JOURNAL OF VIROLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Dec. 2001, p. 12081–12087 Vol. 75, No. 24
Restoration of Wild-Type Infectivity to Human Immunodeficiency
Virus Type 1 Strains Lacking nef by Intravirion
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
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 incom-
pletely 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
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 histocompati-
bility 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 ap-
pears 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 rele-
vant in explaining Nef’s ability to enhance proviral DNA syn-
thesis 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 vesic-
ular stomatitis virus G (VSV-G) envelope glycoprotein targets
viral entry to the endocytic pathway and suppresses the re-
quirement 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 cyto-
plasm 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 observa-
tion 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 alter-
ing the composition of membrane rafts as a possible mecha-
nism 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 mecha-
nism. It has also been noted that there is significant overlap in
mutations known to affect cell sorting of CD4 and enhance-
ment 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
* Corresponding author. Mailing address: Dept. of Microbiology/
Biochemistry/Immunology, Morehouse School of Medicine, 720 West-
view Dr. S.W., Atlanta, GA 30310. Phone: (404) 752-1582. Fax: (404)
752-1179. E-mail: firstname.lastname@example.org.
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 pro-
teins that fuse at low pH do not require Nef for enhanced
infectivity (5). One method that can be used to uncouple dis-
assembly 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 infec-
tivity (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
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 parti-
cles with the amphotrophic murine leukemia virus (MLV) envelope glycopro-
tein. The transfections were done using Effectene (Qiagen) according to the
manufacturer’s protocol. Transfections were carried out in six-well plates using 1
?g 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 Pro-
gram). Viral particles were treated with 20 ?g 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 ?g of streptomycin per ml.
Assays were done in six-well plates seeded with 2 ? 105cells per well the day
before the assay. Each well was infected with pseudotyped virus at a concentra-
tion 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 ferrocya-
nide–2 mM MgCl2in 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 im-
munoglobulin (AIDS Reagent Program, catalog no. 3957) and protein A conju-
gated 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 ?g/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 ?l 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 transcrip-
tion (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 dithio-
threitol, 5 mM MgCl2, 5 ?g of poly(rA)-oligo(dT) (Calbiochem) per ml, 0.05%
NP-40, 1 mM EDTA, and 10 ?Ci of [?-32P]dTTP (Amersham) per ml. For each
assay, 5 ?l of supernatant from each transfection was removed and mixed with 25
?l of RT assay mixture. Each sample was then incubated at 37°C, and 6-?l
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 micro-
coccal nuclease (Roche) for 1 h at 37°C in 50 ?l of MN buffer (50 mM Tris HCl
[pH 7.8], 5 mM NaCl, 2.5 mM CaCl2). To inactivate the micrococcal nuclease but
not viral RT, EGTA was added to a final concentration of 2 mM and then 50 ?l
of endogenous buffer (50 mM Tris HCl [pH 7.5], 60 mM KCl, 5 mM MgCl2, 10
12082 KHAN ET AL.J. VIROL.
mM dithiothreitol, 10 ?Ci 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 ?M spermidine [pH 7.2] [Sigma], 2.5 mM MgCl2) 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
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 gen-
erally 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.
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) tem-
plate (Fig. 2A). In this assay the particles are disrupted by
treatment with NP-40 and tested using an exogenously added
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 pro-
teins 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.
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 exog-
enously 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.
VOL. 75, 2001 NERT TREATMENT RESTORES INFECTIVITY TO Nef?VIRUS12083
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
RT activities after NERT treatment of Nef?and Nef?viri-
ons 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 al-
lowed 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 ear-
liest 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.
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 com-
pared to Nef?virions (2) (Fig. 4A). After treatment by NERT
the infectivity of Nef?virions was restored to Nef?levels (Fig.
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.
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 en-
hancement 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 Mate-
rials 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.
12084KHAN ET AL.J. VIROL.
4A). As previously reported, we saw a modest increase in
infectivity of NERT-treated Nef?virions over nontreated viri-
ons (13). The increase in infectivity was mirrored by an in-
crease in the amount of proviral DNA present in the infected
cells (Fig. 4B). This was evident both qualitatively by tradi-
tional PCR and upon quantitation by real-time PCR (Fig. 4B).
The quantitation showed that Nef?virions increased approx-
imately 50% in infectivity, while Nef?virions increased ap-
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 pat-
tern 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?viri-
ons, and there was no apparent effect of the presence of Nef in
these experiments (Fig. 5B).
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 mecha-
nism 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 pro-
cess of reverse transcription of viral RNA. However, from
evidence presented in this study, Nef does not appear to di-
rectly 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 tran-
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 re-
cent study (42) demonstrated an increase in cytoplasmic deliv-
ery of Nef?virions over Nef?virions as measured by cytoplas-
mic p24 antigen content. One explanation offered for this effect
is that the expression of Nef in producer cells somehow mod-
ifies 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 phosphor-
ylation of matrix protein (MA) (46) and phosphorylated forms
of MA could alter the function of envelope protein. An inter-
esting 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 wasaccomplished by
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 surpris-
ing observation from this study was that expression of Nef
during the production of target virions had no effect on infec-
tivity 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 ad-
dition, 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
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.
VOL. 75, 2001 NERT TREATMENT RESTORES INFECTIVITY TO Nef?VIRUS 12085
is somehow envelope protein dependent, and yet they were not
completely explained by an enhancement of virus-cell fusion
In the present study we show that NERT treatment stimu-
lates the infectivity of Nef?virions. Two directly observable
things happen to NERT-treated virions: proviral DNA is elon-
gated 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 disas-
sembly 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 infec-
tious (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 viri-
ons 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 micros-
copy 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 transcrip-
tion 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.
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
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VOL. 75, 2001NERT TREATMENT RESTORES INFECTIVITY TO Nef?VIRUS12087