Fig. 2B) show that the RT activities after NERT were essentially the
same for both types of virions. The kinetics of this reaction over
12 h were also essentially identical (data not shown).
To see whether the progress of ERT or NERT reactions was influenced by
the presence of Nef, we performed PCR analysis of both
ERT and NERT
reactions using primers that would detect early and
late proviral DNA
products (Fig.
3A). The progress of
replication
appears to be the same in Nef
+ and
Nef

virions in both the ERT and NERT reactions.
The earliest time
that late products could be detected was 4 h. At
this time there
were no late products visible in the ERT reaction
mixture and
only minimal products in the NERT reaction mixture.
Unfortunately,
at this time point the early NERT products had already
reached
saturation (Fig.
3A). To confirm that the amounts of proviral
DNA formed by KFS and Nef

virions were the same
at earlier times points, we performed PCR
on samples removed from the
NERT reaction mixture at 45, 120,
and 240 min (Fig.
3B). Results from
the time course confirm that
the amounts of early products at earlier
time points were also
the same. Together, these data confirm that the
presence of Nef
does not appear to influence directly the course of
reverse transcription
on the actual viral template. These data also
confirm that the
NERT reaction proceeds to similar extents in both
Nef
+ and Nef

virions and
does not introduce a bias in the later MAGI assay
for infectivity.

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FIG. 3.
The progression of reverse transcription is not affected
by nef deletion. (A) The progress of reverse
transcription in ERT and NERT reactions was monitored by PCR with
primers to detect both early and late proviral DNA products. In both
cases the relative amount of proviral DNA appears to be the same. Both
sets of samples were run for 40 cycles. (B) To confirm that the early
NERT products were the same at earlier time points, samples were also
included at 45, 120, and 240 min, using the same PCR protocol for 40 cycles. Samples were run in triplicate, and a representative experiment
is shown.
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NERT treatment restores WT infectivity to Nef
virions.
Using the MAGI cell assay, we determined the
infectivities of Nef+ and
Nef
virions. As has been noted by others, we
saw about a fivefold decrease in infectivity of
Nef
virions compared to
Nef+ virions (2) (Fig.
4A). After treatment by NERT the
infectivity of Nef
virions was restored to
Nef+ levels (Fig. 4A). As previously reported, we
saw a modest increase in infectivity of NERT-treated
Nef+ virions over nontreated virions
(13). The increase in infectivity was mirrored by an
increase in the amount of proviral DNA present in the infected cells
(Fig. 4B). This was evident both qualitatively by traditional PCR and
upon quantitation by real-time PCR (Fig. 4B). The quantitation showed
that Nef+ virions increased approximately 50% in
infectivity, while Nef
virions increased
approximately 500%.

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FIG. 4.
The infectivity of virions with nef
deleted can be restored after NERT treatment. (A) Nef-containing
virions (KFS) and virions lacking Nef [( )Nef] were tested for
infectivity by the MAGI cell assay both before and after NERT
treatment. After NERT treatment the virions lacking Nef
[( )Nef+NERT] were restored to levels similar to those of
Nef-containing virions (KFS+NERT). Note that some enhancement of
infectivity occurs even in Nef-containing virions. The results
represent three independent experiments, and the error bars are the
standard deviations from those experiments. (B) Proviral DNA was
quantitated by PCR using the early primer set. -Globin was used as a
control for total cellular DNA added to each reaction mixture. The
numbers between the lanes are values obtained from real-time PCR using
the early primer set in the presence of SYBR green. In this case a
-actin Taqman probe was used to control cell number (see Materials
and Methods). Samples were run in triplicate, and the data shown are
the means ± one standard deviation. The traditional PCR was run
for 40 cycles, and real-time data were collected for 50 cycles.
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NERT enhancement of virions pseudotyped with MLV or VSV-G.
To
determine the effect of different types of envelope glycoproteins on
the enhancement of infectivity, Nef+ and
Nef
virions were pseudotyped with either MLV or
VSV-G envelope glycoprotein and treated by NERT. The virions
pseudotyped with MLV envelope glycoprotein showed a pattern of
infectivity similar to that of virions with HIV-1 envelope (Fig.
5A). The Nef
virus was about fourfold less infectious than
Nef+ virus before NERT treatment (Fig. 5A). After
NERT treatment, the Nef
virus showed a
substantial increase in infectivity, although it did not restore
infectivity to the levels seen with the Nef+
virus (Fig. 5A) in repeated experiments. As might be expected, virions
pseudotyped with VSV-G envelope glycoprotein showed no difference in
infectivity between Nef+ and
Nef
virions (Fig. 5B). NERT treatment resulted
in about a 3.5-fold increase in infectivity for both
Nef+ and Nef
virions, and
there was no apparent effect of the presence of Nef in these
experiments (Fig. 5B).

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FIG. 5.
Effect of pseudotyping with other envelope
glycoproteins. KFS and Nef virions were pseudotyped with
either amphotrophic MLV envelope glycoprotein (A) or VSV-G envelope
glycoprotein (B) and tested for infectivity with or without NERT
treatment using the MAGI assay. Results represent triplicate
measurements, and the bars represent the standard deviations of those
measurements.
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DISCUSSION |
The goal of this study was to better understand the role
that Nef plays in the enhancement of infectivity and proviral DNA formation. Many possible mechanisms could account for such effects, and
it is entirely possible that more than one mechanism may be involved.
One of the most direct ways that Nef could influence proviral DNA
formation would be to enhance the catalytic activity or amount of RT
present in the virion. Alternatively, Nef could enhance the progression
of the process of reverse transcription of viral RNA. However, from
evidence presented in this study, Nef does not appear to directly
modulate the process of reverse transcription, at least as far as can
be measured in these assays. This suggests that Nef may act on a step
in replication independent of reverse transcription.
It is known that Nef can affect the infectivity of virions in a
CD4-dependent manner by both enhancing virion release (41) and relieving a potential block of Env incorporation by CD4
(31). However, in the present study,
Nef+ and Nef
viral
particles were produced in cells which lack CD4, which should eliminate
these CD4-dependent effects. Therefore, NERT treatment appears to act
on CD4-independent effects of Nef.
Another possibility is that Nef could be acting to enhance the delivery
of replicating complexes to the cytoplasm. A recent study
(42) demonstrated an increase in cytoplasmic delivery of
Nef+ virions over Nef
virions as measured by cytoplasmic p24 antigen content. One explanation offered for this effect is that the expression of Nef in producer cells
somehow modifies the viral envelope protein to allow it to become more
efficient during attachment and entry. A possible mechanism is given by
the observation that Nef can enhance the phosphorylation of matrix
protein (MA) (46) and phosphorylated forms of MA could
alter the function of envelope protein. An interesting observation from
this study is that mutations which are known to affect CD4
downregulation (LL164, WL57, and DD174) or diminished binding to SH3
domains (P69, P72, and P75) also affect the enhancement of infectivity
even if the viral particles are produced in cells that lack CD4.
More evidence of Nef's action is given in another recent study in
which Nef+ and Nef
virions were allowed to undergo intravirion fusion prior to infection of cells (56). The fusion of virions was accomplished by
producing donor virions pseudotyped with gp160 and target virions that
were pseudotyped with CD4. In this study, the infectivity of
Nef
virions could be restored by fusion to
Nef+ virions. A surprising observation from this
study was that expression of Nef during the production of target
virions had no effect on infectivity of virions, while expression of
Nef during production of donor virions increased infectivity. The
effect of Nef appeared to be dependent on the presence of envelope
protein. In addition, when donor particles were pseudotyped with both
HIV-1 and VSV-G envelope proteins and allowed to infect cells that lack
CD4, there was still some residual effect of Nef on infectivity. These
observations suggest that the effect of Nef is somehow envelope protein
dependent, and yet they were not completely explained by an enhancement
of virus-cell fusion alone.
In the present study we show that NERT treatment stimulates the
infectivity of Nef
virions. Two directly
observable things happen to NERT-treated virions: proviral DNA is
elongated and the core particle partially disassembles. Although
elongation of proviral DNA gives virions a "head start" in the
process of reverse transcription, it appears that the progress of
reverse transcription is the same for Nef+ and
Nef
virions. Therefore, it seems more likely
that the partial disassembly of the core is involved in the
enhancement of infectivity. We know very little about what cues are
used to trigger the disassembly process. It is possible that core
disassembly and the envelope protein could be linked such that Nef must
interact with the envelope protein to help trigger the process of
disassembly. The observation that HIV-1 virions can be pseudotyped
with other envelope proteins and remain infectious (1, 2)
suggests that disassembly can occur independently of the type of
envelope present. Indeed, the observation that during the NERT reaction
cores can disassemble and yet virions remain infectious is evidence
that entry and disassembly can be uncoupled. Yet, it is evident from
electron microscopy studies that it is difficult to find intact cores
even early after attachment and entry (20). This suggests
that core disassembly must occur very early after or concurrently with
attachment and fusion of envelope and cell membrane. Electron
microscopy studies have revealed the presence of a structure know as
the CEL (24, 52). This structure is a physical attachment
of the smaller end of the core and the inner surface of the viral
envelope. Although the function of the CEL is unknown, it does provide
for a physical link between the core and envelope with which Nef could
interact. If the CEL represented a type of switch to sense attachment
and entry, it could provide a signal for disassembly to occur. Nef could play a role in mediating this reaction and thus aid the process
of core disassembly. In this fashion Nef could be indirectly aiding in
core disassembly in a manner that is dependent on the presence of
envelope protein. In the case of the NERT-treated virions,
disassembly is started while the virions are still intact. This
premature disassembly could overcome the need for Nef during the entry
process. A similar situation would exist when HIV-1 particles are
pseudotyped with VSV-G envelope protein. In this case, targeting to the
endosomal pathway could trigger disassembly through an alternate
mechanism such as a change in pH, again bypassing the need for Nef to
enhance the trigger process.
We have shown that the influence of Nef on infectivity and proviral DNA
formation can be negated if reverse transcription is allowed to proceed
inside the intact virion. We feel that this suggests a role for Nef in
the processing of the core particle to allow the more efficient
formation of the active reverse transcription complex. One way to
explain this would be if Nef had some influence on the ability of core
particles to disassemble. In light of recent advances in our
understanding of this key process in viral replication, it should be
possible to formulate new tests of the present theories and, in doing
so, increase our overall understanding of HIV-1 replication.
 |
ACKNOWLEDGMENTS |
We thank Eric Freed for pNL4-3KFS, Judith Levin for pNL4-3
nef,
and Jane Burns for pHCMV-G. We also thank Craig Bond for helpful comments and suggestions.
This work was supported by Public Health Service grants G-12-RR03034
(NCRR), K22-HD-1228 (NICHD), and S-06-GM08248 (NIGMS).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Microbiology/Biochemistry/Immunology, Morehouse School of Medicine, 720 Westview Dr. S.W., Atlanta, GA 30310. Phone: (404) 752-1582. Fax: (404) 752-1179. E-mail: powellm{at}msm.edu.
 |
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Journal of Virology, December 2001, p. 12081-12087, Vol. 75, No. 24
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