JVI Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McKnight, A.
Right arrow Articles by Clapham, P. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McKnight, A.
Right arrow Articles by Clapham, P. R.

 Previous Article  |  Next Article 

J Virol, May 1998, p. 4065-4071, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A Broad Range of Chemokine Receptors Are Used by Primary Isolates of Human Immunodeficiency Virus Type 2 as Coreceptors with CD4

Áine McKnight,1,* Matthias T. Dittmar,1 José Moniz-Periera,2 Koya Ariyoshi,3 Jacqueline D. Reeves,1 Sam Hibbitts,1 Denise Whitby,1 Emma Aarons,4 Amanda E. I. Proudfoot,5 Hilton Whittle,3 and Paul R. Clapham1

Section of Virology, Chester Beatty Laboratories, Institute of Cancer Research,1 and Department of GUM, St. Mary's Hospital,4 London, United Kingdom; Faculdade de Farmacia, Universidada de Lisboa, Lisbon, Portugal2; Medical Research Council Laboratories, Fajara, The Gambia3; and Geneva Biomedical Institute, Glaxo Wellcome Research and Development SA, Geneva, Switzerland5

Received 29 September 1997/Accepted 21 January 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Like human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), HIV-2 requires a coreceptor in addition to CD4 for entry into cells. HIV and SIV coreceptor molecules belong to a family of seven-transmembrane-domain G-protein-coupled receptors. Here we show that primary HIV-2 isolates can use a broad range of coreceptor molecules, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Despite broad coreceptor use, the chemokine ligand SDF-1 substantially blocked HIV-2 infectivity of peripheral blood mononuclear cells, indicating that its receptor, CXCR4, was the predominant coreceptor for infection of these cells. However, expression of CXCR4 together with CD4 on some cell types did not confer susceptibility to infection by all CXCR4-using virus isolates. These data therefore indicate that another factor(s) influences the ability of HIV-2 to replicate in human cell types that express the appropriate receptors for virus entry.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CD4 is the primary cell surface receptor for entry of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) into cells. Coreceptors for HIV and SIV have been identified as G-protein-coupled receptors with seven transmembrane domains (reviewed in references 14, 42, 59, and 60). Early evidence suggested that there may be more than one coreceptor, reflecting the extended tropism of syncytium-inducing (SI) HIV-1 strains for CD4-positive T-cell lines compared to non-syncytium-inducing (NSI) strains, which infect mainly primary macrophage and T-cell cultures. CXCR4 was the first HIV type 1 (HIV-1) coreceptor to be defined (25) and was shown to be the major coreceptor for T-cell-line-adapted and primary SI HIV-1 isolates (21, 25, 40, 54, 61). CCR5 is the main coreceptor for primary NSI HIV-1 isolates (3, 10, 18, 21, 22, 61). HIV-1 viral entry is inhibited in the presence of the ligands to these chemokine receptors. Thus, RANTES, MIP-1alpha , and MIP-1beta , the ligands for CCR5, inhibit macrophage-tropic isolates, while SDF-1, the specific ligand for CXCR4, inhibits entry of T-cell-tropic isolates (5, 15, 34, 44).

CCR5 is important for transmission. Individuals with a CCR5 gene deletion are largely protected from HIV-1 infection (17, 31, 33, 37, 46, 50). CCR5-using strains are present throughout the course of infection in an individual, while CXCR4-using viruses often develop late in disease (16). Some HIV-1 isolates have the ability to exploit other chemokine receptor molecules, such as CCR3, CCR2b, BOB, and BONZO (4, 10, 19, 21, 24, 36), in addition to CCR5 and CXCR4. The efficiency of these other coreceptors for HIV entry is controversial.

Phylogenetic analysis has shown that HIV-1 clusters with SIV isolated from chimpanzees (SIVcpz) and that HIV-2 clusters with SIV from sooty mangabeys (SIVsm) (7, 26, 27, 43). HIV is likely to have originated as a result of cross-species transmission of SIV from African apes or monkeys to humans (53). Like HIV, SIV also requires a coreceptor in addition to CD4 for viral entry, since several nonhuman cell types expressing human CD4 fail to support SIV viral entry (39). Chen et al. reported that SIV isolates (SIVmac, SIVsmSL92a, SIVsmLib-1, and SIVcpz GAB) could use human or rhesus monkey CCR5 for entry but not human CXCR4, CCR1, CCR2b, CCR3, or CCR4 (8). SIV, however, could infect several cell lines (now known to be CCR5 negative) that resist HIV-1 infection, indicating that other coreceptors may be exploited by SIV (12, 32). Several groups have recently reported three likely candidates for this unknown receptor(s). BOB/gpr15 (19, 24, 29) and BONZO/STRL33 (4, 36) both assist entry of SIVmac and HIV-2 as well as certain SI and NSI strains of HIV-1. Another orphan receptor, GPR1, allows entry of SIVmac but not HIV-1 into CD4-positive cells (24).

Coreceptors used by the laboratory-adapted isolate HIV-2 ROD were recently studied by Brön et al. (6). In cell-to-cell fusion assays, ROD was particularly promiscuous and induced fusion of CD4-positive cells expressing CCR1, CCR2b, CCR3, CCR5, CXCR2, and CXCR4. In contrast, in cell-free infectivity assays, ROD used CCR3 and CXCR4 but not CCR5. A variant of HIV-2, ROD/B, that efficiently infects some cell types in the absence of CD4 (13) also used CXCR4 and CCR3 or an orphan receptor, V28, in the absence of CD4 (23, 48). Recently, Sol et al. (56) showed that CCR3, CCR5, and CXCR4 were used by HIV-2 primary strains, while Heredia et al. (30) reported that some primary isolates of HIV-2 could use CCR1, CCR2b, and CCR5 as well as CXCR4 but not CCR3 or CCR4. However, there was no indication of the relative efficiencies of these infections. Deng et al. (19) also showed that some primary HIV-2 isolates used several coreceptors, including the newly identified BOB/gpr15 and BONZO/STRL33.

Here we report on the coreceptor use of various primary isolates of HIV-2 isolated mainly from symptomatic patients and cultured in peripheral blood mononuclear cells (PBMCs). We find that these primary HIV-2 isolates can use a broad range of coreceptors, including CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4. Not all human cell types that express an appropriate coreceptor supported virus replication, indicating the presence of other factors influencing viral tropism.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cells. U87/CD4 cells expressing chemokine receptors CCR1, CCR2b, CCR3, CCR5, and CXCR4 were a gift from Dan Littman and have been previously described (18). CCC/CD4 cells derived from cat kidney (CCC S+ L-), RD/CD4 cells derived from human rhabdomyosarcoma, and HeLa/CD4 cells have been previously described (12, 39). SCL/CD4 cells are from human skin (9), and WI-38/CD4 cells are simian virus 40-transformed human lung fibroblasts (28). Human CD4 was stably expressed on these cell types by using an amphotropic retroviral vector (9). MT-2 (41), C8166 (11), Molt 4, H9, and Sup T1 cells are all human CD4+ T-cell lines and have been described elsewhere (13, 39). Peripheral blood PBMCs were stimulated for 2 days with phytohemagglutinin (PHA) (0.5 µg/ml) and then cultured in RPMI plus 10% fetal calf serum (FCS) supplemented with interleukin-2 (IL-2) (20 U/ml).

Chemokines and coreceptor ligands. A recombinant form of SDF-1 that retains the N-terminal methionine residue was used. This, MCP-1, and AOP-RANTES (55) were provided by Glaxo Wellcome. vMIP-II, a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (35), was provided by T. W. Schwartz, University of Copenhagen. AMD3100 is a bicyclam derivative that reacts with CXCR4 (51).

Isolation of virus and preparation of stocks. CBL-20, CBL-23, and V9 were isolated from individuals from The Gambia. CBL-20 and CBL-23 have been described before (52). CBL-20 and V9 were isolated from individuals with AIDS, whereas CBL-23 is from an individual with AIDS-related complex. prCBL-20 was propagated only in primary PBMC cultures, while CBL-20 was adapted for growth in T-cell lines. MIR was isolated from an AIDS patient who died in 1987. A-ND was from a Portuguese individual with progressive disease. ST was from a Senagalese asymptomatic individual (53). Viruses were prepared as follows. PBMCs from HIV-2-infected patients were cocultured with PHA-stimulated uninfected PBMCs in the presence of recombinant IL-2 (20 IU/ml). Supernatant from a day 28 culture was passaged into a culture of fresh uninfected PBMCs, which was monitored weekly for reverse transcriptase (RT) activity. When the culture became positive for RT activity (>50 pg/ml), fresh PBMCs were added and virus was allowed to grow for a further week before the supernatant was harvested. Stocks of ST were made from Molt 4-derived virus passaged into PBMCs. Virus aliquots were stored under liquid N2.

Plasmids. The chemokine receptors CCR1, CCR2b, CCR3, CCR4, CCR5, and CXCR4 were subcloned into the vector pcDNA3.1 (Invitrogen) for transfection and transient expression in CCC/CD4 cells.

Transfection of CCC/CD4 cells. Cells were plated overnight in six-well trays at 2 × 105 cells per ml. For each well, 1 µg of plasmid DNA in 100 µl of FCS-free medium was added to a solution of 7 µl of Lipofectamine (GIBCO BRL) (1 mg/ml) in 100 µl of FCS-free medium and allowed to stand on the bench for 45 min. Cells were washed twice before addition of 0.8 ml of FCS-free medium followed by the plasmid and Lipofectamine mixture. Control transfections (mock) were done by using the vector pcDNA3.1 without insert. After 5 h of incubation, the medium was replaced, and after overnight incubation, the cells were set up in 24-well trays for infection the following day.

Syncytium induction assays. High-titer virus stocks were added to 0.5 ml of target cells (2 × 105 cells/ml for MT-2, C8166, Molt 4, and Sup T1 cells or 1 × 106 cells/ml for PBMCs) in a 48-well tray. Cells were passaged twice weekly for 2 weeks and scored for syncytia as follows: -, no syncytia; +, at least one syncytium in cultured cells but fewer than 5% of nuclei in syncytia; ++, >5% but <19% of nuclei in syncytia, +++, 20 to 40% of nuclei in syncytia; and ++++, >40% of nuclei in syncytia. After 2 weeks, RT activity in the PBMC cultures was measured to assess the presence of virus.

Determination of RT activity. Virus production into the supernatant was assayed by measurement of RT activity by a sensitive nonradioactive method (Retrosys RT activity kit; Cavidi Tech, Uppsala, Sweden).

Virus infectivity and inhibition assays. Cells were plated at 5 × 104 per well in a 24-well plate or at 1 × 104 per well in a 48-well dish. A total of 0.1 ml (48-well dishes) or 0.2 ml (24-well plates) of 10-fold serial dilutions of virus was added to cells and incubated at 37°C for 1 h before washing in growth medium and reincubation for 3 to 5 days. Infected cells were detected by specific immunostaining. For titration of virus on PBMCs, virus (50 µl) was added to 105 cells per well of a 96-well tray. For inhibition-of-infection studies with PBMCs, cells were first treated with SDF-1 at 400 or 1,600 ng/ml, with AOP-RANTES at 200 or 800 ng/ml, or with MCP-1 at 800 ng/ml for 20 min before addition of virus.

Immunostaining of HIV-infected cells. The immunostaining method has been described before (39). Briefly, HIV-infected cells were washed in serum-free phosphate-buffered-saline (PBS) and fixed in methanol-acetone (1:1) at -20°C. After washing of the cells in PBS-1% FCS (PBS-FCS), HIV-2-positive serum was added at 1/5,000 (in PBS-FCS) to detect virus antigen. After three washes in PBS-FCS, the cells were incubated for 1 h with mouse anti-human immunoglobulin G conjugated with beta -galactosidase (Southern Biotech). After a further three washes in serum-free PBS, 0.5 mg of X-Gal (5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside) substrate (Novolabs) per ml in PBS containing 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, and 1 mM magnesium chloride was added. Clusters of blue cells were counted as foci of infection (focus-forming units [FFU]) to estimate the virus titer.

PCR amplification of CCR5 genes. Oligonucleotide primers spanning the CCR5 32-bp deletion (Delta 32) were used to amplify DNA fragments from PBMC cultures (37). This procedure produces a PCR DNA fragment of 183 bp from wild-type CCR5 DNA and a fragment of 151 bp of DNA carrying the 32-bp CCR5 deletion. Genomic DNA was derived from PBMCs by using a Nucleon Biosciences (Coatbridge, United Kingdom) kit. The primer pair used was 759+ CTT CAT TAC ACC TGC AGC TCT and 941- ACC AGC CCT GTG CCT CTT CTT. The samples were initially denatured at 95°C for 5 min, followed by 30 cycles of a denaturing step at 95°C for 30 s, an annealing step at 45°C for 30 s, and an extension step at 72°C for 30 s. A final step at 72°C for 5 min was done. PCR products were visualized by electrophoresis on a 3% agarose gel.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Coreceptor use by primary isolates of HIV-2. We determined the coreceptor use of six primary isolates of HIV-2 grown in PBMCs. ST came from an asymptomatic Senegalese individual (53). The remaining isolates were from symptomatic patients. MIR and A-ND were isolated from two symptomatic Portuguese patients. Three isolates from Gambian individuals were also included. Two of these Gambian isolates, V9 and prCBL-20, were from patients with AIDS, while prCBL-23 was from an individual with AIDS-related complex. To determine which coreceptors these viruses used, we challenged U87/CD4 cells stably expressing each of the chemokine receptors, CCR1, CCR2b, CCR3, CCR5, and CXCR4. Virus isolates MIR, ST, V9, prCBL-20, and prCBL-23 showed efficient use of all five chemokine receptor molecules in cell-free infection (Fig. 1). No infection of the parental U87/CD4 cells was detected. MIR, prCBL-20, and prCBL-23 plated most efficiently on cells expressing CXCR4, while ST preferentially infects cells expressing CCR5. Interestingly, A-ND showed no detectable activity on CCR5-expressing cells.


View larger version (18K):
[in this window]
[in a new window]
  		FIG. 1.   Titration of primary HIV-2 isolates on chemokine receptor-expressing U87/CD4 cells. After incubation for 3 to 5 days, cells were fixed and immunostained and infection (FFU per milliliter) was estimated. Error bars indicate standard deviations.

To verify the infectivity results gained from the set of U87/CD4 cells expressing coreceptors, we tested three isolates, MIR, V9, and prCBL-20, on cat CCC/CD4 cells transfected with and transiently expressing each of the chemokine receptors tested on U87/CD4 cells. Although this procedure yielded variable results, we were able to confirm that these viruses were capable of using a broad range of coreceptors. Table 1 shows a representative set of results from one experiment using MIR. We also tested whether these isolates used CCR4 (not included in the U87/CD4 experiments) in transiently transfected CCC/CD4 cells. Although MIR used CCR4 consistently, low levels of infectivity were observed with the two other strains, V9 and prCBL-20.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   HIV-2 MIR infection of CCC/CD4 cells expressing different chemokine receptors after transfection

U87 and CCC cells express undefined coreceptors that can be used by some HIV-2 strains (12, 39). It was possible that the chemokine receptor-positive subclones of U87/CD4 may express such undefined coreceptors at higher levels than parental U87/CD4 cells and therefore confer infection independently of the expressed recombinant chemokine receptor. To rule out this possibility, we tested whether specific coreceptor ligands could inhibit A-ND infection of U87/CD4 cells expressing CCR1, CCR3, or CXCR4. vMIP-II is a chemokine encoded by Kaposi's sarcoma-associated herpesvirus that preferentially blocks HIV-1 infection via CCR3 (35). vMIP-II (1 µg/ml) reduced A-ND infectivity for CCR3+ U87/CD4 cells by over 90% but had no effect on infection via CCR1 or CXCR4. Similarly, a 100-ng/ml concentration of AMD3100, a bicyclam derivative that binds CXCR4, blocked A-ND infection of CXCR4+ U87/CD4 cells by over 90% but did not inhibit CCR1- or CCR3-dependent infection (data not shown).

Coreceptor use by T-cell-line-adapted HIV-2 isolates. One isolate, prCBL-20, was adapted for replication in a T-cell line (H9). The virus strain derived, CBL-20 (52), was then tested for coreceptor use on the U87/CD4 cells expressing different chemokine receptors. Figure 2 shows that selection of primary prCBL-20 to grow in T cells resulted in a virus variant which could infect only CXCR4+ U87/CD4 cells. Unlike the parental prCBL-20 virus, CBL-20 had lost the capacity to infect U87/CD4 cells via CCR1, CCR2b, CCR3, or CCR5. Thus, selection into T-cell lines resulted in restricted coreceptor use for this virus.


View larger version (10K):
[in this window]
[in a new window]
  		FIG. 2.   Selection of a primary isolate to grow in T-cell lines restricts coreceptor use. prCBL-20 was selected to grow in C8166 cells, and stocks were made in H9 cells (52). The resulting virus was titrated on either the parental U87/CD4 cells or those stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4. Infection (FFU per milliliter) was calculated after immunostaining of 5-day-old infected cultures. Error bars indicate standard deviations.

Phenotypes of HIV-2 strains used. All of the isolates studied here could use CXCR4 efficiently as a coreceptor molecule. For HIV-1, use of CXCR4 generally correlates with an SI phenotype (54, 61), whereas all NSI isolates use at least CCR5 (3, 10, 18, 21, 22). We tested whether our isolates could be categorized as NSI or SI according to the conventional criteria used for HIV (1, 58). Each isolate was plated on MT-2 cells, PBMCs, and a number of T-cell lines. Syncytium induction on these cell types indicates the presence of an SI virus. The results are shown in Table 2. At least two of these isolates, V9 and prCBL-20, did not induce syncytia in any of the cell types tested. MIR, A-ND, ST, prCBL-23, and CBL-20, however, induced syncytia in at least one of the T-cell lines tested.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Phenotype determination of primary HIV-2 isolatesa

Infection of PBMCs from an individual who is homozygous for the Delta 32 CCR5 gene. We assessed whether the primary HIV-2 strains could infect PHA- and IL-2-stimulated PBMCs derived from an individual who was homozygous for the Delta 32 CCR5 gene. Figure 3 shows a smaller PCR-amplified DNA fragment from PBMCs derived from an individual homozygous for deletions of CCR5. These cells were resistant to the CCR5 with HIV-1 strain SF-162 (55a). All but one of the primary HIV-2 strains used CCR5 (see above), while all could use several other coreceptors, including CXCR4. Table 3 shows that all strains tested replicated as efficiently in Delta 32/Delta 32 CCR5 PBMCs as in wild-type CCR5 PBMCs and therefore do not depend on CCR5 for infection of PBMCs.


View larger version (70K):
[in this window]
[in a new window]
  		FIG. 3.   Detection of homozygous deleted CCR5. CCR5 DNA was amplified from genomic DNA derived from the PBMCs of an individual homozygous for the CCR5 deletion and an individual homozygous for the wild-type CCR5 gene. PCR products were visualized by electrophoresis on a 3% agarose gel. Lanes: M, 1-kb marker; 1, negative control; 2 and 3, 200 and 400 ng of homozygous Delta 32 CCR5 PBMC DNA, respectively; 4, negative control; 5 and 6, 200 and 400 ng of wild-type CCR5 PBMC DNA, respectively.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   HIV-2 infection of wild-type and Delta 32/Delta 32 PBMCsa

Inhibition of infection of primary HIV-2 isolates by the ligands to specific chemokine coreceptors. We next investigated the coreceptor molecules used by A-ND, prCBL-20, and CBL-20 to infect PBMCs in vitro. PBMCs were derived either from individuals homozygous for Delta 32 CCR5 or from wild-type donors. We tested for infectivity of cells treated with SDF-1 (400 and 1,600 ng/ml), AOP-RANTES (200 and 800 ng/ml), and MCP-1 (800 ng/ml). AOP-RANTES is an analog of RANTES and a potent inhibitor of HIV-1 infection (55) and is also a ligand for CCR5, CCR3, and CCR1 (47). Figure 4 shows that whatever the source of PBMCs, SDF-1, the natural ligand for CXCR4, inhibited the infection of all three viruses by more than 90% (at 1,600 ng/ml). In contrast, MCP-1, a natural ligand for CCR2b and CCR4, had no effect on the infectivity of PBMCs by prCBL-20. In some experiments AOP-RANTES showed some inhibition early on when RT activity was first detected in control infections after infection of wild-type PBMCs (data not shown). This inhibition was not sustained. These results therefore indicate that CXCR4 is the predominant coreceptor used in vitro by the three HIV-2 strains tested for PBMC infection.


View larger version (40K):
[in this window]
[in a new window]
  		FIG. 4.   Inhibition of infection by prCBL-20, CBL-20, and A-ND with chemokines on wild-type CCR5 and Delta 32 CCR5 PBMCs. Infectivity assays with prCBL-20 and A-ND were performed in the absence and presence of the chemokines SDF-1, AOP-RANTES, and MCP-1 either alone or together at the concentrations indicated. Cell targets were either homozygous wild-type or Delta 32 CCR5 PBMCs. RT activity in the supernatant on day 5 was determined, and the results are shown as percent inhibition with respect to the control (con.) value. Error bars indicate standard deviations.

Tropism of primary HIV-2 isolates for CD4-expressing cell lines. We determined the abilities of the HIV-2 isolates to infect various human cell lines transfected with and stably expressing human CD4. Each of these cell types (HeLa/CD4, RD/CD4, WI-38T/CD4, SCL/CD4, and U87/CD4/CXCR4) was tested for expression of CCR5 or CXCR4 by using specific monoclonal antibodies followed by immunostaining and flow cytometric analysis (data not shown). As reported previously, HeLa/CD4 and RD/CD4 cells both express CXCR4 to high levels (40) but do not express CCR5 (data not shown). The SCL/CD4 or WI-38/CD4 cells did not express either CXCR4 or CCR5 at the cell surface. All seven virus isolates were titrated on these cell types as well as on U87/CD4/CXCR4 cells (Table 4). MIR, A-ND, V9, prCBL-20, and CBL-20 can efficiently infect HeLa/CD4 or RD/CD4 cells, presumably through CXCR4, which is expressed on both of these cell types. Interestingly, ST and prCBL-23, both of which efficiently infect CXCR4+ U87/CD4 cells, did not infect either CXCR4+ HeLa/CD4 or RD/CD4 cells even when the appropriate coreceptor was expressed. Thus, there is a further restriction to infection of some isolates on these cell lines. Furthermore, the ST isolate, unlike the other isolates tested, infected SCL/CD4 and WI-38/CD4 cells. Since ST, like other strains tested here, can use CCR1 to -5 and CXCR4, these results suggest that another cell surface receptor may be expressed on these two cell types.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 4.   Tropism of primary HIV-2 isolates for cell lines expressing human CD4a

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study determined the abilities of six primary isolates and one laboratory-adapted isolate of HIV-2 to use six chemokine receptors as coreceptor molecules for fusion and infection of human cells expressing CD4. The results presented here show that, unlike HIV-1 and SIV, the primary isolates of HIV-2 tested used a wide range of chemokine receptors. Five of the isolates, MIR, ST, V9, prCBL-20, and prCBL-23, could use CCR1, CCR2b, CCR3, CCR5, and CXCR4 expressed on U87/CD4 cells. This broad coreceptor use was confirmed for MIR, V9, and prCBL-20 on CCC/CD4 cells transiently transfected with these chemokine receptors. One isolate, A-ND, is peculiar in that we could not detect any infectivity on CCR5-expressing cells yet it used CCR1, CCR2b, CCR3, and CXCR4. MIR, V9, and prCBL-20 were also tested for infection of CCC/CD4 cells transfected with and expressing CCR4 (not tested in the U87/CD4 panel). We found that all of these virus isolates could also use CCR4 at least to some extent. This is the first report of any HIV or SIV isolates using CCR4.

Recently, Heredia et al. (30) also showed that primary HIV-2 isolates used several coreceptors. However in contrast to the viruses we tested, several isolates that used only CCR5 were identified, as well as some that additionally used CXCR4, CCR1, and CCR3 expressed by HOS cells. It is therefore possible that HIV-2 coreceptor use broadens as disease progresses, as suggested for HIV-1 by Connor et al. (16). Apart from ST, all of the HIV-2 strains tested here were derived from symptomatic patients. Our results therefore support the hypothesis of Connor et al.

For transmission of HIV-1, CCR5 is clearly the most important coreceptor. The majority of isolates thus far reported use CCR5 either exclusively (for NSI isolates) or in addition to CXCR4 (for SI isolates). Early after HIV-1 infection and in the asymptomatic phase, NSI CCR5-using viruses are usually isolated (16), suggesting that there is a strong selective pressure for the spread of CCR5-using viruses. As disease develops, CXCR4-using viruses can be more frequently isolated, from about 50% of patients with AIDS. How the broad coreceptor use by HIV-2 strains shown here influences their transmission remains to be determined.

We tested the coreceptor use of prCBL-20 after its adaptation to culture in H9 cells. The isolate derived (CBL-20) showed a marked loss in its ability to use the broad range of coreceptors used by the parental virus and could use only CXCR4 efficiently. Furthermore, T-cell-line-adapted CBL-20 could use CXCR4 expressed on HeLa/CD4 cells, whereas prCBL-20 could not. In contrast, prCBL-20 but not CBL-20 infected primary macrophages (not shown) even though they express CXCR4 (40). Recently Brön et al. (6) showed that the T-cell-line-adapted ROD strain of HIV-2 infected CXCR4- and CCR3-expressing CD4+ cells. Interestingly, while cell-free infection was restricted to particular coreceptors, cell-to-cell fusion was induced by a broad range of chemokine receptors, including CCR1, CCR2b, and CXCR2. The results of Brön et al. suggest that cell culture adaptation for CXCR4 use preferentially restricts coreceptor use for virion-to-cell fusion compared with cell-to-cell fusion and support the notion that the physical requirements for these processes are subtly different (6, 39).

So far, all primary and T-cell-line-adapted SI isolates of HIV-1 use CXCR4 (2, 25, 54, 61). We tested the phenotypes of the HIV-2 isolates described here by criteria used first for HIV-1 (58) and later for HIV-2 (1). Of the primary isolates, only MIR induced syncytia in PBMCs, while both MIR and prCBL-23 induced syncytia in MT-2 cells. Of the four other primary strains, ST and A-ND induced syncytia in at least one of the T-cell lines tested, whereas V9 and prCBL-20 showed no evidence of syncytium formation in any of the cell types tested. Thus, we did not observe a clear correlation between CXCR4 use and syncytium induction in CXCR4+ T cells by the HIV-2 strains used here.

The determination of coreceptor use by HIV-1 and HIV-2 strains in vitro, using CD4+ cell lines, can be misleading (20). Primary cell types that are targets for HIV infection in vivo may express several coreceptors that can be used by HIV strains in such in vitro assays. It is therefore hard to assess the predominant coreceptor used on these cell types. It has been shown previously that stimulated primary lymphocytes express CCR1 (57), CCR2b (45), CCR3 (49), CCR4 (38), CCR5 (14), CXCR4 (40), and BONZO (4, 36) as determined by mRNA expression. In addition we have shown that CCR5 (unpublished data) and CXCR4 (40) are expressed at the cell surface as detected by specific monoclonal antibodies. We assessed the coreceptors used by HIV-2 strains A-ND, prCBL-20, and CBL-20 on primary PHA- and IL-2-stimulated PBMCs by testing whether the natural ligands for different coreceptors could block infection. SDF-1 inhibited infection of PBMCs by all three virus strains, while AOP-RANTES or MCP-1 had little effect. Since SDF-1 has so far been shown to bind only to CXCR4, these results suggest that this coreceptor is predominantly used for PBMC infection.

We have shown that six primary strains of HIV-2 can use a broad range of different coreceptors. Although the more recently described coreceptors BOB, BONZO, and GPR1 were not included in this study, it seems likely that they too will be used by primary HIV-2 strains, at least for some virus-coreceptor combinations. How such a broad coreceptor use influences the cell types infected by HIV-2 strains in vivo remains to be elucidated. Although HIV-2 is often described as less pathogenic than HIV-1, many HIV-2-infected individuals do progress to full-blown AIDS and suffer from encephalitis. It will be of interest to establish whether the use of a wide spectrum of coreceptors available correlates with any disease state.

    ACKNOWLEDGMENTS

We thank Robin Weiss and Graham Simmons for critical discussion and reading of the manuscript. Dan Littman generously provided U87/CD4 cells expressing different chemokine receptors. Erik de Clercq and Dominic Schols kindly provided AMD3100. We also thank the European Community Concerted Action Group on HIV Variability for providing an intellectual forum for collaboration and discussion.

This work is funded by the Medical Research Council, United Kingdom, and supported partly by an EC Biomed II grant.

    FOOTNOTES

* Corresponding author. Mailing address: Section of Virology, Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB, United Kingdom. Phone: 44-171-352-8133. Fax: 44-171-352-3299. E-mail: aine{at}icr.ac.uk.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Albert, J., P. Stalhandske, S. Marquina, J. Karis, R. A. Fouchier, E. Norrby, and F. Chiodi. 1996. Biological phenotype of HIV type 2 isolates correlates with V3 genotype. AIDS Res. Hum. Retroviruses. 12:821-828[Medline].
2. Alkhatib, G., C. C. Broder, and E. A. Berger. 1996. Cell type-specific fusion cofactors determine human immunodeficiency virus type 1 tropism for T-cell lines versus primary macrophages. J. Virol. 70:5487-5494[Abstract/Free Full Text].
3. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955-1958[Abstract].
4. Alkhatib, G., F. Liao, E. A. Berger, J. M. Farber, and K. W. Peden. 1997. A new SIV co-receptor, STRL33. Nature 388:238[Medline].
5. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark Lewis, J. Sodroski, and T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382:829-833[Medline].
6. Brön, R., P. J. Klasse, D. Wilkinson, P. R. Clapham, A. Pelchen-Matthews, C. Power, T. N. C. Wells, J. Kim, S. Peiper, J. A. Hoxie, and M. Marsh. 1997. Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 ROD envelope protein. J. Virol. 71:8405-8415[Abstract].
7. Chen, Z., P. Telfier, A. Gettie, P. Reed, L. Zhang, D. D. Ho, and P. A. Marx. 1996. Genetic characterization of new West African simian immunodeficiency virus SIVsm: geographic clustering of household-derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse viruses from a single feral sooty mangabey troop. J. Virol. 70:3617-3627[Abstract].
8. Chen, Z., P. Zhou, D. D. Ho, N. R. Landau, and P. A. Marx. 1997. Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J. Virol. 71:2705-2714[Abstract].
9. Chesebro, B., and K. Wehrly. 1988. Development of a sensitive quantitative focal assay for human immunodeficiency virus infectivity. J. Virol. 62:3779-3788[Abstract/Free Full Text].
10. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, C. R. Mackay, G. LaRosa, W. Newman, N. Gerard, C. Gerard, and J. Sodroski. 1996. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148[Medline].
11. Clapham, P. R., R. A. Weiss, A. G. Dalgleish, M. Exley, D. Whitby, and N. Hogg. 1987. Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: receptor modulation and differentiation induced by phorbol ester. Virology 158:44-51[Medline].
12. Clapham, P. R., D. Blanc, and R. A. Weiss. 1991. Specific cell surface requirements for the infection of CD4-positive cells by human immunodeficiency virus types 1 and 2 and by simian immunodeficiency virus. Virology 181:703-715[Medline].
13. Clapham, P. R., Á. McKnight, and R. A. Weiss. 1992. Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4. J. Virol. 66:3531-3537[Abstract/Free Full Text].
14. Clapham, P. R. 1997. HIV and chemokines: ligands sharing cell surface receptors. Trends Cell Biol. 7:264-268. [Medline]
15. Cocchi, F., A. L. DeVico, A. Garzino Demo, S. K. Arya, R. C. Gallo, and P. Lusso. 1995. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science 270:1811-1815[Abstract/Free Full Text].
16. Connor, R. I., K. E. Sheridan, D. Ceradini, S. Choe, and N. R. Landau. 1997. Change in coreceptor use correlates with disease progression in HIV-1-infected individuals. J. Exp. Med. 185:621-628[Abstract/Free Full Text].
17. Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R. Allikmets, J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, S. Donfield, D. Vlahov, R. Kaslow, A. Saah, C. Rinaldo, R. Detels, and S. J. O'Brien. 1996. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science 273:1856-1862[Abstract/Free Full Text].
18. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, S. Marmon, R. E. Sutton, C. M. Hill, C. B. Davis, S. C. Peiper, T. J. Schall, and D. R. Littman. 1996. Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666[Medline].
19. Deng, H., D. Unutmaz, V. N. KewalRamani, and D. R. Littman. 1997. Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388:296-300[Medline].
20. Dittmar, M., Á. McKnight, G. Simmons, P. R. Clapham, R. A. Weiss, and P. A. Simmonds. 1997. HIV-1 tropism and co-receptor use. Nature 385:495-496[Medline].
21. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158[Medline].
22. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima, C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, and W. A. Paxton. 1996. HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673[Medline].
23. Endres, M. J., P. R. Clapham, M. Marsh, M. Ahuja, J. D. Turner, Á. McKnight, J. F. Thomas, B. Stoebenau Haggarty, S. Choe, P. J. Vance, T. N. Wells, C. A. Power, S. S. Sutterwala, R. W. Doms, N. R. Landau, and J. A. Hoxie. 1996. CD4-independent infection by HIV-2 is mediated by fusin/CXCR4. Cell 87:745-756[Medline].
24. Farzan, M., H. Choe, K. Martin, L. Marcon, W. Hofmann, G. Karlsson, Y. Sun, P. Barrett, N. Marchand, S. Nancy, C. Gerard, and J. Sodroski. 1997. Two orphan seven-transmembrane segment receptors that are expressed in CD4-positive cells support immunodeficiency virus infection. J. Exp. Med. 186:405-411[Abstract/Free Full Text].
25. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877[Abstract].
26. Gao, F., L. Yue, A. T. White, P. G. Pappas, J. Barchue, A. P. Hanson, B. M. Greene, P. M. Sharp, G. M. Shaw, and B. H. Hahn. 1992. Human infection by genetically diverse SIVSM-related HIV-2 in west Africa. Nature 358:495-499[Medline].
27. Hahn, B. H., L. I. Kong, S. Lee, P. Kumar, M. E. Taylor, S. K. Arya, and G. M. Shaw. 1987. Relation of HTLV-4 to simian and human immunodeficiency associated viruses. Nature 300:184-186.
28. Hayflick, L. 1965. Limited in vitro lifetime of human diploid cell strains. Exp. Cell Res. 37:614-636[Medline].
29. Heiber, M., A. Marchese, T. Nguyen, H. H. Heng, S. R. O. George, and B. F. Dowd. 1996. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3. Genomics 32:462-465[Medline].
30. Heredia, A., A. Vallejo, V. Soriano, J. S. Epstein, and I. K. Hewlett. 1997. Chemokine receptors and HIV-2. AIDS 11:1198-1199[Medline].
31. Hill, C. M., and D. R. Littman. 1996. Natural resistance to HIV? Nature 382:668-669[Medline].
32. Hoxie, J. A., B. S. Haggarty, S. E. Bonser, J. L. Rackowski, H. Shan, and P. J. Kanki. 1988. Biological characterization of a simian immunodeficiency virus-like retrovirus (HTLV-IV): evidence for CD4-associated molecules required for infection. J. Virol. 62:2557-2568[Abstract/Free Full Text].
33. Huang, Y., W. A. Paxton, S. M. Wolinsky, A. U. Neumann, L. Zhang, T. He, S. Kang, D. Ceradini, Z. Jin, K. Yazdanbakhsh, K. Kunstman, D. Erickson, E. Dragon, N. R. Landau, J. Phair, D. D. Ho, and R. A. Koup. 1996. The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nat. Med. 2:1240-1243[Medline].
34. Jansson, M., M. Popovic, A. Karlsson, F. Cocchi, P. Rossi, J. Albert, and H. Wigzell. 1996. Sensitivity to inhibition by beta-chemokines correlates with biological phenotypes of primary HIV-1 isolates. Proc. Natl. Acad. Sci. USA 93:15382-15387[Abstract/Free Full Text].
35. Kledal, T. N., M. M. Rosenkilde, F. Coulin, G. Simmons, A. H. Johnson, S. Alouani, C. A. Power, H. R. Lüttichau, J. Gerstoft, P. R. Clapham, I. Clark-Lewis, T. N. C. Wells, and T. W. Schwartz. 1997. A broad spectrum chemokine antagonist encoded by Kaposi's sarcoma-associated herpesvirus. Science 277:1656-1659[Abstract/Free Full Text].
36. Liao, F., G. Alkhatib, K. W. Peden, G. Sharma, E. A. Berger, and J. M. Farber. 1997. STRL33, a novel chemokine receptor-like protein, functions as a fusion cofactor for both macrophage-tropic and T cell line-tropic HIV-1. J. Exp. Med. 185:2015-2023[Abstract/Free Full Text].
37. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E. MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau. 1996. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86:367-377[Medline].
38. Mackay, C. R. 1996. Chemokine receptors and T cell chemotaxis. J. Exp. Med. 184:799-802[Free Full Text].
39. McKnight, Á., P. R. Clapham, and R. A. Weiss. 1994. HIV-2 and SIV infection of nonprimate cell lines expressing human CD4: restrictions to replication at distinct stages. Virology 201:8-18[Medline].
40. McKnight, Á., D. Wilkinson, G. Simmons, S. Talbot, L. Picard, M. Ahuja, M. Marsh, J. A. Hoxie, and P. R. Clapham. 1997. Inhibition of human immunodeficiency virus fusion by a monoclonal antibody to a coreceptor (CXCR4) is both cell type and virus strain dependent. J. Virol. 71:1692-1696[Abstract].
41. Miyoshi, I., I. Kubonishi, S. Yoshimoto, and Y. Shiraishi. 1981. A T-cell line derived from normal human cord leukocytes by co-culturing with human leukemic T-cells. Gann 72:978-981[Medline].
42. Moore, J. P., A. Trkokla, and T. Dragic. 1997. Co-receptors for HIV-1 entry. Curr. Opin. Immunol. 9:551-562[Medline].
43. Myers, G., K. MacInnes, and B. Korber. 1992. The emergence of simian/human immunodeficiency viruses. AIDS Res. Hum. Retroviruses. 8:373-386[Medline].
44. Oberlin, E., A. Amara, F. Bachelerie, C. Bessia, J. L. Virelizier, F. Arenzana Seisdedos, O. Schwartz, J. M. Heard, I. Clark Lewis, D. F. Legler, M. Loetscher, M. Baggiolini, and B. Moser. 1996. The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1. Nature 382:833-835[Medline].
45. Oin, S., G. LaRosa, J. J. Campbell, H. Smith Heath, N. Kassam, X. Shi, L. Zeng, E. C. Buthcher, and C. R. Mackay. 1996. Expression of monocyte chemoattractant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential. Eur. J. Immunol. 26:640-647[Medline].
46. Paxton, W. A., T. Dragic, R. A. Koup, and J. P. Moore. 1996. The beta-chemokines, HIV type 1 second receptors, and exposed uninfected persons. AIDS Res. Hum. Retroviruses 12:1203-1207[Medline].
47. Premack, B., and T. J. Schall. 1996. Chemokine receptors: gateways to inflammation and infection. Nat. Med. 2:1174-1178[Medline].
48. Reeves, J. D., A. McKnight, S. Potempa, G. Simmons, P. W. Gray, C. A. Power, T. Wells, R. A. Weiss, and S. J. Talbot. 1997. CD4-independent infection by HIV-2 (ROD/B): use of the 7-transmembrane receptors CXCR-4, CCR-3, and V28 for entry. Virology 231:130-134[Medline].
49. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005-2007[Abstract/Free Full Text].
50. Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber, S. Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, G. Muyldermans, C. Verhofstede, G. Burtonboy, M. Georges, T. Imai, S. Rana, Y. Yi, R. J. Smyth, R. G. Collman, R. W. Doms, G. Vassart, and M. Parmentier. 1996. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382:722-725[Medline].
51. Schols, D., J. A. Este, G. Henson, and E. De Clercq. 1997. Bicyclams, a class of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/CXCR-4. Antiviral Res. 35:147-156[Medline].
52. Schulz, T. F., D. Whitby, J. G. Hoad, T. Corrah, H. Whittle, and R. A. Weiss. 1990. Biological and molecular variability of human immunodeficiency virus type 2 isolates from The Gambia. J. Virol. 64:5177-5182[Abstract/Free Full Text].
53. Sharp, P. M., D. L. Robertson, and B. H. Hahn. 1995. Cross-species transmission and recombination of `AIDS' viruses. Philos. Trans. R. Soc. London B 349:41-47[Medline].
54. Simmons, G., D. Wilkinson, J. D. Reeves, M. T. Dittmar, S. Beddows, J. Weber, G. Carnegie, U. Desselberger, P. W. Gray, R. A. Weiss, and P. R. Clapham. 1996. Primary, syncytium-inducing human immunodeficiency virus type 1 isolates are dual-tropic and most can use either Lestr or CCR5 as coreceptors for virus entry. J. Virol. 70:8355-8360[Abstract].
55. Simmons, G., P. R. Clapham, L. Picard, R. E. Offord, M. M. Rosenkilde, T. W. Schwartz, R. Buser, T. N. C. Wells, and A. E. Proudfoot. 1997. Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276:276-279[Abstract/Free Full Text].
55a. Simmons, G. Personal communication.
56. Sol, N., F. Ferchal, J. Braun, O. Pleskoff, C. Tréboute, and M. Alizon. 1997. Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2. J. Virol. 71:8237-8244[Abstract].
57. Su, S. B., N. Mukaida, J. Wang, H. Nomura, and K. Matsushima. 1996. Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes. J. Leukocyte Biol. 60:658-666[Abstract].
58. Tersmette, M., R. E. de Goede, B. J. Al, I. N. Winkel, R. A. Gruters, H. T. Cuypers, H. G. Huisman, and F. Miedema. 1988. Differential syncytium-inducing capacity of human immunodeficiency virus isolates: frequent detection of syncytium-inducing isolates in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. J. Virol. 62:2026-2032[Abstract/Free Full Text].
59. Weiss, R. A., and P. R. Clapham. 1996. Hot fusion of HIV. Nature 381:647-648[Medline].
60. Weiss, R. A. 1996. HIV receptors and the pathogenesis of AIDS. Science 272:1885-1886[Free Full Text].
61. Zhang, L., Y. Huang, T. He, Y. Cao, and D. D. Ho. 1996. HIV-1 subtype and second receptor use. Nature 383:768[Medline].

J Virol, May 1998, p. 4065-4071, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McKnight, A.
Right arrow Articles by Clapham, P. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McKnight, A.
Right arrow Articles by Clapham, P. R.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Mol. Cell. Biol. Microbiol. Mol. Biol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS