Proc. Natl. Acad. Sci. USA

Vol. 89, pp. 7963-7967, September 1992 Immunology

CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1 B. F. HAGUE*, S. SAWASDIKOSOL, T. J. BROWN, K. LEEt, D. P. RECKERt, AND T. J. KINDT Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Twinbrook II Facility, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852

Communicated by Richard M. Krause, April 29, 1992

rabbit cells with HIV-1 is not dependent on expression of RbCD4, although it appears to serve as receptor in a cell line expressing it. Unpredictably, productive HIV-1 infections were obtained with lines that were RbCD4-negative and this infection was only partially inhibited by recombinant soluble HuCD4 (rsHuCD4), implying the presence of an additional, non-CD4, HIV-1 receptor in the rabbit.

Human CD4 (HuCD4) is the principal recepABSTRACT tor for human immunodeficiency virus ype 1 (HIV-1) in human cell infection. Susceptibility of rabbit cell lines to infection with HIV-1 raised questions concerning whether a CD4 homolog serves as HIV-1 receptor on rabbit cells. Sequence comparisons of rabbit CD4 (RbCD4) cloned from a rabbit thymus cDNA library showed that 6 of the 18 residues implicated in HIV-1 binding by CD4 differ between the human and rabbit proteins. No correlation between RbCD4 expression by rabbit cell lines and their ability to support HIV-1 infection was seen. Transfection of RbCD4-negative, HTLV-I-transformed cell lines with HuCD4 significantly enhanced HIV-1 infectivity, suggesting that these lines lack a receptor present on other RbCD4-negative lines that produce high levels of p24 in their native state. Inhibition of HIV-1 infection with soluble HuCD4 was demonstrated for all rabbit lines tested, but complete inhibition was obtained only with a rabbit T-cell line expressing RbCD4 and with HuCD4 transfectants. The results suggest that HIV-1 infection of the RbCD4-positive line proceeds through a receptor similar to HuCD4 but that an additional receptor or receptors may serve this purpose in RbCD4-negative lines.

MATERIALS AND METHODS Cloning and Sequence Determination of RbCD4. A custom cDNA library was constructed in the Uni-Zap XR vector by Stratagene, using thymus RNA from an 8-week-old rabbit. The library was probed with a 675-base-pair fragment amplified by PCR from rabbit thymus cDNA (see Fig. 1A for primer sequences) (17-19). Plaque hybridization was carried out for 18 hr at 45°C using Hybrisol I (Oncor, Gaithersburg, MD) and membranes were washed at 55°C prior to autoradiography. The cDNA was sequenced (20) using Sequenase (United States Biochemical). Data base searches, sequence alignments, and comparisons were carried out using the MacVector program (IBI). Detection ofRbCD4 RNA Transcripts. RNA was isolated by the RNAzol B method (Tel-Test, Friendswood, TX) (21). Northern blot analysis was carried out (14) using the RbCD4 probe as described above. Alternatively, RbCD4 message was detected in tissues and cell lines by PCR amplification of rabbit CD4 cDNA. First, cDNA was synthesized from total rabbit RNA by using the rabbit CD4-specific oligonucleotide 5'-TCATAGTGTTGGGCTTTCCA-3' as reverse primer for first-strand synthesis by reverse transcriptase in 20 ,ul of 1 x PCR buffer (Perkin-Elmer) containing dNTPs and RNase inhibitor. This reaction employed the thermal cycler under the following conditions: 42°C for 15 min, 99°C for 5 min, and 5°C for 5 min. Next, 80 ,ul of the reaction mixture containing the CD4 forward primer 5'-ACAAGAAGATGGAGGTGGAACTGCT-3', water, and Taq polymerase was added to the existing 20 Al of the first-strand reaction. The reaction mixture was then subjected to 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec. Amplified products were analyzed by electrophoresis in 1.5% agarose gel. Rabbit Cell Lines. Cell line RL-5 was obtained by culture of a lymph node following in vivo infection of a rabbit with herpesvirus ateles (22). Other lines were transformed by human T-lymphotropic virus type I (HTLV-I). The human

Infection of human cells with human immunodeficiency virus type 1 (HIV-1) is mediated by the cell surface receptor CD4, which binds with high affinity to the viral envelope glycoprotein gpl20 (1, 2). In HIV-1-infected individuals, depletion of T helper cells, which bear the CD4 molecule on their surface, heralds the onset of severe immunodeficiency and development of AIDS. The interaction of HIV-1 envelope glycoprotein gpl20 with CD4 has been well studied (refs. 3-5; reviewed in ref. 1) and is being exploited in the design of agents intended to block HIV-1 infection by preventing interaction of virus with the CD4 receptor on human cells (6) or by use of toxic substances coupled to soluble CD4 in order to specifically target and destroy cells that express HIV-1 gpl20 (7). Development of CD4-based therapies could be facilitated by the availability of suitable small animal models for AIDS (8, 9). Reported data indicate that the laboratory rabbit supports HIV-1 infection, although no consistent clinical manifestation has been seen in infected animals (10-13). Studies of in vitro HIV-1 infection in rabbit cells (14) have been limited by a lack of stable cell lines and by the paucity of knowledge concerning cell surface markers for T-cell subpopulations in this species (15). Reports of enhanced HIV-1 infection following transfection of rabbit cells with human CD4 (HuCD4) suggest that receptors on certain rabbit cells are not as efficient as HuCD4 (16), but no information concerning the rabbit homolog (RbCD4) of HuCD4 has been reported. The present report describes the structure of RbCD4§ and compares it with those of other species. The infection of

Abbreviations: HuCD4, human CD4; rsHuCD4, recombinant soluble HuCD4; RbCD4, rabbit CD4; HIV, human immunodeficiency virus; HTLV, human T-lymphotropic virus; TCID5o, median tissue culture infective dose (i.e., amount of viral stock that will infect 50%o of target cell cultures). *To whom reprint requests should be addressed. tPresent address: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261. tPresent address: United States Congress, 1414 Longworth, Washington, DC 20515. §The sequence reported in this paper has been deposited in the GenBank data base (accession no. M92840).

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Immunology: Hague et A

HTLV-I-transformed cell line MT-2 (23, 24) was injected into a rabbit and the cell line RHT-16 was derived from peripheral blood mononuclear cells of this rabbit. RH/K34 was derived from rabbit peripheral blood mononuclear cells by coculture with irradiated (104 rads; 1 rad = 0.01 Gy) RHT-16 cells. The cell lines RH/K30, RH/66A through E, and RH/45 were derived in the same manner but with irradiated MT-2 cells as a source of virus. Transfection and Selection of Rabbit Cell Lines. Rabbit cell lines stably expressing human CD4 were constructed by 18 hr of cocultivation of the rabbit cell lines with irradiated (104 rads) DAMP/T4 cells (25). Forty-eight hours after transfection, G418 (Life Technologies, Gaithersburg, MD) was added to 800 ig/ml (active concentration). Fifteen days after transfection, CD4+ cells were selected by magnetic sorting with anti-Leu3a+3b (Becton Dickinson). Flow cytofluorimetric analyses of transfectants and parent lines were carried out with fluorescein-conjugated anti-Leu3a+3b. HIV-1 Infection and Inhibition of Infection. HIV-lBu was grown in A3.01 cells (26) and the median tissue culture infective dose (TCID5o) was determined by titration on phytohemaggutinin-stimulated human peripheral blood mononuclear cells (Applied Biosystems). Rabbit cell lines were infected with HIV-1 by incubating 5 x 106 cells with 1 ml of a 1:1000 dilution of HIV-1 stock (TCID50 = 107/ml). After a 2-hr exposure to virus, cells were then diluted to a concentration of 106 per ml and, after 18 hr, washed extensively to remove free viral particles and suspended at 2 x 105 cells per ml in RPMI 1640 containing 10% fetal bovine serum, 5 mM glutamine, and 25 gg of gentamicin per ml. Culture supernatants were withdrawn and HIV-1 p24 was determined by ELISA (Coulter) on the specified days postinfection. When rsHuCD4 (American Biotechnologies, Cambridge, MA) was used as a potential inhibitor of infection, cells (5 x 106) were incubated with the virus in the presence of rsHuCD4 at 2 or 10 pug/ml for the first 2 hr, and this was diluted to 0.8 or 4 Mg/ml for the next 18 hr. The monoclonal antibodies OKT4 and OKT4a (Ortho Diagnostics) were tested for ability to inhibit infection by incubating 5 x 106 cells with antibody (10 gg/ml) at 40°C for 30 min and then adding the virus.

RESULTS AND DISCUSSION Structure of the RbCD4 Molecule. The fact that rabbit cell lines support infection with HIV-1 (14, 16), along with reports documenting the role of CD4 as the HIV-1 receptor in humans (1, 2), prompted a study of RbCD4. PCR was used to amplify a region of RbCD4 cDNA flanked by sequences identical among those reported for human (17), mouse (18), and rat (19) CD4 molecules. The chosen primer pair (Fig. 1A) amplified a 675-base-pair sequence from rabbit thymus RNA consistent with that predicted from the HuCD4 sequence. The amplified fragment, which corresponds to codons 194419, hybridized with a 3.5-kilobase RNA transcript from thymus and spleen but was unreactive with RNA from liver or kidney. The fragment showed =70%o nucleotide sequence identity with human, rat, and mouse CD4. With the amplified fragment as probe, a rabbit thymus cDNA library yielded a clone corresponding to a full-length transcript fot RbCD4. An open reading frame extends from an initiation sequence at position 66 to a stop codon at position 1443 (Fig. 1A). The mature protein is deduced to begin at position 141 following a leader sequence of 25 amino acids (29). The size of the deduced mature protein is 48 kDa and it has an estimated isoelectric point of 9.5. Analysis of hydrophobicity shows an extended hydrophobic region spanning amino acids 373-396 followed by a markedly hydrophilic sequence. Comparison of RbCD4 hydrophilicity plots with those for CD4 molecules of mouse, cat (28), and human

Proc. Nadl. Acad. Sci. USA 89 (1992)

shows overall conservation in this property (data not shown). The intracytoplasmic serine residues that serve as phosphorylation targets and the p56LCK-binding region (27) are also conserved in RbCD4. Alignment of the deduced RbCD4 amino acid sequence with reported CD4 sequences reveals strong homology but also several unique features. Comparisons with human, mouse, and cat CD4 for domains 1 and 2 (V1 and V2) of CD4 (Fig. 1B) reveal an area of insertion around amino acid 42; a repeat of TCC results in a string of four additional serine residues in RbCD4. These residues, in the second complementarity-determining region (CDR), are numbered 42a-e in order to maintain maximum alignment with HuCD4. Such length differences in the CDRs of immunoglobulin molecules are not unusual, and inserted regions are normally given alphabetical designations as proposed here (30). A second domain points out a second difference between HuCD4 and RbCD4 that involves the disulfide bridge formed between residues 130 and 159 in HuCD4 and mouse CD4 (29). RbCD4 lacks the cysteine at position 130, but a cysteine at position 144 could be involved in a bridge with the cysteine at 159. Cat CD4 also differs in having cysteines at positions 157 and 159 (28). The cat CD4 sequence also contains a large insertion near the beginning of the second (V2) domain. The RbCD4 sequence was next examined for sequence motifs important in binding HIV-1 gpl20 (31-34). Six of the 18 residues identified as critical for the binding of HuCD4 to gpl20 (1) (+ in Fig. 1B) differ between RbCD4 and HuCD4; 2 of these involve conservative substitutions of lysine for arginine. Furthermore, the inserted stretch of serine residues in CDR2 of RbCD4 interrupts one of the major areas (positions 40-50) implicated in gpl20 binding, making it unlikely (5) that RbCD4 would bind gpl20 with an affinity equal to that of HuCD4. The glutamate at position 87 of HuCD4, potentially involved in syncytium formation, is a glycine in rabbit as it is in chimpanzee (34). Rabbit cells do not form syncytia as readily as human cells, but syncytia can be observed in heavily infected rabbit cell cultures. Expression of RbCD4 in Tissues and Cells. Northern blot analyses of RNA from various tissues with a RbCD4 probe showed a 3.4-kilobase transcript in the thymus, spleen, and mesenteric lymph node (data not shown) (5, 35). RNA PCR was used to detect RbCD4 at levels not detectable by Northern blot. By this analysis, peripheral blood mononuclear cells and appendix were found to be positive; bone marrow was negative as were nonlymphoid organs. Brain was found to be negative by this procedure even though a primer pair designed to detect the truncated brain transcript was used (29). A group of rabbit cell lines obtained by transformation with HTLV-I, or in the case of RL-5, Herpesvirus ateles, were tested for expression of RbCD4 by Northern blot analysis. All were negative by this analysis (data not shown). When RNA PCR was used to test for CD4 expression by the lines a strong signal was obtained for RL-5, but all other lines were negative. Infection of Rabbit Cell Lines with HIV-1. A series of rabbit cell lines were infected with HIV-lBr,, and the production of viral gag protein p24 in the culture supernatants was monitored for 7 days. Production of p24 is a more reliable indicator of rabbit cell infection than determination of reverse transcriptase because certain rabbit lines produce a nuclease activity that interferes with the assay for reverse transcriptase (36). One of the HTLV-1 transformants, RH/K30, produced the highest amounts of p24 although no transcripts for RbCD4 were detected for this line (Fig. 2). The one RbCD4-positive line, RL-5, was definitely positive for p24, although its p24 production did not match that of the RbCD4negative line RH/K30. Levels of p24 varied severalfold in replicate experiments and with different viral stocks. When

Immunology: Hague et al. A ~~~~~~~AGCCC AGGGCCCAGAGGCTCAGGTCCTTGAAGTCT AATGAACCGGAGAATCTACTTCCAGTGCCTG CTGCTGGTGCTGCCGCTGGCACTACTCCCA M NR

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Proc. Nati. Acad. Sci. USA 89 (1992) CAGGCCCCTGCCCGCCTCAGCAAAGCCACC GCGGCCACCTGGGGGAAGACCGTGGTGAGA G K

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ATCCTGGGAAACCAGGGCTCCTCCTCCTCC TCCTTCTGGCTTAAAGGGAACTCCCCGCTG AGCAATCGCGTTGAATCCAAAAAAAACATG S N R V E S K K N M S F W L K G N S P L I L G N Q G S S S S

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TGGGACCAAGGATCTTTCCCCCTGGTCATC AAGGATCTCAGGATGGACGACTCGGGGACT TATATCTGCGAAGTGGGGGACAAGAAGATG Y I C E V G D K K N K D L R N D D S G T N D Q G S F P L V I

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GAGGTGGAACTGCTGGTGTTCAGATTGACT GCCAACCCGAACACCCGCCTGCTACATGGA CAGTCACTGACCCTCACCTTGGAAGGCCCC Q S L T L T L E G P A N P N T R L L N G E V E L L V F R L T

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TCTGTGGGGAGCCCCTCCGTGCAATGGAAG AGTCCAGAAAATAAAATCATAGAAACCGGG CCGACTTGCTCCATGCCCAAGCTGAGGCTC P T C S N P K L R L S P E N K I I E T G 126- S V G S P 5 V Q N K

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CAGGACAGTGGCACCTGGTCCTGCCACCTG TCGTTCCAGGACCAGAACAAACTGGAGTTA GACATAAAAATCATAGTGTTGGGCTTTCCA D I K I I V L G F P S F Q D Q N K L E L Q D S G T N S C N L

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CCTGACCTCAAGATCCAGATGAGCAAGGGG CTCCCGCTCAGCCTCACCCTGCCCCAGGCC CTGCATCGCTATGCTGGTTCCGGAAACCTG L N R Y A G S G N L L P L S L T L P Q A P D L K I Q M S K G

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AGCCTGACCCTTGATAAGGGAAAGTTGCAT CAGCAAGTGAGCCTGGTGATGCTGAAAGTG ACTCAGGTAAAGAACAAGTTGACTTGTGAG 1055 T Q V K N K L T C E Q Q V S L V M L K V 5 L T L D K G K L N

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GTGCTGGGACCCATTGACCCCAAGATGAAG CTGAGCTTGAAGCTGGAGGACCAGGAAGCA AAGGTCTCCACCCAGAAGATGGTGCAGGTG 1145 K V S T Q K N V Q V L S L K L E D Q E A V L G P I D P K N K

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