JOURNAL

OF

Vol. 65, No. 2

VIROLOGY, Feb. 1991, p. 852-860

0022-538X/91/020852-09$02.00/0 Copyright © 1991, American Society for Microbiology

Differential Loss of Envelope Glycoprotein gpl20 from Virions of Human Immunodeficiency Virus Type 1 Isolates: Effects on Infectivity and Neutralization JANE A. McKEATING,* AINE McKNIGHT, AND JOHN P. MOORE Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, United Kingdom Received 20 June 1990/Accepted 23 October 1990

Several parameters which may affect the infectivity of human immunodeficiency virus type 1 in tissue culture were analyzed. In particular, we used gel exclusion chromatography to investigate how the loss of the surface glycoprotein gpl20 from virions of the HTLV-IIIB (IIIB), HTLV-IIIRF (RF), and SF-2 isolates modulates infectivity. In IIIB and RF cultures, a high proportion of the total gpl20 was virion bound initially but was gradually lost from the virions over time. In contrast, most of the gpl20 (and p24) in SF-2-infected cultures was soluble and the few particles present had a fivefold-lower level of virus-bound gpl20. However, this reduced level of virion-bound gpl20 was more resistant to shedding. Loss of a major proportion of gpl20 from IIIB and RF virions resulted in reduced infectivities, and in addition, the resulting accumulation of soluble gpl20 in the cultures could competitively inhibit viral infection, especially with SF-2. Increased shedding of virion gpl20 also affected the neutralization of IIIB and RF particles. However, the high sensitivit to human serum neutralization characteristic of SF-2 was unaffected by soluble gpl20 in cultures, suggesting that the epitopes responsible are not present on soluble gpl20.

The env gene of human immunodeficiency virus type 1 (HIV-1) encodes a glycoprotein precursor (gpl60) that is proteolytically cleaved within infected cells to two mature proteins, gpl20 and gp4l (33, 42, 46). These are incorporated into virions as oligomeric complexes (31, 34) of a transmembrane glycoprotein (gp4l) and a surface glycoprotein (gpl20) (22), the latter containing the receptor-binding site (17, 18) for the cell surface antigen CD4 (3, 15). The interaction between gpl20 and gp4l of HIV-1 is noncovalent and does not involve disulfide bonds (46). The surface glycoprotein spikes are easily lost from the virion surface, leaving in extreme cases virus particles extensively denuded of their glycoprotein receptors (7, 8, 35). Loss of gpl20 occurs as virions age and has been suggested to correlate with a reduction in virion infectivity (7, 8). There has, however, been no quantitative study of how infectivity varies with virion spike retention. We therefore determined the infectivity/particle ratio for three established laboratory isolates of HIV-1 and measured the retention of gpl20 on the virion particles using gel exclusion chromatography to separate virion-bound from soluble gpl20. One of the isolates studied, SF-2 (originally called ARV-2) (21), was selected because of its extreme sensitivity to neutralization by HIV-positive human serum (1, 11, 45). The other strains, HTLV-IIIB (IIIB) and HTLV-IIIRF (RF) (32), have also been comprehensively studied and are widely available.

chronically infected H9 cells. Extracellular virus was harvested every 24 h after cocultivation of infected cells with uninfected cells in a 1:4 ratio. HIV infectivity and RT assays. Tenfold serial dilutions of virus (50 pA) were incubated with 100 RI1 of C8166 cells (2 x 105/ml) in microtiter plates for 7 days. The plates were scored for the presence or absence of syncytia, and the 50% tissue culture infective doses (TCID50) were determined by the Karber method as described previously (23). Extracellular virus was also monitored for virion-associated reverse transcriptase (RT) activity essentially as outlined elsewhere (10). Neutralization assays. HIV neutralization was assessed by established methods (23). Human sera were from a panel selected by the World Health Organization for standardization of neutralization assays (23). Polyclonal antisera to the V3 loops of IIIB, RF, and SF-2 were raised against synthetic peptides and were gifts from S. Putney and T. Palker. Chromatography of HIV on Sephacryl S-1000. Gel exclusion chromatography on Sephacryl S-1000 (Pharmacia, Milton Keynes, United Kingdom) was used to separate infectious HIV particles from free gpl20 and p24 in the supernatants from virus-infected cell cultures (29). All chromatography procedures were done within a class I safety cabinet. Supernatant medium (100 ,ul) was applied to S-1000 (2-ml packed volume, 4-cm height) in a disposable plastic column (Bio-Rad, Watford, United Kingdom), and proteins were eluted with TBS (144 mM NaCl, 25 mM Tris, pH 7.5) under gravity. Fractions (2 drops; average volume, 85 RI) were collected manually, or in some experiments, the entire infectious virus peak (elution volume, 0.6 to 1.2 ml) was collected as a single fraction. After removal of aliquots for assay of infectious virus, Empigen-BB dipolar ionic detergent was added to 1% (vol/vol) to inactivate HIV prior to immunoassay. Viral antigens were recovered quantitatively from the chromatography columns within the error of en-

MATERIALS AND METHODS Virus stocks and cell lines. The CD4-positive lymphocytic leukemia cell line H9 (32) and the HTLV-I-transformed T-cell line C8166 (2, 38) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum. All virus stocks were prepared from the culture supernatants of *

Corresponding author. 852

VOL. 65, 1991

gpl20 LOSS FROM HIV-1 VIRIONS AND INFECTIVITY IlII

1000

106

U

gpl20,

TCID50

p24 ng/ml

U

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gpl20.

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ng/ml

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gpl20, p24

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96

72

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tHouns FIG. 1. Detection of extracellular gpl20 and p24 released from HIV 1-infected H9 cells: comparison with RT activity and infectivity. H9 cells chronically infected with the HIV-1 strains IIIB (a), RF (b), and SF-2 (c) were cocultivated with uninfected H9 cells (infected/uninfected, 1:4). Extracellular virus was harvested every 24 h for 96 h and monitored for gpl20 (0, nanograms per milliliter), p24 (U, nanograms per milliliter), RT activity (V, cpm per milliliter), and infectious virus (A, TCID50 per milliliter).

100 t 0

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zyme-linked immunosorbent assay (ELISA) determinations (see legend to Fig. 2 and reference 29). Detection and quantitation of gpl2O and p24 by ELISA. gp120 and p24 from virions disrupted with 1% Empigen were detected and quantitated by twin-site ELISA essentially as described previously (27, 29, 30). The use of immunoassays to quantitate p24 and especially gp120 from different HIV-1 strains is potentially hindered by immunological variation in the detected proteins. We minimized this by selecting as the capture antibody in the gpl20 ELISA an affinity-purified sheep polyclonal antibody (D7324; Aalto BioReagents, Dublin, Eire) to a peptide APTKAKRRVVQREKR from the C terminus of gpl20 (IIIB). The corresponding sequence in SF-2 is identical, and there is a single conservative K-to-R change in the RF sequence. Bound gpl20 was detected with a pool of sera (1:2,000 dilution) from HIV-positive humans of European origin, followed by alkaline phosphatase-conjugated goat anti-human immunoglobulin G (SeraLab). Using a pool of HIV-positive human sera as detection antibody also reduces the extent of systematic error in quantitating gpl20 from different strains. Nonetheless, absolute quantitations of RF and SF-2 gp120 must be regarded as estimates with an unknown, but probably small, systematic error. Under the assay conditions used, p24 was not detected in the gpl20 assay.

854

McKEATING ET AL.

a

1.6

- - - -

-

-

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gpl2O, ng

J. VIROL. 16

-

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p24, ng

gp120, ng 0

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FIG. 2. Analysis of IIIB, RF, and SF-2 by gel exclusion chromatography on Sephacryl S-1000. Extracellular IIIB (a), RF (b), and SF-2 (c) viruses harvested 48 h after cocultivation were fractionated on a Sephacryl S-1000 column. Input virus (100 pAl) for IIIB, RF, and SF-2 contained 4.9, 5, and 6 ng of gpl20 and 90, 72, and 286 ng of p24, respectively. Eluted fractions were assayed for gp120 (0) and p24 (U). Viral antigens in the infectious virus peaks were recovered as follows: IIIB, 4.8 ng of gpl20, 86 ng of p24; RF, 4.0 ng of gpl20, 57 ng of p24; SF-2, 0.57 ng of gpl20, 36 ng of p24. The soluble antigen peaks contained the following: IIIB, 1.2 ng of gpl20, 18 ng of p24; RF, 0.7 ng of gpl20, 7.8 ng of p24; SF-2, 3.5 ng of gp120, 299 ng of p24. Fractions from the IIIB column (a) were also assayed for infectious virus, and the presence of this is indicated by a cross in the strip above the elution profile.

20

(see reference 28). p24 (IIIB) was expressed in and purified from yeast Ty particles (9) and was a gift from S. E. Adams (British Biotechnology Ltd., Oxford, United Kingdom). The sensitivity limit for these assays is 3 to 10 pg of recombinant gpl20 or p24 per 100-pul microplate well. We could not detect virion gp4l before or after S-1000 chromatography because none of the assays attempted was sufficiently sensitive; it is, however, likely that gp4l will be found exclusively in the virion peak (because it is a transmembrane protein) except when extensive disintegration of particles has occurred. program

o0 0

0-4

0-8

1-2 1-6 2-0 2-4 ELUTION VOLUME, ml

2-8

For the same reasons, the capture antibody (D7320; Aalto BioReagents) in the p24 ELISA was raised against three peptides from the LAV-1 (i.e., IIIB) sequence. These were SALSEGATPQDLNTML, GQMREPRGSDIA, and LDIRQ GPKEPFRDYV. These sequences are absolutely conserved in RF and SF-2. Bound p24 was detected with an alkaline phosphatase-conjugated mouse monoclonal antibody (EH 12E1-AP) that was raised (39) against the CBL-1 isolate (44) of HIV-1. The alkaline phosphatase conjugate was prepared by Novo Nordisk (Cambridge, United Kingdom) and is available from the U.K. Medical Research Council resources program. EH12E1 recognizes a complex epitope incorporating amino acids NPPIPVGEIYKRWII and GHQ AAMQMLKETINEEAAEWDRVHPVHAGPIAPGQ (6), also completely conserved in each of the three strains. In each assay, bound alkaline phosphatase was detected with the AMPAK ELISA amplification system (Novo Nordisk) (13, 40) as described previously (27, 30). The assays were calibrated with recombinant gp120 and p24. gpl20 (IIIB, BH10 clone) was expressed in and purified from CHO cells by Celltech Ltd. (Slough, United Kingdom) and was provided by the U.K. Medical Research Council resources

RESULTS Rate of virus production from HIV-1 strains IIIB, RF, and SF-2. Release of extracellular HIV-1 from H9 cells infected with the IIIB, RF, or SF-2 strain was assessed by measuring gpl20 and p24 antigen levels, RT activity, and the concentration of infectious virus present (TCID50) (Fig. 1). To minimize the effect of different primary infection rates for the three isolates, we analyzed the rate of production from chronically infected H9 cells when mixed with uninfected cells. Under these conditions, a secondary round of infection is established leading to peak virus production in which >90% of the cells are virally infected as determined by immunofluorescence analysis (data not shown). The amounts of gpl20 and p24 in the cultures increased rapidly from 24 to 96 h after cocultivation with uninfected H9 cells and were maximal at about 96 h (72 h for RF). Although there were slight differences in the rates of antigen produc-

VOL. 65, 1991

gpl20 LOSS FROM HIV-1 VIRIONS AND INFECTIVITY

tion for the three HIV-1 strains, the maximum levels of HIV antigens recorded in the cultures were similar in the experiment shown (60 ng of gp120 per ml, 1,000 ng of p24 per ml). Levels of HIV antigens produced in cultures of all three viruses do, however, vary and are dependent on the precise culture conditions used. In general, the ratio of total gpl20 to total p24 (envlgag ratio) in SF-2 cultures is about fivefold lower for SF-2 than for IIIB and RF. This is not, however, a particularly informative parameter for assessment of infectious virus production (see below). In contrast to the similar amounts of total viral antigen in the three cultures, the maximum RT activity in the SF-2 culture (3,000 cpm/ml) was 15- to 30-fold lower than in the IIIB (45,000 cpm/ml) and RF (90,000 cpm/ml) cultures. The low RT levels in the SF-2 culture reflected the 33- to 66-fold-lower levels of infectious HIV-1 produced (7,000 TCID50/ml for SF-2, but 100,000 to 200,000 TCID50/ml for IIIB and RF). Other SF-2 cultures analyzed contained similar low levels of infectious virus and RT activity. The paucity of RT activity in SF-2 cultures may be due to rapid denaturation of the RT enzyme when it is not within a virion; thus, in a IIIB culture, all the detectable RT activity recovered from a Sephacryl S-1000 column (see Fig. 2 below) was in the particulate, infectious virus peak and none was present as soluble enzyme (data not shown). We could, however, detect by ELISA (21a) RT protein in the soluble antigen fraction that was presumably not enzymatically active (data not shown). Many of the virus particles in a culture may be poorly infectious or noninfectious. We therefore defined the specific infectivity, or infectivity per particle, of a culture as the ratio of TCID50 to RT activity (arbitrary units). Specific infectivities (48-h harvests) were 0.85, 1.51, and 7.10 for IIIB, RF, and SF-2, respectively, for the experiment described in Fig. 2. In two other experiments of similar design, specific infectivities for IIIB were 1.10 and 0.95; for RF, 1.34 and 1.25; and for SF-2, 7.56 and 7.82. Thus, SF-2 virions containing an active RT enzyme are of comparable infectivity to IIIB and RF virions; the low total infectivity (TCID50) of an SF-2 culture is probably due, at least in part, to the relatively small number of infectious virions present. To obtain an estimate of the amount of envelope glycoprotein associated with virus particles, we determined gpl20-to-RT ratios (picograms/counts per minute). At 48 h after infection, this ratio was 0.5, 0.2, and 100 for IIIB, RF, and SF-2, respectively. The SF-2 culture therefore contains 200- to 500-fold more gpl20 than the IIIB and RF cultures for each virus particle present that contains an active RT enzyme, and there is also a similar excess of p24 in the SF-2 culture (data not shown). Taken together, the above observations suggested that much of the SF-2 viral protein was nonparticulate. Gel exclusion chromatography of HIV-1. The physical state of the viral antigens in HIV-1 cultures was characterized by gel exclusion chromatography on Sephacryl S-1000 columns (29). Conventional biochemical methods (e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western immunoblotting) were insufficiently sensitive for quantitation of the small amounts of gpl20 and p24 present in the maximum volume of viral culture supernatant (100 ,ul) that could be analyzed by chromatography under viral containment conditions, so we detected eluted proteins by ELISA. The particle exclusion limit of the S-1000 gel matrix is 300- to 400-nm diameter (Pharmacia technical handbook), close to the estimated diameter of HIV (100 to 150 nm) (7), so we anticipated that virions would be almost completely ex-

855

cluded from the matrix and elute close to the void volume. This was confirmed in preliminary experiments: infectious HIV was recovered in a single peak corresponding to an elution volume of 0.6 to 1.2 ml (Fig. 2a) (data not shown) (see reference 29). In contrast, recombinant gpl20 and p24 were retained in the gel matrix and eluted quantitatively in a broad peak from 1.2 to 2.4 ml, with the larger gpl20 molecules eluting slightly earlier than p24 (data not shown, but see Fig. 2c). Thus, using this technique we could cleanly separate virions (108 molecular weight) from soluble viral antigens (104 to 105 molecular weight) and recover the viral proteins from the columns without systematic loss. Culture supernatants harvested 48 h after cocultivations of TIIB-, RF-, and SF-2-infected H9 cells with uninfected cells were fractionated on Sephacryl S-1000 columns (Fig. 2). The majority of the viral antigens in IIIB and RF virus preparations coeluted with the first, infectious virus peak, and there was little soluble p24 and gpl20 (Fig. 2a and b) (see reference 29). Thus, in the experiment shown, 68% of the total gpl20 recovered from the column and 83% of the total p24 were present on IIIB virions. For RF, the corresponding percentages were 86 and 88%. In marked contrast, chromatography showed that most gpl20 and p24 in SF-2 cultures was present as nonparticulate soluble antigen and that there were relatively few intact virions present; only 14 and 9% of the total gpl20 and p24, respectively, were recovered in the SF-2 virus peak. The envlgag ratio in the infectious virus peak (which we define as spike density) for SF-2 was 0.016, somewhat less than for IIIB and RF (0.05 to 0.1). This suggests that each SF-2 virion, on average, has approximately fivefold less gpl20 bound than virions from the IIIB and RF isolates harvested under similar conditions 48 h after cocultivation. When we estimated the ratio of virion-bound gpl20 or p24 to RT activity in the cultures of the three viruses studied, it was clear that SF-2 particles contained significantly more viral antigen per unit of RT activity present. For example, the virion p24/RT ratio (picograms/counts per minute) was 0.27 for IIIB and 0.13 for RF but 0.91 for SF-2. This observation has several possible explanations among which we are as yet unable to discriminate: many of the SF-2 virus particles may physically lack the RT protein; many of the particles may contain an inactive enzyme; or all the particles may contain an enzyme of low specific activity compared with the IIIB and RF enzymes. The last explanation is, perhaps, the least probable because of the high infectivity per particle ratios (TCID5dRT) in SF-2 cultures (see above). Loss of HIV infectivity with time: effects of virion-bound and soluble gpl20. To analyze how infectivity of a virus particle varied with its gpl20 content, we measured virion spike density and infectivity during the aging of RF and SF-2 virus preparations. H9 cells chronically infected with RF and SF-2 were extensively washed to remove extracellular virus and then cocultivated with uninfected H9 cells. Extracellular virus was harvested 24 h later and incubated at 37°C for 3, 6, 24, and 28 h (Fig. 3a). At each time point, an aliquot of virus was removed and assayed for particulate (i.e., virus-bound) gpl20 and p24 by S-1000 chromatography and for infectivity (TCID50). Initially, 60% of the RF gpl20 was virion bound, but gpl20 was gradually lost from the virions so that after 28 h only 12% was bound (Fig. 3a). This reduction was attributable predominantly to loss of gpl20 from the particles since the virion-bound p24 levels declined by less than 30% (data not shown). Consequently, the envlgag ratio in the virion fraction declined fourfold from 0.035 initially to 0.009 after 28 h. Over the same period, there was a more than

J. VIROL.

McKEATING ET AL.

856

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0 m

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HOURS FIG. 3. Effect of loss of gpl20 on virion infectivity. Extracellular RF (a) and SF-2 (b) viruses were harvested 24 h after cocultivation of infected H9 with uninfected H9 cells and then incubated at 37°C for 3, 6, 24, and 28 h. Aliquots of virus were removed at these times and assayed for virion-bound gpl20 (0) and p24 (0) and for their

infectivity (TCID50 per milliliter) for C8166 cells before (U) and after (O) removal of soluble gpl20. The gpl20 and p24 data are plotted as percentages of the amount of each antigen that was virion bound initially: RF, 22 ng of gpl20 per ml and 646 ng of p24 per ml; SF-2, 6.6 ng of gpl20 per ml and 880 ng of p24 per ml.

3,000-fold reduction in viral infectivity, from

an

102 0

.

.

50

100

.

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150 200 gpW2M

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FIG. 4. Effect of recombinant gpl20 on HIV infectivity. Extracellular IIIB (0), RF (U), or SF-2 (A) virus (harvested 48 h after cocultivation) was mixed with the indicated concentrations of recombinant IIIB gpl20 and incubated with C8166 cells for 1 h at 37°C. Unbound virus and gpl20 were washed away, and the amount of infectious extracellular virus produced 7 days later was determined as TCID50 for C8166 cells. The data for SF-2 (A) are also replotted (A) taking into account the concentration of endogenous, soluble gpl2O present in the culture (71 ng/ml). For IIIB and RF, the concentrations of endogenous gpl20 were negligible compared with that of the added recombinant gpl20.

initial

TCID50 of 250,000/ml to 80/ml after 28 h (Fig. 3a). Similar data were obtained for IIIB (data not shown). Thus, a major loss of infectivity can occur in association with only a relatively small loss of gp120 from the virions. We noted above that SF-2 virions had a lower spike density (envigag ratio) than those from IIIB and RF. As SF-2 virions aged over 28 h, the envigag ratio remained approximately constant, however, at a value of 0.007 (Fig. 3b), which contrasts with the gradual loss of gpl20 from RF and IIIB virions. There was also a slight decrease in the recovery of virion-bound p24 as SF-2 cultures aged (Fig. 3b), which probably reflects gradual disintegration of virions. In contrast to the catastrophic reduction in IIIB and RF infectivity over 28 h at 37°C, the infectivity (TCID50) of SF-2 declined only 175-fold from 14,000/ml initially to 80/ml after 28 h. One factor that might be relevant to loss of viral infectivity over time is the gradual accumulation of soluble gp120 in the cultures. To assess how infectivity was affected by soluble gpl20, we added recombinant (IIIB) gpl20 (30 to 250 ng/ml,

equivalent to 0.25 to 2.1 nM) to target cells simultaneously with input virus (48-h harvest). Recombinant gpl20 caused a dose-dependent inhibition of the amount of infectious virus produced 7 days later, maximal inhibition occurring at exogenous gpl20 concentrations in the range of 60 to 250 ng/ml (Fig. 4). SF-2 appeared initially to be more sensitive to inhibition by exogenous recombinant gpl20, but when the estimated amount of endogenous gpl20 in the SF-2 culture was taken into consideration and the data were replotted accordingly, it was apparent that each of the three strains had comparable sensitivity to inhibition by soluble gpl20 (Fig. 4). The data also suggested that the infectivity of SF-2 is compromised to a certain extent by the high levels of endogenous, soluble gp120 that build up in the cultures. We addressed the above possibility by analyzing the ability of RF and SF-2 viruses to infect C8166 cells before and after removal of soluble gpl20 by S-1000 chromatogra-

gpl20 LOSS FROM HIV-1 VIRIONS AND INFECTIVITY

VOL. 65, 1991

TABLE 1. Effect of endogenous and exogenous soluble gp120 on HIV neutralizationa Reciprocal neutralization titer

Strain

Serum

24 h Fresh

IIIB RF

QC1 QC5 QC1 QC5

SF-2 QC1 QC5 Anti-V3 loop 1577 RF Anti-V3 loop 1577 SF-2 Anti-V3 loop 1577 IIIB

24aged aged

Aged

Fresh

+gpl20 -gpl2O +gpl20 -gpl2O 160 320 160 160

160 320 160 160

40 80 40 80

320 640 640 640

5,120 5,120 5,120 2,560 2,560 2,560

5,120 2,560

5,120 2,560

5,120 2,560

20 10 80 10 40 20

20 10 80 10 80 20

10 10 40 10 40 20

80 10 160 10 80 20

160 320 160 160

40 80 40 80

a The same extracellular virus harvests from cocultivations of chronically infected H9 cells with uninfected cells used in the experiments described in the legend to Fig. 3 were assayed for sensitivity to neutralization by two human HIV-positive sera (QC1, QC5; see reference 24) before and after incubation for 24 h at 37°C. The input virus dose at each time point was 100 TCID50. The neutralization titers are the reciprocal of the greatest serum dilution capable of blocking infection by >90% as judged by syncytium formation in C8166 cells. The sensitivities of fresh and aged (24 h, 37C) IIIB, RF, and SF-2 viruses to neutralization by anti-V3 loop antisera (specific for each viral strain) and a gp4l-specific monoclonal antibody (1577) or by human HIV-positive sera (QC1, QC5) are shown before (+) and after (-) removal of soluble gpl20 by S-1000 chromatography. Prior to aging, the IIIB, RF, and SF-2 cultures contained, respectively, 15, 14, and 49 ng of soluble gpl20 per ml. After incubation for 24 h at 37°C, the soluble gpl20 levels were 43, 40, and 42 ng/ml, respectively. Similar data for IIIB were obtained with monoclonal antibody 1105 (14, 37) to the V3 loop.

phy. The viral inocula contained identical levels of RT activity (1,000 cpm/ml) but very different absolute amounts of both virion-bound and soluble gpl20 and p24 (see above). The envlgag ratios for the input virus particles (determined as described above) were 0.03 and 0.007 for RF and SF-2, respectively, in this experiment, so the SF-2 virions in the initial inoculum had on average approximately fourfold less gpl20 than the RF virions. Despite this, the amounts of progeny virus in the RF and SF-2 cultures after 7 days, assessed by TCID50, were similar: 1,000/ml for RF and 1,260/ml for SF-2. This suggests that a major determinant of infectivity is the RT activity in the inoculum, irrespective of the amount of viral protein present. However, removal of soluble gpl20 from the inocula (RF, 15 ng/ml; SF-2, 50 ng/ml) by S-1000 chromatography prior to initiation of infection led to a fourfold increase in the amount of infectious virus produced after 7 days in the SF-2 culture (TCID50; 5,000/ml), but only a much smaller increase for RF (TCID50; 1,260/ml). Taken together, the data suggest that the higher concentration of soluble gpl20 in the SF-2 inocula compromises infectivity to a substantially greater extent than the lower level of gpl20 present on the SF-2 particles. Effect of soluble gpl20 on HIV-1 neutralization. HIV-1 is neutralized in vitro by serum antibodies that bind to epitopes on the gpl20 and gp4l components of the virion's spike glycoproteins (reviewed in reference 24). To assess any effect of gpl20 loss on the neutralization process, we compared the sensitivity to neutralization of IIIB, RF, and SF-2 virions before and after incubation of cell-free virus supernatants for 24 h at 37°C. Aged IIIB and RF viruses were significantly (fourfold) less sensitive than fresh ones to

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neutralization by HIV-positive human sera (Table 1) and slightly (twofold) less sensitive to neutralization by polyclonal antisera to peptides from their V3 loops (gifts from S. Putney, Repligen, and T. Palker, Duke University) (Table 1). The reduced neutralization sensitivity of aged IIIB and RF virions could be due in principle either to an inherent difficulty in neutralizing particles with a low complement of bound spikes or to the accumulation of soluble gpl20 in cultures buffering out a proportion of the available neutralizing antibodies. These possibilities were tested by examining the neutralization sensitivity of fresh and aged IIIB and RF virions before and after removal of soluble gpl20 (Table 1). A monoclonal antibody (1577) to an epitope on gp4l (5) was used as a control. None of the procedures for any of the viruses affected neutralization by this monoclonal antibody (Table 1). Removal of soluble gpl20 (15 ng/ml) from IIIB and RF viruses prior to aging did not significantly affect the neutralization titers for the anti-V3 loop antisera and human sera (Table 1). After aging for 24 h at 37°C, a major proportion of virion gpl20 had dissociated from IIIB and RF (Fig. 3) so that the free gpl20 concentration rose approximately threefold to 43 ng/ml (IIIB) and 50 ng/ml (RF). The aged viruses were more difficult to neutralize by the anti-V3 loop antisera and human sera (Table 1). The neutralization sensitivity of the aged IIIB and RF viruses was, however, more than restored by removal of gpl20. After removal of soluble gpl20, the aged viruses were more sensitive than fresh ones to neutralization (Table 1). Thus, the reduced neutralization sensitivity after aging is a consequence of the increased concentration of soluble gpl20 in the culture. Loss of gpl20 from the virions probably has two effects. The soluble gpl20 chelates neutralizing antibodies and reduces the quantity available to bind to and neutralize the virus; opposing this to an extent, the lower levels of virion gpl20 make the virus easier to neutralize. The SF-2 isolate is abnormally sensitive to neutralization by human sera (1, 11, 45), but the neutralization-sensitive epitope(s) on (presumably) the SF-2 envelope has not been defined. Aging of SF-2 for 24 h had no effect on its neutralization by either human sera or antiserum to the V3 loop (Table 1). This is consistent with the reduced loss of gpl20 over 24 h from SF-2 virions compared with those from IIIB and RF (Fig. 3). Furthermore, removal of soluble gpl20 had no effect on neutralization of fresh SF-2 cultures by human sera but slightly increased their neutralization sensitivity to anti-V3 loop antiserum (Table 1). The concentration of soluble gpl20 in the SF-2 culture was initially 50 ng/ml, comparable with the concentrations reached in IIIB and RF cultures after 24 h of aging, which did modulate the neutralization titers for these viruses. The epitope(s) on the envelope glycoproteins of SF-2 that is responsible for neutralization by human serum antibodies may, therefore, not be present on soluble, endogenous gpl20; removal of this material from SF-2 cultures did not affect the neutralization sensitivity of the virus under the assays conditions used. DISCUSSION Analysis of viral cultures by gel exclusion chromatography showed that the amounts of particle-bound gpl20 and p24 differed greatly for SF-2 compared with IIIB and RF; in a typical culture of SF-2, only approximately 10% of the gpl20 and p24 was present as virus particles, whereas for IIIB and RF, this proportion could be up to 90% (although it was usually somewhat lower). The low level of virion-bound

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gp120 in SF-2 cultures was observed with six independent biological clones of the virus and three cell lines (H9, C8166, and MT4) (data not shown), implying that it is a stable phenotype of the strain and independent of the cell type. We do not know, however, whether the large excess of soluble antigen in SF-2 cultures is a consequence of inefficient particle formation or whether virus particles form but are very unstable and disintegrate rapidly. The envlgag ratio in the virus particle fraction eluted from Sephacryl S-1000 columns allows a rough estimate of the average density of gp120 spikes on the virion surfaces. Typical envigag ratios that we measured were 0.03 to 0.1 for IIIB and RF but approximately fivefold less for SF-2. We are unaware of any estimate of the p24 content of an HIV virion, but a value of 5 x 10-16 g of core protein for avian tumor virus has been reported (43) and we assume that HIV is similar. At a maximum of 70 glycoprotein spikes per HIV (IIIB) virion (7) and three to four gpl20 molecules per spike (31, 34), the amount of gpl20 per virion can be calculated as approximately 5 x 10-17 g. The envlgag ratio from these values is 0.1, in good agreement with our measurements for IIIB. From the experimental values and theoretical calculations, we can estimate that SF-2 virions contain on average about 15 glycoprotein spikes. We do not know, however, how the spectrum of spike density varies within a virus population (see below). Analysis by electron microscopy (7, 8) has indicated that shedding of gpl20 from IIIB virions occurs predominantly as the virus particle matures and buds from the infected cell. Maturation of a virion possibly destabilizes the gpl2O-gp4l complex. We showed that the efficiency of gpl20 spike retention on mature virions from different HIV-1 isolates is variable; as the cultures aged, SF-2 virions retained well the low levels of spikes that they possessed at the earliest time point at which they were analyzed. In contrast, mature IIIB and RF virions continuously lost gpl20 over time from an initial level that was greater than that for SF-2. There was, however, no significant increase in the proportion of p24 in the IIIB and RF cultures that was nonparticulate, indicating that there was no disintegration of viral particles. This is consistent with electron microscopy data (7, 8). A number of factors may influence HIV-1 infectivity as the virions age. We identified the accumulation of soluble gpl20 and the reduction in virion gpl20 as two relevant, and obviously interconnected, parameters. Thus, endogenous gpl20 shed from SF-2 virions accumulated in cultures to concentrations of up to 40 to 50 ng/ml and inhibited HIV-1 infectivity. Recombinant gpl20 added to viral cultures at 60 to 250 ng/ml also reduced viral infectivity by up to 50-fold (Fig. 4), presumably by competitively blocking virus binding to a proportion of the available CD4 receptors on the target cells. We doubt, however, that transfer of gpl20 from virions to the medium is the only factor to be taken into consideration. For example, lability of the reverse transcriptase enzyme at 37°C (even within a virus particle) may be very important. Our data showing that the low concentrations of infectious virions in SF-2 cultures are associated with high virion p24/RT ratios allude to this possibility. They further suggest that measurement of RT activities alone does not provide an unambiguous indication of viral particle numbers, which has often been assumed (12, 25, 41, 47). RT activity does, however, probably provide a good measure of infectivity potential. Factors determined by the viral genotype independent of the spike density can clearly influence infectivity (25, 41), but it has been reported that infection proceeds at a rate

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proportional to the number of free gpl20 spikes on a virion's surface (19, 20). Our data are not inconsistent with this notion; as virions age they lose both gpl20 and infectivity. It is clear, however, that there is not a linear relationship between the two parameters, as reduction in IIIB and RF infectivity over time was many-fold greater than suggested solely by their loss of gpl20, and SF-2 lost infectivity without significant loss of gpl20. It may be that there is a critical gpl20 surface density below which virion infectivity is catastrophically compromised (19, 20) and that as IIIB and RF virions age they soon drop below the critical value. Consistent with this, of those virus preparations we have examined, the one (RF) with the highest infectivity (>107 TCID50/ml) had a very high percentage (>80%) of total gpl20 that was virion bound (29). Thus, the most labile fraction of dissociable gpl20 may be that which most dramatically influences infectivity. We do not know the minimal spike density on a virion that allows productive infection, but from soluble CD4 (sCD4)-blocking experiments (19), a value of 50% of the maximal level can be inferred. Ultimately, loss of all the spikes from a virion will render it uninfectious. We have also noticed that aged IIIB and RF viruses are unusually sensitive to neutralization by sCD4 and that sCD4 removes most of the residual gpl20 from the aged virions (29; unpublished data). This suggests that viruses with a low initial gpl20 complement are easily prevented by sCD4 from binding to and/or fusing with CD4-positive cells. The SF-2 data are perhaps best understood if it is assumed that the lower spike density we measured in the population is the consequence of a small number of infectious particles with a high level of surface gpl20, diluted with a much greater number of virions that have lost most of their spikes and are considerably less infectious. We suggested above that SF-2 virions are abnormally unstable compared with IIIB and RF; the biological half-life for SF-2 may be very short, and freshly budded, infectious virions may be at a premium in the cultures. The alternative explanation for SF-2, that perhaps only 10 to 15% of the maximum spike density is sufficient for infectivity, is incompatible with the massive loss of IIIB and RF infectivity associated with only a fourfold reduction in spike density and also with data in reference 19. There are, nonetheless, infectious virions in SF-2 cultures, and the spike density is approximately constant over 28 h. This may indicate spike heterogeneity on SF-2 virions; a very labile fraction of SF-2 gpl20 may be shed rapidly from the virions and compromise infectivity, but additional gpl20 appears to be very stable. We have shown elsewhere (29) that treatment of IIIB and RF virions with sCD4 causes the rapid dissociation of most of their gpl20 from gp4l into the medium. However, a fraction of the virion gpl20 (10 to 20%) is resistant to sCD4-induced shedding, and this fraction is similar in magnitude to that which appears most resistant to slow, spontaneous shedding as IIIB and RF cultures age and also similar to the low basal levels of spikes on SF-2. The origin of this potential spike heterogeneity is not known, but we have suggested that it has a function in the virus-cell fusion reaction (29). Loss of spikes is most probably a phenotype that is determined genetically by variation in the amino acids involved in the noncovalent interaction between gpl20 and gp4l. For example, it has been reported that the introduction of a number of insertions into the aminoterminal half of gpl20 resulted in increased spontaneous dissociation of gpl20 from gp4l (17). Additional unknown factors may also be involved. The neutralization sensitivity of HIV-1 to HIV-positive

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human sera and anti-V3 loop antisera declines as the virions age. In principle, two factors can be relevant here: the increase in soluble gpl20 and the consequential decrease in spike density on the virions. The neutralization sensitivity of IIIB and RF decreased as the cultures aged but was more than restored when soluble gpl20 was removed. The likely effect of soluble gpl20 is chelation of neutralizing antibodies and reduction of their effective concentration. The greater ease of neutralization of aged virions freed from soluble gpl20 compared with fresh virions under the same conditions also suggests that particles with fewer virion-bound gpl20 spikes are easier to neutralize. In vitro, this is probably outweighed by the greater, opposing effect of soluble gp120. In contrast to IIIB and RF, neutralization of SF-2 by HIV-positive human sera was unaffected by high levels of soluble gp120. Note that the input virus dose (1,000 TCID50) for SF-2 corresponds to fivefold fewer virus particles than for IIIB and for RF, because its specific infectivity is fivefold greater. This may contribute to the enhanced group-specific neutralization of SF-2 by human serum (Table 1) (1, 11, 45) but is probably not the only important factor. The relevant epitope(s) may therefore be located on gp4l, or be dependent on the tertiary and/or quaternary structure of gpl20 while on the virion surface, or be exposed only after CD4 binding. The lack of a potent neutralization epitope on SF-2 soluble gpl20 is further suggested by the observation that in contrast to IIIB and RF, antisera raised against the SF-2 V3 loop sequence only neutralize SF-2 weakly compared with HIV-positive sera (Table 1). Furthermore, removal of soluble SF-2 gpl20 slightly increased the sensitivity of SF-2 virus to neutralization mediated by the V3 loop but not by human serum (Table 1). In vivo, gpl20 shed from vinons (or infected cells) may have pathogenic effects (7, 8). For example, by binding to CD4 on uninfected CD4-positive cells, soluble gpl20 may cause those cells to be targeted for autoimmune destruction (36). Interaction of gpl20 with CD4 has also been reported to stimulate quiescent T cells to reenter the proliferative pathway (16), and soluble gpl20 has been shown to perturb neural cell function by a CD4-independent mechanism (4). In addition, except during the early stages and very late stages of human infection by HIV, shed gpl20 is likely to be bound by serum antibodies; the combination of soluble gpl20 and serum anti-gpl20 antibodies has been reported to block T-cell activation in vitro synergistically (26). Whether in vitro observations of glycoprotein shedding are relevant in vivo is not known, but the amount of free gpl20 produced may be very dependent on the phenotype of the infecting virus. Furthermore, the detection of p24 antigen in human serum may be very poorly indicative of viral particles if, like SF-2, most of the p24 produced is nonparticulate. The methods described above provide simple, nondestructive ways to characterize parameters of viral stocks that influence infectivity. They may be of particular value for analysis of viral inocula intended for use in immunization and challenge protocols in trials of candidate AIDS vaccines, especially those involving viruses like SF-2 which rapidly lose gp120 and/or disintegrate. ACKNOWLEDGMENTS Robin Weiss is thanked for his support and advice throughout this project, and we are also grateful to him and Paul Clapham for reading the manuscript critically and making many suggestions. We thank Harvey Holmes (resources manager of the MRC AIDS Directed Programme reagent program) for several reagents, Bridget

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Ferns and Richard Tedder (University College and Middlesex Hospital School of Medicine) for monoclonal antibody EH12E1, Sally Adams (British Biotechnology Ltd.) for recombinant p24, Clive Loveday (University College and Middlesex Hospital School of Medicine) for RT ELISA reagents, and Scott Putney (Repligen) and Tom Palker (Duke University) for anti-gpl20 antisera. This work was supported by grants from the MRC AIDS Directed Programme and by the Cancer Research Campaign. REFERENCES 1. Cheng-Mayer, C., J. Homsy, L. A. Evans, and J. A. Levy. 1988. Identification of human immunodeficiency virus subtypes with distinct patterns of sensitivity to serum neutralisation. Proc. Natl. Acad. Sci. USA 85:2815-2819. 2. Clapham, P. R., R. A. Weiss, A. G. Daigleish, M. Exley, D. Whitby, and N. Hogg. 1987. Human immunodeficiency virus infection of monocytic and T-lymphotropic cells: receptor modulation and differentiation induced by phorbol ester. Virology 158:44-51. 3. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature (London) 312:763-766. 4. Dreyer, E. B., P. K. Kaiser, J. T. Offerman, and S. A. Lipton. 1990. HIV-1 coat protein neurotoxicity prevented by calcium channel antagonists. Science 248:364-367. 5. Evans, D. J., J. A. McKeating, J. M. Meredith, K. L. Burke, K. Katrak, A. John, M. Ferguson, P. D. Minor, R. A. Weiss, and J. W. Almond. 1989. An engineered poliovirus chimaera elicits broadly reactive HIV-1 neutralising antibodies. Nature (London) 339:385-388. 6. Ferns, R. B., J. C. Partridge, R. P. Spence, N. Hunt, and R. S. Tedder. 1989. Epitope location of 13 anti-gag HIV-1 monoclonal antibodies and their cross reactivity with HIV-2. AIDS 3:829834. 7. Gelderblom, H. R., E. H. S. Hausmann, M. Ozel, G. Pauli, and M. A. Koch. 1987. Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology 156:171-176. 8. Gelderblom, H. R., H. Reupke, and G. Pauli. 1985. Loss of envelope antigens of HTLV-III/LAV, a factor in AIDS pathogenesis? Lancet ii:1016-1017. 9. Gilmour, J. E. M., J. M. Senior, N. R. Burns, M. P. Esnouf, K. Gull, S. M. Kingsman, A. J. Kingsman, and S. E. Adams. 1989. A novel method for the purification of HIV-1 p24 protein from hybrid Ty virus-like particles (Ty-VLPs). AIDS 3:717-723. 10. Goff, S., P. Traktman, and D. J. Baltimore. 1981. Isolation and properties of Moloney murine leukemia virus mutants: use of a rapid assay for release of virion reverse transcriptase. J. Virol. 38:239-248. 11. Haigwood, N. L., J. R. Shuster, G. K. Moore, H. Lee, P. V. Skiles, K. W. Higgins, P. J. Barr, C. George-Nascimento, and K. S. Steimer. 1990. Importance of hypervariable regions of HIV-1 gpl20 in the generation of virus neutralizing antibodies. AIDS Res. Hum. Retroviruses 6:855-869. 12. Harada, S., D. T. Purtilo, Y. Koyonagi, J. Sonnabend, and N. Yamamoto. 1986. Sensitive assay for neutralizing antibodies against AIDS-related viruses (HTLV-III/LAV). J. Immunol. Methods 92:177-181. 13. Johannsson, A., D. Ellis, D. L. Bates, A. M. Plumb, and C. J. Stanley. 1986. Enzyme amplification for immunoassays. Detection limit of one hundredth of an attomole. J. Immunol. Methods 87:7-11. 14. Kinney Thomas, E., J. N. Weber, J. McClure, P. R. Clapham, M. C. Singhal, M. K. Shriver, and R. A. Weiss. 1988. Neutralizing monoclonal antibodies to the AIDS virus. AIDS 2:25-29. 15. Klatzmann, D., E. Champagne, S. Chamaret, J. Gruest, D. Guetard, T. Hercend, J.-C. Gluckman, and L. Montagnier. 1984. T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV. Nature (London) 312:767-768. 16. Kornfeld, H., W. W. Cruikshank, S. W. Pyle, J. S. Berman, and D. M. Center. 1988. Lymphocyte activation by HIV-1 envelope glycoprotein. Nature (London) 335:445-448.

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