Human immunodeficiency viruses initiate infections via CCR5 coreceptors and then change their tropism to C-X-C chemokine receptor type 4 (CXCR4), this change being associated with rapid disease progression. HIV-1IIIB, a widely described pure X4-tropic strain, is distinct from R5X4-tropic viruses. In this study, the requirement for amino terminal regions (NTRs) of CXCR4 for entry of HIV-1IIIB virus into host cells was examined and compared to that of R5X4-tropic viruses. CXCR4 and its deletion mutant (CXCR4ΔNTR23; first 23 amino acids removed from NTR) were amplified to examine their coreceptor activities. NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cell lines were prepared accordingly. Indirect immune fluorescence assay (IFA), PCR, and reverse transcriptase (RT) activity were used to compare the process of infection of host cells by HIV-1IIIB virus, one R5-tropic and five other R5X4-tropic viruses. All the R5X4-tropic HIVs were found to utilize both CCR5 and CXCR4 but unable to use CXCR4ΔNTR23 as coreceptors. In contrast, X4-tropic HIV-1IIIB was found to preferentially infect through CXCR4ΔNTR23. Viral antigens in infected NP-2/CD4/CXCR4ΔNTR23 cells were detected by IFA and confirmed by detection of proviral DNA and by performing RT assays on the spent cell-supernatants. In dual tropic viruses, deletion of 23 amino acids from NTR abrogates the coreceptor activity of CXCR4. This observation demonstrates that NTR of CXCR4 have an obligatory coreceptor role for dual tropic viruses. However, HIV-1IIIB may have different requirements for NTR than R5X4 viruses or may infect host cells independent of NTR of CXCR4.
C-C chemokine receptor type 5
cluster of differentiation
C-X-C chemokine receptor type 4
mutant of CXCR4 made by removal of first 23 amino acids from NTR
Dulbecco's modified EMEM
Eagle's minimum essential medium
G protein-coupled receptor
immune fluorescence assay
National Center for Biotechnology Information
National Institute for Biological Standard and Control
amino terminal region
open reading frame
simian immunodeficiency virus
Human immunodeficiency viruses infect and enter into host cells by binding their mature envelop (env) glycoprotein, gp120, to CD4 as primary receptor [1, 2] and to chemokine receptors, CCR5, and CXCR4, exclusively or mutually, as major coreceptors [3, 4]. Combined receptor–coreceptor-bindings trigger gp41 activation and membrane fusion of the infected cells, thus facilitating viral entry [5, 6]. Moreover, several other GPCRs reportedly have coreceptors functions for HIV-1, HIV-2, and SIV [7, 8]. Several GPCRs of primate origin have also been reported to be functional coreceptors for SIV. The phenotypic variability of viruses is based on which coreceptors they utilize when infecting susceptible cells. Their use of the major coreceptors determines the tropism of infectious viruses and results in classification of most HIVs into three groups: R5 viruses (which use CCR5 alone as coreceptor), R5X4 viruses (which use both CCR5 and CXCR4) and X4 viruses (which use CXCR4 alone); the last are rare. However, all primary infections are initiated via CCR5 receptors and then change their tropism to CXCR4, probably expanding subsequently to more GPCR-usage . Evolution of viruses from R5-tropism to X4-tropism can lead to accelerated disease progression with rapid decline of CD4+ and CD8+ T cells [10, 11]. In addition, a recent study found that the predominant HIVs responsible for mother to child transmission are CXCR4-using viruses . Therefore, studies exploring tropism shifting and alternative coreceptor-utilization are extremely important.
HIV-1IIIB is a widely described laboratory-adapted strain that uses CXCR4 exclusively as its coreceptor and induces syncytia in T-cell lineages [13, 14]. This virus is distinct from R5X4 tropic viruses in that it loses its capacity to utilize CCR5 after adapting to CXCR4. Therefore, the role of CXCR4 in infection by HIV-1IIIB and other dual tropic viruses demands further investigation. Earlier studies demonstrated the critical roles of extracellular amino terminal regions (NTRs) for the coreceptor functions of GPCRs . In this study, we prepared a mutant of CXCR4 designated as CXCR4ΔNTR23by deleting 23 amino acids from NTR. To discriminate the coreceptor requirements of X4-tropic and dual-tropic viruses, we compared the coreceptor function of CXCR4ΔNTR23 for HIV-1IIIB with R5X4 tropic HIV strains/isolates. We observed that all dual tropic viruses became incapable of utilizing CXCR4ΔNTR23 as coreceptor whereas, interestingly, HIV-1IIIB used the deletion mutant with ease to infect cells.
1 MATERIALS AND METHODS
1.1 Preparation of C-X-C chemokine receptor type 4-deletion mutant
To examine their coreceptor activities, CXCR4 was amplified and a deletion mutant of it (CXCR4ΔNTR23) prepared by removing the first 23 amino acids from the NTR. Because it is reportedly a dependable cell-system tool for coreceptor examination study , the human glioma cell line, NP-2 was used to prepare NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cell lines. The amino acid sequence for the coding region of CXCR4 was obtained from the UniProtKB/Swiss-Prot protein database (accession number: P61073) and the corresponding DNA sequence obtained from GenBank database (accession number: AY242129.1). Oligonucleotide primers for detecting reverse-transcribed mRNA by PCR were designed and synthesized to cover the CXCR4-ORF (Proligo K.K., Tokyo, Japan). The nucleotide sequences of PCR primers, their orientations and the positions of primers in the respective ORFs of the genes were as follows: X4-F, 5′ATGGAGGGGATCAGTATATACACTTC3′ (sense: 1–26) and X4-R, 5′TTAGCTGGAGTGAAAACTTGAAGACTC3′ (antisense: 1033–1059). For the deletion mutant, a forward primer was designed from the corresponding position of the third methionine from NTR (5′ATGAAGGAACCCTGTTTCCGTGAAGA3′, sense: 70–95), keeping the same reverse primer as described above. Therefore, the expected PCR products for CXCR4 ORF and the deletion mutant were 1059 bp and 990 bp, respectively. As a control, mRNA expression of GAPDH was examined.
Total RNA was isolated from human cell lines using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. Contaminated DNA in RNA preparations was removed by digestion with 1 U/µL RNA qualified 1 RNase-free DNase (Promega, Madison, WI, USA) at 37°C for 1 hr. cDNA was prepared by RT reaction using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The expressions of mRNA for CXCR4 and CXCR4ΔNTR23 were detected by PCR from cDNA preparations using the sense and antisense primer pairs. Amplified cDNA was examined by electrophoresis through 1% (w/v) agarose gel.
1.2 Cloning of C-X-C chemokine receptor type 4 and its deletion mutant as coreceptors
DNA fragments for the full-length CXCR4 ORF and the mutant were cloned into a TA-cloning plasmid, pGEM-T Easy (Promega), and the derivative plasmids designated as pGEM-T-Easy/CXCR4 and pGEM-T Easy/CXCR4ΔNTR23. DNA sequencing of TA cloned derivatives was performed by a 5500 DNA-sequencer (Hitachi, Tokyo, Japan). The ligated DNA fragments were separated from TA plasmids by digestion with suitable restriction enzymes and recloned into an expression plasmid, pMX-puro . Transfection to NP-2/CD4 cells was performed with expression plasmids using FuGENE 6 (Roche, Basel, Switzerland) according to the manufacturer's protocol. Transfected cells were selected by maintaining them in culture medium containing 1 µg/mL puromycin for 2–3 weeks, as described elsewhere .
C-C chemokine receptor type 5-transduced human T-cell line, C8166/CCR5 was used for the preparation of HIV-1 stocks, as described previously . Varieties of established human cell lines were used to prepare mRNA and to amplify cDNA for the ORF of CXCR4 and its deletion mutant. T-cell lines, ATL-1K and a B-cell line, Daudi were cultured in RPMI 1640 medium (Nissui, Tokyo, Japan) containing 10% FBS. EMEM (Nissui) with 10% FBS was used to maintain NP-2 and its derivative cell lines. The amphotropic packaging cell line, Phoenix-Ampho  was maintained in DMEM containing 10% FBS.
Three laboratory-adapted strains, GUN4wt , IIIB  and BaL , and four primary isolates of HIV-1 were examined for their ability to utilize both CXCR4 and CXCR4ΔNTR23 as coreceptors in collaboration with CD4. Laboratory adapted HIV-2CBL was also used to a limited degree in this study. Primary isolates of HIV were obtained from the NIBSC (London, UK). Their origins, subtypes and NIBSC-reference codes are as follows: HIV-1 strains, MVP-5180 (Cameroon, subtype O, EVA167), 93BR020 (Brazil, subtype F, ARP179.25) and HAN2 (Germany, subtype B, EVA158). The primary isolate, GUN11, was isolated in Gunma University Hospital and included in this study.
1.5 HIV infection assay
NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cells were seeded separately into wells of 24-well plates at 25,000 cells/well in 500 µL of culture medium. On the following day, 400 µL of culture medium was drained off and subsequently replaced by respective HIV-1 inoculums. After overnight incubation, the cells were washed three times with EMEM containing 10% FBS to remove free virus, after which they were cultured in 500 µL fresh medium at 37°C. The cells were passaged every 3–5 days and maintained for up to 4 weeks. Expression of HIV antigen in the infected cells was detected by IFA after each passage, as described previously . RT activities in culture supernatants of cells harvested at the final time points of infection was also detected by a method described elsewhere .
1.6 Viral DNA amplification and sequencing
Polymerase chain reaction to detect viral DNA for the Gag-coding regions was also performed using the genomic DNA of infected cells as templates. The selected primer pair was HIV1-gagF (forward 5′-TCTATAAAAGATGGATAATCATGGG-3′) and HIV1-gagR (reverse 5′-AAGCACTTAACAGTCTTTCTTTGG-3′), which are located between positions 1571–1595 and 1944–1967, respectively (nucleotide positions were derived according to HIV-1-US88WR27, accession number, AF286365). DNA sequences obtained were blasted in the NCBI nucleotide database (http://blast.ncbi.nlm.nih.gov).
2.1 Amplification and cloning of C-X-C chemokine receptor type 4 and its deletion mutant as coreceptors
Human CXCR4 is a seven-transmembrane (7TM) receptor consisting of 352 amino acids, the extracellular amino terminal containing 39 amino acids as shown in a schematic diagram (Fig. 1a). The cDNA for CXCR4 was amplified in ATL-1K, C8166, Daudi and HeLa-cell lines. The NP-2 cell line did not express CXCR4 naturally (Fig. 1b), making it a prudent host for CXCR4 cloning and relevant coreceptor studies. The mutant-CXCR4 was amplified from the third methionine (M) position by PCR from ATL-1K cell lines and used for cloning. cDNAs of wild type and mutant-CXCR4 were transduced into NP-2/CD4 cells to produce NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cell lines. NP-2/CD4/CCR5 cells were prepared as positive controls for examining CCR5 functions. mRNA expressions of coreceptor candidates in cloned cells were detected accordingly.
2.2 Examination of the coreceptor activities of C-X-C chemokine receptor type 4 and its deletion mutant
NP-2/CD4/CCR5, NP-2/CD4/CXCR4, NP-2/CD4/CXCR4ΔNTR23 and NP-2/CD4 cells were seeded and inoculated with the virus strains and isolates, cultured for up to 4 weeks and examined upon cell passage for whether IFA could detect HIV antigen-positive cells. Except for HIV-1IIIB, all HIV strains and isolates replicated well in NP-2/CD4/CCR5 cells: most cells becoming HIV-antigen positive 3–7 days after inoculation (Table 1), upon which a large number of fused cells were detectable by light microscopy (data not shown). Similarly, NP-2/CD4/CXCR4 cells were found to support all viral propagations except for HIV-1BaL (exclusively R5-tropic). Thus, all R5X4-tropic HIV-1 were found to utilize both CCR5 and CXCR4 coreceptors. However, no R5X4-tropic viral antigen was detected in virus-exposed NP-2/CD4/CXCR4ΔNTR23 cells, indicating that CXCR4ΔNTR23 does not function as a coreceptor for any dual tropic strains or isolates (Table 1 and Fig. 2). Thus, in dual tropic viruses, deletion of 23 amino acids from NTR abrogates the coreceptor activity of CXCR4. In contrast, pure X4-tropic HIV-1IIIB was found to infect NP-2/CD4/CXCR4ΔNTR23 cells readily (Fig. 2). Of note, HIV-1IIIB showed a slower infection course through 23 amino acids-deleted CXCR4 than through native CXCR4. Whereas about 90% of NP-2/CD4/CXCR4 cells became HIV-1IIIB viral antigen positive with 1 week of inoculation, the virus took more than 3 weeks to reach a similar level of infection in NP-2/CD4/CXCR4ΔNTR23 cells (Fig. 3a,b). According to IFA, HIV-2CBL23 was more strongly infective via CXCR4 than via CCR5 but was unable to infect NP-2/CD4/CXCR4ΔNTR23 cells (Table 1). NP-2/CD4 cells were found to be completely resistant to infection by all HIV strains/isolates tested here. Concurrent with IFA, RT activities were assayed in spent culture media of NP-2/CD4/CXCR4 on day 7, when 70–90% cells were viral antigen-positive, and in those of NP-2/CD4/CXCR4ΔNTR23 cells on day 24, when 60% of cells were found to be antigen-positive for HIV-1IIIB but < 0.1% antigen-positive for other R5X4 viruses. All viruses except HIV-1BaL produced infection via CXCR4, thereby generating high RT activities. The RT values ranged from 1.4 × 105 to 2.9 × 105 H-cpm/mL (Fig. 3c). On the other hand, CXCR4ΔNTR23 allowed only HIV-1IIIB to produce infection with RT activity of 1.2 × 105 H-cpm/mL. Other dual tropic viruses produced negligible to background level RT activities (2.0 × 103 H-cpm/mL) via CXCR4ΔNTR23 (Fig. 3c). Therefore, the results of RT activities were concordant with the corresponding percentages of viral antigen positive cells as detected by IFA.
|Virus||Infection to NP-2/CD4 cells using coreceptor of:|
|IIIB||−||++ + +†||+++|
|BaL||++ + +||−||−|
|GUN4wt||++ + +||+++||−|
|CBL23||+++||++ + +||−|
|93BR020||++ + +||+++||−|
|GUN11||++ + +||+++||−|
|HAN2||++ + +||++ + +||−|
|MVP5180||++ + +||++ + +||−|
2.3 Proviral DNA and sequence analyses
Providing additional evidence of infection of NP-2/CD4/CXCR4ΔNTR23 cells, proviral DNA was detected for only HIV-1IIIB by PCR (Fig. 3d), whereas no dual-tropic viral DNA was detected. The proviral DNAs of HIV-1IIIB propagated in NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cells were sequenced and blasted in the NCBI nucleotide database. Both DNAs showed homogeneity to the gag region of reference strains. The IIIB-sequences obtained through CXCR4 and CXCR4ΔNTR23 were aligned to each other and had two common nucleotide substitutions (Supplementary Data 1). However, because both nucleotide substitutions were synonymous, no amino acid substitutions were detected in the amplified gag domain, (Supplementary Data 2).
All primary HIV infections were thought to be initiated through CCR5 coreceptors ; CXCR4-using viruses not usually being found during early phases of infection . However, in a recent study CXCR4-tropic viruses were detected during a recently-diagnosed infection, indicating that CXCR4 can play a role in initiating viral transmission . Thus, the coreceptor role of CXCR4 is quite complex and demands further detailed investigation. Tyrosines in the amino terminal domain of CCR5 play a critical role in viral entry . However, the role of tyrosine residues in the N-terminal tail of CXCR4 as HIV-coreceptor has not been clarified . In this study, we investigated the contribution of the first 23 amino acids of NTR of CXCR4, which contains tyrosine in the seventh, 12th, and 21st position, to CXCR4 performing as a coreceptor for several R5X4 tropic HIV-1 strains and compared them with an X4 tropic HIV-1IIIB strain. During examination of the ability of dual tropic HIV strains and isolates to infect through CXCR4ΔNTR23 as coreceptor, we found NP-2/CD4/CXCR4ΔNTR23 cells to be completely uninfected 4 weeks after inoculation (Fig. 3b). This observation demonstrates that the NTR of CXCR4 has an obligatory role as coreceptor for dual tropic viruses. In this study, in addition to HIV-1 strains and isolates, we examined laboratory-adapted HIV-2CBL23, which is also a dual tropic virus with strong replication capacity through CXCR4, and found it to be incapable of replicating through CXCR4ΔNTR23 (Table 1). We predicted that the deleted region plays critical roles in the formation of the structure necessary for the coreceptor activity of CXCR4 for those R5X4 viruses.
The deleted 23 amino acids include three tyrosines and three aspartic acids; these may be responsible for the coreceptor activities of CXCR4 for infection-incapable R5X4 viruses. The 16 amino acids remaining attached to the NTR of CXCR4ΔNTR23 do not include any tyrosine or aspartic acid (Fig. 1), supporting a previous conjecture that absence of tyrosine/aspartic acid precludes coreceptor activity [15, 27]. Thus, we predicted possible functional roles for tyrosine/aspartic acids in the NTR of CXCR4. Wild type CXCR4 contains 39 amino acids in its NTR versus16 amino acids to the deletion mutant (Fig. 1a). Thus, CXCR4ΔNTR23's loss of coreceptor function indicates that the first 59% of amino acids (23/39) of the NTR are more critical than the remaining 41% (16/39) in determining coreceptor activity. Accurate identification of the number(s) and positions of specific amino acids responsible for coreceptor function requires further detailed studies. In our study, HIV-1IIIB, unlike R5X4 viruses, showed a distinct CXCR4 utilization pattern. We found that HIV-1IIIB was incapable of initiating infection of NP-2/CD/CXCR4ΔNTR23 cells; < 1% cells were found to be antigen-positive 2 weeks after inoculation. Subsequently, the virus propagated strongly through CXCR4ΔNTR23 (Fig. 3b). Both RT assay and proviral DNA detection supported the findings of IFA. Thus, we conclude that exclusive X4-tropic HIV-1IIIB may have different requirement for NTR than R5X4 viruses. The HIV-1IIIB virus may infect the host cells using the residual NTR region (from 24 to 39) of CXCR4 or independent of the NTR of the coreceptor. Our observations are in agreement with a previous study in which replacement of aspartic acid at transmembrane domain 4 and 6 of 7TM was found to hamper the coreceptor function of CXCR4 for HIV-1IIIB and other pure X4-tropic viruses . Moreover, HIV-1IIIB may have a different coreceptor-binding domain in gp120 than other viruses that interact typically with the V3 region while entering target cells . Therefore, identifying sequences of the variable regions of gp120 of HIV-1IIIB and comparing them to those of other viruses could be vital to understanding its coreceptor utilization.
The findings of this study may contribute to understanding the pathogenesis of X4-tropic viruses as well as the mechanism of infection via CXCR4 utilization. This investigation would be more significant and complete if more X4 viral strains and/or primary isolates were studied as well as HIV-1IIIB. Further studies are required to determine the clinical relevance of these findings.
This work was supported in part by grants-in-aid from the 21st Century COE Program, “Biomedical Research using Accelerated Technology” and Gunma University Graduate School of Medicine.
Authors have no conflicts of interest to disclose.