JOURNAL OF VIROLOGY, June 1992, p. 3531-3537
Vol. 66, No. 6
0022-538X/92/063531-07$02.00/0 Copyright C) 1992, American Society for Microbiology
Human Immunodeficiency Virus Type 2 Infection and Fusion of CD4-Negative Human Cell Lines: Induction and Enhancement by Soluble CD4 PAUL R. CLAPHAM, AINE McKNIGHT,
AND
ROBIN A. WEISS*
Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, England Received 2 December 1991/Accepted 18 February 1992
We describe human immunodeficiency type 2 (HIV-2) strains which induce cell-to-cell fusion and infect certain CD4- human cell lines. Soluble CD4 (sCD4) induces or enhances fusion by most HIV-2 strains tested. Soluble CD4-immunoglobulin G chimeras and conjugates of sCD4 and antibody to the third domain of CD4 block HIV-2 fusion of CD4- cells. We conclude that HIV-2 can enter CD4- cells via an alternative cell surface receptor to CD4. While some strains entered efficiently, others retained a dependency on an interaction with sCD4 to initiate changes in the virion envelope required for membrane fusion.
SIVmac25i and SIVagmTyo strains and HIV-1 strains IIIB,
CD4 acts as the primary cell attachment receptor for human and simian immunodeficiency viruses (HIV and SIV) (15, 20, 28). Binding of HIV or SIV to CD4 at the cell surface is not sufficient for infection, and several examples of CD4+ human and nonhuman cells that bind HIV or SIV without proceeding to membrane fusion have been reported (4, 6, 8, 11, 22). These observations suggest that cell surface components other than CD4 are important in entry. Furthermore, CD4 not only acts to attach HIV and SIV to cells but also induces conformational changes in the virus envelope glycoproteins (29) which may be necessary for viral and cellular membranes to fuse together. This double dependency on CD4 for HIV and SIV entry may explain why HIV-1 infection of CD4- human cells is so inefficient (10) despite efficient binding of gpl20 to some neural cell types (16). Here we report the selection of an HIV-2 strain, LAV-2/B, which directly induces cell-to-cell fusion and efficient infection of certain CD4- human cell lines. Other HIV-2 strains, however, retain a dependency on an interaction with CD4 for fusion, which is induced or greatly enhanced for CD4cells by soluble CD4 (sCD4). These HIV-2 strains presumably bind to an alternative cell surface receptor but proceed to fuse with the cell membrane only after treatment with sCD4.
RF, MN, SF-2, CBL-4, Z34, Z84, Z129, U455, and NDK were propagated as previously described (11). MAbs. CD4 monoclonal antibodies (MAbs) Q4120 (domain 1 [D1]), L122, OKT4, Q425 (D3), and L120 (D4) have been described elsewhere (18). The MAbs and the Fab fragment of Q425 were kindly provided by Q. Sattentau and P. Kwong. sCD4 constructs. sCD4, derivative protein products (Dl and D1D2), and mutants (5) were kindly provided by R. Sweet (SmithKline Beecham). DlD2-immunoglobulin G (IgG) (7) was a kind gift from P. Berman (Genentech). Cell fusion assays. Syncytium induction assays were carried out by mixing 50 ,ul of virus-producing cells (106 cells per ml) with 50 ,ul of appropriate indicator or test cells (106 cells per ml). After overnight incubation, the cells were washed once in serum-free phosphate-buffered saline (PBS) and were fixed and stained in methanol containing 1% methylene blue and 0.25% basic fuchsin for 10 min. Cell layers were then washed in tap water and examined for syncytia by low-power microscopy. An estimate of the percentage of nuclei incorporated into syncytia in adherent cells was recorded as follows: -, 50. Immunostaining of HIV-infected cells. The immunostaining method was similar to that described by Chesebro and Wehrly (9) but adapted for a 1-galactosidase conjugate instead of a peroxidase conjugate. Briefly, HIV-infected cells were washed once in serum-free medium and fixed in 50% methanol-50% acetone for 2 min at -20°C. After washing in PBS containing 1% fetal calf serum, appropriate HIV-positive human serum was used to detect virus antigen (HIV-1+ serum or HIV-2+ serum). The sera were added at a 1:400 dilution in PBS-1% fetal calf serum (FCS) for 1 h at room temperature and then washed three times in PBS-1% FCS. Sheep anti-human IgG-p-galactosidase conjugate (Amersham) at a 1:100 dilution was added for 1 h at room temperature and washed three times in serum-free PBS; 0.5 mg of 5-bromo-4-chloro-3-13-D-galactopyranoside (X-Gal) substrate (Novolabs) per ml in PBS containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride was added. Infected cells stained blue within 1 to 2 h of addition of substrate.
MATERLALS AND METHODS Cells. The RD human rhabdomyosarcoma cells were the TE671 subline (12), previously thought to be medulloblastoma cells (35). Human T-cell lines (C8166 and MOLT4), B-cell lines (BJAB, Daudi, and Raji), carcinoma lines (HeLa, Heb7a, HT-29, Rsb, osteosarcoma HOS, and glioma U87) and rhesus monkey kidney FRhK and African green monkey kidney Vero and BGM cell lines used in this laboratory were described previously (11, 32, 33). Viruses. The HIV-2 strains used were LAV-2ROD (13), SBL6669 (1), CBL-20 to CBL-23 (30), and ST (21). The LAV-2/A and LAV-2/B substrains differed in passage history in our laboratory; LAV-2/A is the prototype ROD isolate, and LAV-2/B was derived from chronically infected C8166 cells that had been extensively passaged. The *
Corresponding author. 3531
3532
CLAPHAM ET AL.
J. VIROL.
+sCD4
Ant i-HIV-2
+sCD4
HIV-2 LAV-2/BI '., i 4
i
,41
4wJ, HIV-2 CBL-20
S'
W,.;i,
..h0
i
At
HIV-1 RE
A40
X
A _
,
_
:
-AV Mib
FIG. 1. Fusion of CD4- RD cells by HIV-2. HIV-producing H9 cells were mixed with RD cells as described in Materials and Methods and stained after overnight incubation. Soluble CD4 was added where indicated at 10 ,ug/ml, and human anti-HIV-2 serum was added at a 1:200 dilution.
Cell-free virus titrations. Virus supernatants were serially diluted in 10-fold steps and added in 0.2-ml amounts to appropriate cell types in 24-well 30-mm-diameter trays. Cells were set up at 10 cells per well 1 day prior to addition of virus. Infected cells were passaged as required, and productive infection was assessed by observing syncytia either in the cell culture or in a cocultivation with appropriate indicator cells such as C8166 or MOLT4 (24). HIV-2 1251-gp10 binding assay. Recombinant, baculovirus-expressed LAV-2RoD gpllO (27) was labelled with 1251, using Enzymobeads (Amersham); 106 cpm was added to cells in 100 ,ul of complete PBS-1% FCS for 2 h at 37°C at subsaturation levels. After multiple washing, cells were solubilized and counted as described previously (11).
RESULTS HIV-2 fusion of CD4-negative RD cells. Figure 1 illustrates the fusion properties of the HIV-2 strains LAV-2/B and CBL-20 and of HIV-1 RF for human RD cells previously found to have no detectable cell surface CD4 (11). LAV-2/B induced extensive syncytia among these cells, whereas CBL-20 and RF were negative. When soluble CD4 was added to the cocultivations, however, CBL-20 induced a level of fusion similar to that induced by LAV-2/B, while RF remained negative. Syncytia induced by CBL-20 and LAV2/B in the presence or absence of sCD4 were blocked in the presence of HIV-2+ human antiserum. Table 1 shows that syncytium induction was highly sensitive to neutralization by two human HIV-2 antisera that had previously been shown to neutralize infection of CD4+ T cells. Interestingly, HIV-2 syncytia were also weakly neutralized by HIV-1 human antisera. Cell lines susceptible to HIV-2 fusion. Thirteen CD4primate cell lines were tested for susceptibility to syncytium induction by the LAV-2/B and CBL-20 strains of HIV-2 and
by the RF strain of HIV-1 in the presence of sCD4 (Table 2). None of the cell lines was susceptible to fusion by HIV-1 RF even though RF induced massive fusion in all of the CD4+ human cell lines tested, including RD cells transfected with CD4. Only RD and the B-cell lines Raji and Daudi were sensitive to LAV-2/B fusion; these cell lines were also permissive to CBL-20 fusion provided sCD4 was added. SIVmac251 failed to induce fusion of any CD4- cell line. Also, RD, Daudi, and Raji cells remained nonpermissive to fusion by SIVmac251 when transfected with and expressing human CD4. HIV-2 strains fusigenic for RD cells. To determine whether fusion of CD4- cells was a property specific to HIV-2 strains, 20 diverse HIV-1, HIV-2, and SIV isolates were tested in fusion assays with and without sCD4 (Table 3). We observed that SBL6669 and CBL-22 directly induced fusion of RD cells though less efficiently than did LAV-2/B. sCD4 enhanced fusion by these two strains and activated fusion by
TABLE 1. Neutralization of HIV-2 fusion of RD cells Neutralization titer of serum
against HIV-2 strains
Human sera
LAV-2/B
CBL-20 + sCD4
G7 G33 HIV-1
320 640
1,280 2,560
QC2 QC6
80 40
40 10
Vol. 66, No. 6
0022-538X/92/063531-07$02.00/0 Copyright C) 1992, American Society for Microbiology
Human Immunodeficiency Virus Type 2 Infection and Fusion of CD4-Negative Human Cell Lines: Induction and Enhancement by Soluble CD4 PAUL R. CLAPHAM, AINE McKNIGHT,
AND
ROBIN A. WEISS*
Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, England Received 2 December 1991/Accepted 18 February 1992
We describe human immunodeficiency type 2 (HIV-2) strains which induce cell-to-cell fusion and infect certain CD4- human cell lines. Soluble CD4 (sCD4) induces or enhances fusion by most HIV-2 strains tested. Soluble CD4-immunoglobulin G chimeras and conjugates of sCD4 and antibody to the third domain of CD4 block HIV-2 fusion of CD4- cells. We conclude that HIV-2 can enter CD4- cells via an alternative cell surface receptor to CD4. While some strains entered efficiently, others retained a dependency on an interaction with sCD4 to initiate changes in the virion envelope required for membrane fusion.
SIVmac25i and SIVagmTyo strains and HIV-1 strains IIIB,
CD4 acts as the primary cell attachment receptor for human and simian immunodeficiency viruses (HIV and SIV) (15, 20, 28). Binding of HIV or SIV to CD4 at the cell surface is not sufficient for infection, and several examples of CD4+ human and nonhuman cells that bind HIV or SIV without proceeding to membrane fusion have been reported (4, 6, 8, 11, 22). These observations suggest that cell surface components other than CD4 are important in entry. Furthermore, CD4 not only acts to attach HIV and SIV to cells but also induces conformational changes in the virus envelope glycoproteins (29) which may be necessary for viral and cellular membranes to fuse together. This double dependency on CD4 for HIV and SIV entry may explain why HIV-1 infection of CD4- human cells is so inefficient (10) despite efficient binding of gpl20 to some neural cell types (16). Here we report the selection of an HIV-2 strain, LAV-2/B, which directly induces cell-to-cell fusion and efficient infection of certain CD4- human cell lines. Other HIV-2 strains, however, retain a dependency on an interaction with CD4 for fusion, which is induced or greatly enhanced for CD4cells by soluble CD4 (sCD4). These HIV-2 strains presumably bind to an alternative cell surface receptor but proceed to fuse with the cell membrane only after treatment with sCD4.
RF, MN, SF-2, CBL-4, Z34, Z84, Z129, U455, and NDK were propagated as previously described (11). MAbs. CD4 monoclonal antibodies (MAbs) Q4120 (domain 1 [D1]), L122, OKT4, Q425 (D3), and L120 (D4) have been described elsewhere (18). The MAbs and the Fab fragment of Q425 were kindly provided by Q. Sattentau and P. Kwong. sCD4 constructs. sCD4, derivative protein products (Dl and D1D2), and mutants (5) were kindly provided by R. Sweet (SmithKline Beecham). DlD2-immunoglobulin G (IgG) (7) was a kind gift from P. Berman (Genentech). Cell fusion assays. Syncytium induction assays were carried out by mixing 50 ,ul of virus-producing cells (106 cells per ml) with 50 ,ul of appropriate indicator or test cells (106 cells per ml). After overnight incubation, the cells were washed once in serum-free phosphate-buffered saline (PBS) and were fixed and stained in methanol containing 1% methylene blue and 0.25% basic fuchsin for 10 min. Cell layers were then washed in tap water and examined for syncytia by low-power microscopy. An estimate of the percentage of nuclei incorporated into syncytia in adherent cells was recorded as follows: -, 50. Immunostaining of HIV-infected cells. The immunostaining method was similar to that described by Chesebro and Wehrly (9) but adapted for a 1-galactosidase conjugate instead of a peroxidase conjugate. Briefly, HIV-infected cells were washed once in serum-free medium and fixed in 50% methanol-50% acetone for 2 min at -20°C. After washing in PBS containing 1% fetal calf serum, appropriate HIV-positive human serum was used to detect virus antigen (HIV-1+ serum or HIV-2+ serum). The sera were added at a 1:400 dilution in PBS-1% fetal calf serum (FCS) for 1 h at room temperature and then washed three times in PBS-1% FCS. Sheep anti-human IgG-p-galactosidase conjugate (Amersham) at a 1:100 dilution was added for 1 h at room temperature and washed three times in serum-free PBS; 0.5 mg of 5-bromo-4-chloro-3-13-D-galactopyranoside (X-Gal) substrate (Novolabs) per ml in PBS containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride was added. Infected cells stained blue within 1 to 2 h of addition of substrate.
MATERLALS AND METHODS Cells. The RD human rhabdomyosarcoma cells were the TE671 subline (12), previously thought to be medulloblastoma cells (35). Human T-cell lines (C8166 and MOLT4), B-cell lines (BJAB, Daudi, and Raji), carcinoma lines (HeLa, Heb7a, HT-29, Rsb, osteosarcoma HOS, and glioma U87) and rhesus monkey kidney FRhK and African green monkey kidney Vero and BGM cell lines used in this laboratory were described previously (11, 32, 33). Viruses. The HIV-2 strains used were LAV-2ROD (13), SBL6669 (1), CBL-20 to CBL-23 (30), and ST (21). The LAV-2/A and LAV-2/B substrains differed in passage history in our laboratory; LAV-2/A is the prototype ROD isolate, and LAV-2/B was derived from chronically infected C8166 cells that had been extensively passaged. The *
Corresponding author. 3531
3532
CLAPHAM ET AL.
J. VIROL.
+sCD4
Ant i-HIV-2
+sCD4
HIV-2 LAV-2/BI '., i 4
i
,41
4wJ, HIV-2 CBL-20
S'
W,.;i,
..h0
i
At
HIV-1 RE
A40
X
A _
,
_
:
-AV Mib
FIG. 1. Fusion of CD4- RD cells by HIV-2. HIV-producing H9 cells were mixed with RD cells as described in Materials and Methods and stained after overnight incubation. Soluble CD4 was added where indicated at 10 ,ug/ml, and human anti-HIV-2 serum was added at a 1:200 dilution.
Cell-free virus titrations. Virus supernatants were serially diluted in 10-fold steps and added in 0.2-ml amounts to appropriate cell types in 24-well 30-mm-diameter trays. Cells were set up at 10 cells per well 1 day prior to addition of virus. Infected cells were passaged as required, and productive infection was assessed by observing syncytia either in the cell culture or in a cocultivation with appropriate indicator cells such as C8166 or MOLT4 (24). HIV-2 1251-gp10 binding assay. Recombinant, baculovirus-expressed LAV-2RoD gpllO (27) was labelled with 1251, using Enzymobeads (Amersham); 106 cpm was added to cells in 100 ,ul of complete PBS-1% FCS for 2 h at 37°C at subsaturation levels. After multiple washing, cells were solubilized and counted as described previously (11).
RESULTS HIV-2 fusion of CD4-negative RD cells. Figure 1 illustrates the fusion properties of the HIV-2 strains LAV-2/B and CBL-20 and of HIV-1 RF for human RD cells previously found to have no detectable cell surface CD4 (11). LAV-2/B induced extensive syncytia among these cells, whereas CBL-20 and RF were negative. When soluble CD4 was added to the cocultivations, however, CBL-20 induced a level of fusion similar to that induced by LAV-2/B, while RF remained negative. Syncytia induced by CBL-20 and LAV2/B in the presence or absence of sCD4 were blocked in the presence of HIV-2+ human antiserum. Table 1 shows that syncytium induction was highly sensitive to neutralization by two human HIV-2 antisera that had previously been shown to neutralize infection of CD4+ T cells. Interestingly, HIV-2 syncytia were also weakly neutralized by HIV-1 human antisera. Cell lines susceptible to HIV-2 fusion. Thirteen CD4primate cell lines were tested for susceptibility to syncytium induction by the LAV-2/B and CBL-20 strains of HIV-2 and
by the RF strain of HIV-1 in the presence of sCD4 (Table 2). None of the cell lines was susceptible to fusion by HIV-1 RF even though RF induced massive fusion in all of the CD4+ human cell lines tested, including RD cells transfected with CD4. Only RD and the B-cell lines Raji and Daudi were sensitive to LAV-2/B fusion; these cell lines were also permissive to CBL-20 fusion provided sCD4 was added. SIVmac251 failed to induce fusion of any CD4- cell line. Also, RD, Daudi, and Raji cells remained nonpermissive to fusion by SIVmac251 when transfected with and expressing human CD4. HIV-2 strains fusigenic for RD cells. To determine whether fusion of CD4- cells was a property specific to HIV-2 strains, 20 diverse HIV-1, HIV-2, and SIV isolates were tested in fusion assays with and without sCD4 (Table 3). We observed that SBL6669 and CBL-22 directly induced fusion of RD cells though less efficiently than did LAV-2/B. sCD4 enhanced fusion by these two strains and activated fusion by
TABLE 1. Neutralization of HIV-2 fusion of RD cells Neutralization titer of serum
against HIV-2 strains
Human sera
LAV-2/B
CBL-20 + sCD4
G7 G33 HIV-1
320 640
1,280 2,560
QC2 QC6
80 40
40 10
