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Previous Article | Next Article 
Journal of Virology, November 2000, p. 10074-10080, Vol. 74, No. 21
Departments of
Microbiology,1 Pathology and Laboratory
Medicine,2 and
Medicine,3 University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148
Received 23 June 2000/Accepted 6 August 2000
The study of early events in the human immunodeficiency virus type
1 (HIV-1) life cycle can be limited by the relatively low numbers of
cells that can be infected synchronously in vitro. Although the
efficiency of HIV-1 infection can be substantially improved by
centrifugal inoculation (spinoculation or shell vial methods), the
underlying mechanism of enhancement has not been defined. To understand
spinoculation in greater detail, we have used real-time PCR to
quantitate viral particles in suspension, virions that associate with
cells, and the ability of those virions to give rise to reverse
transcripts. We report that centrifugation of HIV-1IIIB
virions at 1,200 × g for 2 h at 25°C increases
the number of particles that bind to CEM-SS T-cell targets by
~40-fold relative to inoculation by simple virus-cell mixing.
Following subsequent incubation at 37°C for 5 h to allow
membrane fusion and uncoating to occur, the number of reverse
transcripts per target cell was similarly enhanced. Indeed, by
culturing spinoculated samples for 24 h, ~100% of the target
cells were reproducibly shown to be productively infected, as judged by
the expression of p24gag. Because the modest
g forces employed in this procedure were found to be
capable of sedimenting viral particles and because CD4-specific
antibodies were effective at blocking virus binding, we propose that
spinoculation works by depositing virions on the surfaces of target
cells and that diffusion is the major rate-limiting step for viral
adsorption under routine in vitro pulsing conditions. Thus, techniques
that accelerate the binding of viruses to target cells not only promise
to facilitate the experimental investigation of postentry steps of
HIV-1 infection but should also help to enhance the efficacy of
virus-based genetic therapies.
The infectious life cycle of human
immunodeficiency virus type-1 (HIV-1) is initiated when virions bind to
susceptible target cells via the viral surface glycoprotein
gp120/41env. The fusion of the viral membrane
with the target cell membrane is mediated through the interaction of
Env with CD4 and members of the chemokine receptor family of proteins,
most commonly CCR5 or CXCR4 (4). While Env interactions with
these receptors are essential for virus infection, it is now
appreciated that virus-cell binding may also be initiated through
interactions with other cell surface molecules, such as heparan
sulfate-containing proteoglycans (29, 32, 46) and the
dendritic cell-specific intercellular adhesion molecule 3 binding
C-type lectin DC-SIGN (17). Following membrane fusion, the
viral core enters the cytosol, and a mysterious series of events that
are collectively termed uncoating then ensues. These steps, which
appear to include the shedding of p24gag from
the core, are presumably essential for formation of the viral
nucleoprotein complex that mediates reverse transcription, transport to
the nucleus, and integration of viral cDNA into the host genome to
establish the provirus (13). Although these complexes have
been described by various terms, we will refer to them broadly as
postentry nucleoprotein, or reverse transcription (RT), complexes. Once
the provirus is established, it serves as the template for subsequent
virus gene expression, genome synthesis, and progeny virion production.
Importantly, the relative efficiencies of these defined steps in HIV-1
replication have not been determined to a high degree of accuracy. As a
consequence, the underlying reasons for the low infectivity-to-particle
ratios that are usually assigned to HIV-1 (for example, 1 in 3,500 to
60,000 [26, 36]) are not well understood.
In an effort to address these points, we have been exploiting kinetic
(real-time) PCR and RT-PCR to quantitate viral nucleic acids during the
progressive stages of single-cycle HIV-1 infection. Early on in these
studies, we found that our infection efficiencies were
substantially enhanced by spinoculation, or centrifugal infection, techniques. Although such protocols have been used in clinical microbiology since the 1950s (18) to enhance infection by a number of difficult-to-culture pathogens (24), including
HIV-1 (20, 37) and other retroviruses (3, 5), the
mechanism whereby centrifugation leads to an enhanced level of
infection remains controversial (24). Specifically, some
groups have proposed that centrifugation enhances cellular
susceptibility (22, 23) or viral fusion (43),
whereas others have suggested that increased viral deposition is the
most important aspect of centrifugal enhancement of infection (21,
34). With these uncertainties in mind, we decided to extend
our quantitative analyses of virus binding, entry, reverse
transcription, and particle production to investigate how
spinoculation works. In summary, we demonstrate that
centrifugal inoculation increases infection of T cells by HIV-1 mostly
by depositing large numbers of virions on target cell surfaces in a
CD4-dependent manner.
Cell lines and viruses.
The CD4-positive T-cell lines
CEM-SS, HUT 78, and SupT1 were maintained as described previously
(16, 33, 40, 41). Wild-type HIV-1IIIB virus
stocks were initially generated by transfection of 293T cells with the
pIIIB infectious molecular clone (15), whereas the G
glycoprotein of vesicular stomatitis virus (VSV G)-pseudotyped stocks
were produced by cotransfection with pIIIB/ Inoculation with HIV-1 at ambient gravity and at 1200 × g.
As shown in the flow diagram of the centrifugal
inoculation process (see Fig. 1), 2 × 105 cells per
well in flat-bottom 96-well microtiter plates were incubated with 100 µl of viral stock (0.45 to 3.0 µg of
p24gag/ml). Plates were enclosed in plastic bags
and centrifuged in microtiter plate carriers at 1,200 × g for 2 h at 25°C, and the cells were washed five times
with cold medium. Control cells were incubated on a laboratory bench
for the same time period and then washed similarly. Cell-associated
viral RNA, DNA, and p24gag were assayed
immediately (see below). After 5 or 24 h of culture, total viral
DNA and p24gag were also measured. In some
experiments, mixing (25 rotations/s) of virus-cell suspensions at
ambient gravity for 2 h was performed using level 2 on a Vortex
Genie 2 mixer (Scientific Industries Inc.). For other experiments,
virus challenges were performed in the presence of the anti-CD4 mouse
immunoglobulin G1 (IgG1) monoclonal antibody 19, which blocks HIV-1
infection (12), or 8 µg of polybrene/ml (Sigma Biochemicals).
Indirect immunofluorescence.
Cytocentrifuged cells were
prepared from infected cultures 24 h after inoculation and then
fixed and permeabilized with 2% paraformaldehyde-1% Triton X-100.
Samples were incubated with a polyclonal rabbit
p24gag-specific antiserum (38)
followed by a fluorescein isothiocyanate-conjugated F(ab')2
polyclonal goat anti-rabbit IgG antibody (Jackson Laboratories). The
nuclei were counterstained with 4',6'-diamidino-2-phenylindole (Vector
Laboratories), and the samples were visualized at a magnification of
×400 using a Nikon Microphot-SA microscope with a cooled
charge-coupled device camera (Princeton Instruments) and Metamorph
image analysis software (Universal Imaging).
Measurement of cellular and viral RNA.
Total cell-associated
RNA (which includes viral RNA associated with cell surfaces as well as
intracellular RNA) was prepared by lysis of 105 infected or
uninfected cells in 1 ml of TRIZOL reagent (Life Technologies, Gibco
BRL) with excess glycogen as a carrier (Roche Molecular Biochemicals),
according to the manufacturer's instructions (6). RNA was
similarly prepared from viral stocks by adding 10 µl of viral stock
to 1 ml of TRIZOL.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Human Immunodeficiency Virus Type 1 Spinoculation
Enhances Infection through Virus Binding
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
env (39) and pHIT/G (14). At 24 h,
virus-containing supernatants were centrifuged at 500 × g for 10 min, treated with 30 µg of DNase I (Roche
Biochemical)/ml for 30 min at room temperature in the presence of 10 mM
MgCl2, filtered through 0.45-µm-pore-size filters, and
stored in aliquots at 
80°C. CEM-SS cells were then infected with
wild-type virus, and high-titer DNaseI-treated stocks were prepared as
described above at the time of peak virus production. Importantly, we
used HIV-1IIIB stocks that had been passed through CEM-SS
cells in an effort to minimize the presence of contaminating plasmid
DNA. In several experiments, freshly thawed viral stocks were
additionally passed through 0.2-µm-pore-size filters immediately prior to use to ensure the removal of any viral aggregates that might
have formed.
Measurement of cell-associated viral DNA.
Cell-associated
DNA was prepared from 105 infected CEM-SS cells, by lysis
in 100 µl of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA-0.2 mM
CaCl2-0.001% Triton X-100-0.001% sodium dodecyl
sulfate-1-mg/ml proteinase K. The lysates were then incubated at
58°C for 1 h, heat inactivated at 95°C for 15 min, and stored
at 80°C. Kinetic (fluorescence-monitored) PCR was performed on
1.25 × 104 cell equivalents to quantitate viral
gag DNA and cellular 
-globin DNA. The sequences of the
-globin forward and reverse primers were
5'-CCCTTGGACCCAGAGGTTCT-3' and
5'-CGAGCACTTTCTTGCCATGA-3', and the molecular beacon, which
was labeled with JOE
(6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein) and DABCYL,
was 5'-GCGAGCATCTGTCCACTCCTGATGCTGTTATGGGCGCTCGC-3'. The
reaction and cycling conditions were the same as for RT-PCR, except
that the concentration of MgCl2 was 3 mM and reverse
transcriptase and the initial 48°C incubation were omitted. A
standard curve for the HIV-1 DNA copy number was prepared from mixtures
of ACH-2 cells (which harbor two HIV-1 proviruses) and CEM-SS cells,
using the above lysis procedure. In some experiments, a standard curve was also prepared by diluting pIIIB DNA in a CEM-SS cell lysate.
Measurement of cell-associated and supernatant p24gag. Cell suspensions or corresponding supernatants were treated with 0.5% Triton X-100, and p24gag was quantitated using an enzyme-linked immunosorbent assay (ELISA) (NEN Life Science Products). Importantly, cell-associated p24gag represents the sum of the surface-bound and intracellular Gag antigen.
Calculation of virion equivalents from p24gag concentration. Virion equivalents were determined by assuming that there are an average of 1,500 molecules of p24gag present per viral particle (49) and that the molecular mass of p24gag is 25.587 kDa for HIV-1IIIB. This equates to ~15,800 viral particles per pg of p24gag, such that a viral stock of 3.0 µg of p24gag/ml should represent 4.7 × 1010 virions per ml (or 9.4 × 1010 genomes per ml). In agreement with this, RT-PCR analysis (in triplicate) showed that the RNA copy number for a 3.0-µg/ml p24gag stock was 7 × 1010 ± 4 × 1010 genomes per ml.
Calculation of virion sedimentation time.
The time
(t = k/S) required for the sedimentation of HIV-1 viral
particles was calculated using established formulas for calculating k values [k = ln
(rmax/rmin)/
2],
where is the angular velocity of the rotor in radian/s and rmax and rmin are the
maximum and minimum radii (2) (J. Condino, Beckman
publication, Beckman Instruments, Inc., Fullerton, Calif., 1996) and
assuming that HIV-1 virions share with other retroviral particles a
sedimentation coefficient of 600S (600 × 10
13 s)
(48).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Spinoculation dramatically increases the adsorption of
HIV-1 to T cells.
To address the mechanistic basis for
the enhancement of virus infection by centrifugal inoculation, we
devised the experimental scheme outlined in Fig.
1. Here, the human T-cell line CEM-SS was
incubated with a concentrated stock of HIV-1IIIB at 25°C
for 2 h at either 1 × g or 1,200 × g (spin conditions). Of note, these centrifugation conditions had
no effect on cell viability as judged by trypan blue exclusion (data
not shown). After extensive washing to remove unbound virions, the mean
numbers of viral RNA copies, viral cDNA copies (extended minus-strand
reverse transcripts), and virion equivalents of
p24gag associated per cell were measured by
RT-PCR, PCR, and ELISA, respectively (zero time point). The cells were
then cultured at 37°C for 5 h, and the average number of copies
of viral cDNA within each cell was determined. Normalization for the
number of cell equivalents present in each sample was achieved through
measuring GAPDH mRNA and -globin DNA. Finally, after a total of
24 h in culture, the mean p24gag protein
production per cell was determined by ELISA and again converted to the
number of virion equivalents. In most experiments, 95% confidence
intervals were estimated after the assays were performed in triplicate.
|
|
Spinoculation can result in 100% productive infection of
susceptible T cells in a single cycle.
To determine the percentage
of cells that became productively infected with HIV-1 following
spinoculation, cytocentrifuge preparations of the infected cultures
analyzed above were subjected to indirect immunofluorescence using a
Gag-specific antiserum at 24 h postchallenge (Fig.
3). When the target cells were exposed to
concentrated virus (4.7 × 109 virions per 2 × 105 cells in 100 µl) by spin infection, essentially 100%
of the cells were expressing significant levels of viral antigen and
the culture contained numerous syncytia (Fig. 3A). This contrasts with
the infection at 1 × g, for which only 5 to 10% of
the cells were visibly expressing Gag (Fig. 3B). The increased
cell-associated p24 is unlikely to represent input virus, since total
p24gag as measured by ELISA (cell-associated
plus supernatant p24gag) increased 24-fold over
input levels in the spinoculated cultures during 24 h of
culturing.
|
Modest g forces (1,200 × g for 2 h) are sufficient to sediment HIV-1 virions.
As discussed earlier,
a variety of mechanisms have been proposed to explain centrifugal
enhancement of viral infection (21-24, 34, 35, 43). Because
our results show that HIV-1 infection is significantly enhanced by
spinoculation but that subsequent progression to cDNA accumulation is
not further augmented (Fig. 2), it appeared most likely that this
method works by simply depositing virions on target cell surfaces. On
the other hand, and contrary to this notion, there has been some
skepticism concerning the ability of viruses to be sedimented by the
modest g forces used in spinoculation techniques
(22). Because of these uncertainties, it was important for
us to determine whether our centrifugation conditions resulted in
virion sedimentation. Virus-containing supernatants of 100 µl were
therefore incubated at 25°C at 1 × g or
1,200 × g for 2 h, and 20-µl fractions were
then drawn serially from the top and assayed for
p24gag content by ELISA (Fig.
4). Inspection of these data reveals that between 50 and 80% of the viral inoculum became concentrated in the
lower 20% of the well volume following centrifugation. This concentrating effect occurred in the presence or absence of target cells, at 4 or 25°C, and with concentrated (900 ng of
p24gag/ml) and dilute (9 ng of
p24gag/ml) viral stocks (data not shown).
Moreover, effective sedimentation was also maintained when virus stocks
were passed through 0.2-µm-pore-size filters immediately prior to
centrifugation, thus implying that virus aggregation was unlikely to be
contributing to sedimentation (data not shown).
|
Spinoculation selectively enhances CD4-dependent binding.
Although HIV-1 entry generally requires the surface expression of CD4
and one of the chemokine coreceptors (42), a number of
additional cell surface proteins can also serve as binding sites for
HIV-1. In an effort to measure the specific contribution of CD4 to the
binding of HIV-1 to CEM-SS cells under spinoculation conditions, we
inoculated cells in the presence or absence of saturating doses of
blocking anti-CD4 monoclonal antibodies (Fig. 5). Cell-associated
p24gag was reduced by ~90% when the challenge
was performed at 1,200 × g, indicating that
spinoculation enhances CD4-selective binding to T cells to a greater
extent than it enhances CD4-independent interactions. In contrast, CD4
blocking only reduced virus binding by approximately half at ambient
gravity; this is broadly consistent with earlier work showing that a
significant proportion of HIV-1 adsorption to cell surfaces can be CD4
independent under standard experimental conditions (27, 29,
32).
|
Polybrene enhances viral binding less than centrifugal inoculation. Polycations, such as polybrene, have been used for many years to enhance infection by HIV-1 and other viruses. Although this effect is thought to be mediated through increased viral adsorption as a consequence of decreasing repulsive forces (35) between virions and cells, a direct effect on HIV-1 binding has not been demonstrated (8, 44). When 8 µg of polybrene/ml was added to the 1 × g inoculation, the amount of cell-associated p24gag was increased approximately sixfold over that for the control (Fig. 5). Thus, while virus adsorption was increased by polybrene, the effect was relatively minor in comparison to the 30- to 40-fold enhancements seen following spinoculation.
Spinoculation-enhanced binding is temperature dependent.
To
characterize the spinoculation-mediated procedure further, we also
determined the effect of a reduction in temperature on cell-associated
p24gag levels. When all the centrifugation and
washing steps were executed at 4°C, the levels were reduced
approximately sixfold (Fig. 5). Because reducing the temperature to
4°C does not affect the sedimentation characteristics of HIV-1 (see
above), we have concluded that the binding of virus to T cells most
likely occurs more efficiently at the higher temperature (25°C)
a
finding that agrees with the results of Ugolini and colleagues
(45). Given that the majority of centrifugation-enhanced
infection is CD4 dependent, this result might have been predicted,
since earlier reports have demonstrated that the kinetics of HIV-1 Env
binding to CD4 are slower at 4°C than at 37°C (9, 10, 28, 30,
31).
Vigorous mixing at ambient gravity does not enhance viral binding. The preceding experiments suggested to us that viral particle diffusion might be the limiting step for viral binding under standard infection conditions. To address this, virus-cell suspensions were gently vortexed at 25 rotations per s for 2 h at 25°C to assess whether continuous agitation could increase binding. Figure 5 clearly shows that this was not the case compared to results for a conventional stationary inoculation. Although not intuitive, these data are still consistent with diffusion being the major rate-limiting step for viral binding (discussed below).
Centrifugal inoculation enhances virus binding to a variety of T
cells and with two different viral envelope proteins.
Having shown
that spinoculation enhances HIV-1IIIB particle adsorption
to CEM-SS cells, we wished to confirm the generality of this mechanism
by testing two additional T-cell lines, CD4-positive peripheral blood
lymphocytes and HIV-1 cores pseudotyped with VSV G. Figure
6A shows that similar levels of
p24gag were associated with all three T-cell
lines following challenge at 1 × g and that these
levels were increased by similar orders of magnitude following
spinoculation. Although primary T cells bound lower quantities of virus
(perhaps as a consequence of having a smaller surface area), binding
was still substantially increased by spin infection. Finally, we also
challenged CEM-SS cells with virions pseudotyped with VSV G (Fig. 6B).
Once again, cell-associated p24gag levels were
substantially increased by spinoculation (~16-fold in this
experiment), thus demonstrating that this technique can be extended to
viral Env glycoproteins other than gp120/41env.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Here we have utilized contemporary PCR-based methodologies to show that spinoculation enhances the CD4-dependent binding of HIV-1 to susceptible T-cell targets mainly by depositing increased numbers of virions onto the cells (Fig. 2). In other words, viral binding is a major rate-limiting step under routine pulsing conditions. The concomitant stimulation of viral infectivity does not appear to be due to a direct effect of centrifugation on the cells, nor does it appear to be due to an increased fusion efficiency, since spinoculation enhances the accumulation of reverse transcripts and virus binding to similar degrees (Fig. 2). While the initial steps of fusion and reverse transcription appear to be relatively efficient steps in the HIV-1 life cycle, the progression from the presence of reverse transcripts to fulminant viral infection is inefficient. For instance, for cells challenged under standard conditions, we found ~4 extended minus-strand cDNAs per cell (Fig. 2), yet only 5 to 10% of such cells became virus producers (Fig. 3). This equates to a cumulative efficiency of ~1% for the intervening steps of the life cycle and is very consistent with previous calculations made using alternative methods (25). The reasons that underlie this "loss" remain to be determined and may include inefficiencies during the completion of reverse transcription, nuclear transport, integration, or even transcription. Addressing these issues will likely shed light on the nature of the virus-host relationship during the postentry stages of virus infection; for example, it is possible that inefficiencies could be caused by any number of virion defects or cell-mediated antiviral phenotypes.
In experiments using a CD4-specific antibody we were able to demonstrate that the binding of virus to CEM-SS cells under spinoculation conditions is largely (~90%) CD4 dependent, whereas at 1 × g binding is only partially (~50%) CD4 dependent (Fig. 5). We therefore propose that there may be a relatively small number of CD4-independent binding sites on CEM-SS T cells and that these can be readily saturated by centrifugal inoculation. In addition, we also found that the cell-associated levels of p24gag were reduced approximately sixfold when spinoculation was performed at 4°C (Fig. 5), even though viral particles are still sedimented at this lower temperature. As noted earlier, this finding is consistent with previous demonstrations of reduced binding of soluble CD4 to virions at 4°C (30, 31). It is also in keeping with the observation that the binding of Moloney murine leukemia virus to susceptible cells is less efficient at 4°C (50).
There are a number of factors that could, alone or in combination, contribute to this temperature effect. (i) As previously proposed, the binding of virions to cells may occur more slowly at 4°C, such that equilibrium would not be reached during the inoculation times used here. (ii) The efficiency of virus binding may be lower at 4°C. This could be the case if binding were to be stabilized by endothermic conformational changes in Env, CD4, or CXCR4. (iii) Virus binding may also be more stable at higher temperatures because of increased membrane fluidity, which would facilitate the interaction of multiple Env trimers from a single virion with the surface of one target cell. (iv) If virus binding were reversible, this would have a more dramatic effect on cell-associated p24gag levels (i.e., surface-bound and internalized p24Gag) at 4°C where viral entry does not occur: challenges at higher temperatures result in membrane fusion and the uptake of Gag into the target cell cytoplasm.
We confirmed that bulk fluid agitation of virus and cells does not overcome the diffusion-imposed limitations to infection that are operative at ambient gravity (Fig. 5), as previously reported (1, 35, 47). This finding was to be expected, since the laminar boundary layers that are present close to the surfaces of cells in suspension are, like the mean viral displacement distance at 1 × g (i.e., the distance that a virus is expected to move by diffusion within a half-life of 5 to 8 h), ~500 µm in depth (35). In other words, virus particles that are more than 500 µm from their target cells at the initiation of virus challenge do not contribute significantly to infection at 1 × g (7). Thus, spinoculation apparently stimulates virus adsorption by imposing directionality on virus movement; in addition to improving the recovery of infectious virus from low-titer sources, centrifugal infections will be useful for infecting large numbers of cells synchronously, so that kinetic methods can be applied to the analysis of the early stages of HIV-1 infection, and for increasing the efficiency of retrovirus-mediated gene transfer.
Lastly, we speculate that in vivo conditions may surmount the limitation on the efficiency of HIV-1 infection that is imparted by diffusion in culture. In particular, direct contact between uninfected cells and cells producing (or carrying) virus particles will accelerate spread by substantially reducing the time between virus budding and the next round of binding. In keeping with this, it is well known that cell-to-cell infection by HIV-1 is much more efficient than infection with "cell-free" stocks (11). One cell type that is emerging as a critical participant during initial transmissions of HIV-1 and the subsequent establishment of vigorous and productive infections is the dendritic cell. These cells may bind and capture virions at sites of mucosal transmission and then migrate to T-cell-rich lymphoid tissues, where cell-to-cell contact would facilitate efficient viral transfer to highly permissive activated T cells.
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ACKNOWLEDGMENTS |
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We thank Stylianos Andreadis, Paul Bates, Bob Doms, Jim Hoxie, Jeff Dvorin, Nathan Gaddis, John Moore, Farida Shaheen, and Alexandra Trkola for helpful discussions and Jim Hoxie for anti-CD4 antibodies.
The ABI-7700 is maintained through a core grant of the University of Pennsylvania Center for AIDS Research (AI45008), and the work was supported by U.S. Public Service grants AI41933 (to M.H.M.) and HL03984 (to U.O.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, University of Pennsylvania School of Medicine, 347B Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104-6148. Phone: (215) 573-3493. Fax: (215) 573-2172. E-mail: malim{at}mail.med.upenn.edu.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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