Identification of Replication-Competent Strains of Simian Immunodeficiency Virus Lacking Multiple Attachment Sites for N-Linked Carbohydrates in Variable Regions 1 and 2 of the Surface Envelope Protein

Site-specific mutagenesis and subcloning.

To reduce the size of the plasmids to be mutated, a SphI-ClaI fragment of the proviral SIVmac239 DNA containing 1469 nucleotides of env coding sequence (proviral nucleotides 6450 to 8073 in the numbering of Regier and Desrosiers [29]) was subcloned into pSP72 (Promega), resulting in pSP72SC. The SacI-EcoRI fragment containing the 3′ 1,050 bases of the proviral genome was subcloned into pSP72 to create pSP72SE. Mutations of env were created by recombinant PCR mutagenesis (9). Carbohydrates were numbered according to their order of appearance within the envelope sequence of SIVmac239. The following mutagenic primers were used: for g4, (6931 to 6970) 5′-ACTATGAGATGCCAGAAAAGTGAGACAGATAGATGGGGAT-3′ and (6957 to 6919) 5′-TGTCTCACTTTTCTGGCATCTCATAGTAATGCATAATGG-3′; for g5, (7027 to 7068) 5′-GTAGACATGGTCCAGGAGACTAGTTCTTGTATAGCCCAGGAT-3′ and (7053 to 7014) 5′-AGAACTAGTCTCCTGGACCATGTCTACTTTTGCTGATGCT-3′; for g6, (7057 to 7097) 5′-ATAGCCCAGGATCAATGCACAGGCTTGGAACAAGAGCAAAT-3′ and (7084 to 7045) 5′-CCAAGCCTGTGCATTGATCCTGGGCTATACAAGAACTAGT-3′; for M144V, (7026 to 7062) 5′-AGTAGACGTGGTCAATGAGACTAGTTCTTGTATAGCC-3′ and (7045 to 7010) 5′-GTCTCATTGACCACGTCTACTTTTGCTGATGCTGTCG-3′; for g456 (M144V), (7022 to 7055) 5′-CAAAAGTAGACGTGGTCCAGGAGACTAGTTCTTG-3′ and (7042 to 7009) 5′-CCTGGACCACGTCTACTTTTGCTGATGCTGTCGT-3′; for g7, (7103 to 7137) 5′-GCTGTAAATTCCAGATGACAGGGTTAAAAAGAGAC-3′ and (7126 to 7092) 5′-ACCCTGTCATCTGGAATTTACAGCTTATCATTTGC-3′; for g8, (7145 to 7180) 5′-AAGAGTACCAGGAAACTTGGTACTCTGCAGATTTGG-3′ and (7164 to 7130) 5′-CCAAGTTTCCTGGTACTCTTTTTTCTTGTCTCTTTT-3′; for g9, (7186 to 7220) 5′-GAACAAGGGCAGAACACTGGTAATGAAAGTAGATG-3′ and (7206 to 7171) 5′-ACCAGTGTTCTGCCCTTGTTCACATACCAAATCTGC-3′; for g10, (7198 to 7231) 5′-AACACTGGTCAGGAAAGTAGATGTTACATGAACC-3′ and (7218 to 7184) 5′-TCTACTTTCCTGACCAGTGTTATTCCCTTGTTCAC-3′; for g11, (7228 to 7264) 5′-ACCACTGTCAGACTTCTGTTATCCAAGAGTCTTGTG-3′ and (7249 to 7213) 5′-TAACAGAAGTCTGACAGTGGTTCATGTAACATCTACT-3′; and for g12 and g13, (7228 to 7364) 5′-GATGTCAGGACACACAATATTCAGGCTTTATGCCTAA-3′ and (7349 to 7313) 5′-GAATATTGTGTGTCCTGACATCTAAGCAAAGCATAAC-3′. The primers were synthesized on a Cyclone DNA synthesizer (Biosearch, Inc.) or purchased from Genosys Biotechnologies, Inc. (Woodlands, Texas). The SphI-ClaI fragment containing the mutated env sequence was excised and subcloned into the 3′ parental clone, pSP72-239-3′ (15). For transient gene expression, the wild-type envelope sequence was subcloned into the unique XhoI and BamHI sites of the expression vector pSVL (Pharmacia) after creation of a BamHI site 3′ of the env coding sequence by using the mutagenic primers 27 (9268 to 9302) 5′-GTATATGAAGGATCCATGGAGAAACCCAGCTGAAG-3′ and 28 (9286 to 9253) 5′-CCATGGATCCTTCATATACTGTCCCTGATTGTAT-3′. The mutant envelope sequences were subcloned into the resultant pSVLenv via the unique XhoI and ClaI sites.

DNA transfection of cultured cells.

For viral stocks, the 5′ and 3′ clones of SIVmac239 were digested with SphI and heated to 65°C for 15 min. Each right-half clone was ligated with the left-half clone p239SpSp5′ by using T4 DNA ligase. A 3-μg portion of the ligated DNA was used to transfect CEMx174 cells treated with DEAE-dextran (25). The vector pSVL, which expresses the simian virus 40 late promoter, was used for transient transfection of the wild-type and mutant env genes. A 1-μg portion of DNA was combined with DEAE-dextran, and transfection of COS-1 cells at 80% confluence in 35-mm diameter plates (Falcon Primaria) was performed by the procedure of Levesque et al. (19).

Virus stocks and cell culture.

Rhesus monkey peripheral blood mononuclear cells (PBMC), CEMx174, 221, and COS-1 cells were maintained as described previously (21, 23). For virus stocks, CEMx174 cells were transfected as described above. The medium was changed every 2 days, and the supernatants were harvested at or near the peak of virus production. Cells and debris were removed by centrifugation, and virus contained in the supernatant was aliquoted and stored at −70°C. The concentration of p27 antigen was measured by an antigen capture assay (Coulter Corp., Hialeah, Fla.). For virus infections, 5 ng of p27 was used to infect 2.5 million pelleted cells.

DNA sequencing and PCR amplification.

Cloned fragments containing mutated envelope DNA were sequenced in their entirety on an ABI377 automated DNA sequencer by using dye terminator cycle-sequencing chemistry as specified by the manufacturer (Perkin-Elmer Inc., Foster City, Calif.). Total genomic DNA was isolated with the AmpPrep kit (HRI Research, Inc., Concord, Calif.) and used as a template for nested PCR amplification with primers located outside of the viral env sequence. The outer primers were 39 (6320 to 6348) 5′-GAGGGAGCAGGAGAACTCATTAGAATCCTCC-3′ and 40 (9373 to 9405) 5′-GTTCTTAGGGGAACTTTTGGCCTCACTGATACC-3′. The inner mutagenic primers created XhoI and BamHI sites used for cloning the PCR products into pSP72 and were 38 (6423 to 6465) 5′-CTCAGCTATACCGCCCTCGAGAAGCATGCTATAAC-3′ and 32 (9256 to 9288) 5′-CTCCATGGATCCTTCATATACTGTCCCTGATTG-3′. Each 100-μl reaction mix contained 1 μg of total DNA, 2 mM Mg²⁺, 200 μM each deoxynucleoside triphosphate, 0.2 μM each primer, and 2 U of Vent polymerase (New England Biolabs, Beverly, Mass.), and the mixtures were amplified for 30 cycles. Each cycle consisted of denaturation at 93°C for 1 min, annealing at 50°C for 1 min, and elongation at 72°C for 3 min 15 s, ending with a 10-min final extension at 72°C.

Immunoblotting and CD4 binding.

For Western analysis, transfected COS-1 cells were rinsed three times with phosphate-buffered saline (PBS) and lysed in 0.5 ml of lysis buffer (1% Triton X-100, 0.5% sodium deoxycholate, 10 mM NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl [pH 7.4], 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Pefabloc, 1 mg of iodoacetamide). A 20-μl volume of extract was mixed with an equal amount of sample buffer and boiled for 4 min. Following electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore) and incubated sequentially with a rhesus polyclonal antibody generated against SIVmac239 followed by horseradish peroxidase-conjugated anti-rhesus immunoglobulin G (Southern Biotechnology Associates). The membranes were reacted with a chemiluminescent substrate (ECL reagents; Amersham) and placed against film (Kodak BioMax) for 5 to 200 s. For metabolic labeling, COS-1 cells were transfected and then cultured for 3 days. The cells were starved for 30 min by replacing the culture medium with labeling medium (minimum essential medium without methionine or cysteine but with 10% dialyzed fetal calf serum). The cold medium was replaced with 1 ml of the same medium containing 100 μCi of [³⁵S]methionine and [³⁵S]cysteine (Dupont NEN, Boston, Mass.), and the cells were returned to culture at 37°C. The length of the labeling period varied depending on the assay and is indicated in the figure legends. At the end of the labeling period, the cells were washed twice in PBS and lysed in 0.5 ml of lysis buffer. All the lysates were frozen at −20°C, thawed, and vortexed vigorously, and the cell debris was pelleted by centrifugation for 5 min. For CD4 binding assays, 100 μl of each lysate was incubated with either PBS or 250 ng of soluble CD4 as described previously (23). For the shedding assay (see Fig. 9), the transfected cells were cultured for 3 days. The culture medium was replaced with labeling medium, and the cells were metabolically labeled for 44 h. The assay was repeated twice with shorter labeling times of 18 and 30 h and gave similar results. For immunoprecipitations, after the labeling period, supernatants were clarified by centrifugation for 5 min and the labeled proteins in the supernatants or 100 μl of lysates were immunoprecipitated with serum obtained from a rhesus macaque infected with SIVmac239. For the pulse-chase experiment (see Fig. 10), the entire extract was immunoprecipitated. The immune complexes were precipitated with protein A-agarose (Santa Cruz), washed, and resolved on a 10% polyacrylamide–sodium dodecyl sulfate (SDS) gel.

N-Glycosidase F digestions.

Immunoprecipitated proteins were denatured by boiling for 2 min in 20 μl of 0.1% SDS–1% 2-mercaptoethanol–20 mM sodium phosphate buffer (pH 8). The sample volume was brought up to 80 μl in 1% Triton–20 mM sodium phosphate buffer–10 mM EDTA. The samples were split into aliquots, and either 2 U of N-glycosidase F (Boehringer) in a volume of 10 μl was added or, for mock digestions, 10 μl of sodium phosphate buffer was added. The samples were incubated at 37°C for 20 h, dried, and resuspended in 30 μl of sample buffer containing 5% 2-mercaptoethanol.

Replication of mutant viruses involving fourth, fifth, and sixth sites.

The fourth, fifth, and sixth sites containing Asn-X-Ser/Thr in the gp120 sequence of SIVmac239 were initially selected for mutagenesis (Fig. 1). These sites are located in the vicinity of the highly variable region 1 but nonetheless are strongly conserved among SIV sequences in the database (24). The Asn codon at all three sites of SIVmac239 is AAT. The AAT codon at sites 4 and 5 was changed to CAG (Gln), and at site 6 it was changed to CAA (Gln). Glutamine is structurally similar to asparagine, differing only by a single CH₂ group. Since only AAT and AAC can code for Asn, two nucleotide changes would be required for the codon to revert to an Asn codon. All seven possible mutant forms of these sites were created. These are referred to as g4, g5, g6, g45, g46, g56, and g456.

All six single and double mutants (g4, g5, g6, g45, g46, and g56) replicated similarly to the parental virus upon transfection of cloned DNA into CEMx174 cells (data not shown). Normalized amounts of mutant and parental virus stocks produced from CEMx174 transfection were used to analyze viral replication in CEMx174 cells, the rhesus monkey 221 cell line, and primary rhesus monkey PBMC cultures. Again, all single- and double-mutant forms of the virus replicated similarly to the parental virus in CEMx174 cells (Fig. 2A and B), 221 cells (data not shown), and stimulated rhesus monkey PBMC cultures (Fig. 2C). Slight delays or differences in peak heights were observed with the mutants in some experiments, but it is uncertain whether these represent a significant difference.

Utilization of sites.

Utilization of the fourth, fifth, and sixth potential sites was investigated by examining the mobility of the proteins in SDS-polyacrylamide gels. Vectors for transient expression of the wild-type and all mutant env sequences were constructed and transfected into COS-1 cells. Only slight size differences were apparent when the gp160 molecules of the panel of labeled envelope proteins were immunoprecipitated and resolved by gel electrophoresis (Fig. 3A, lanes 10 to 18). When N-glycans were removed by treatment with N-glycosidase F, all precursor molecules comigrated, as expected, at approximately 110 kDa, indicating that any size differences between the envelope proteins were due entirely to differences in N-linked glycosylation (lanes 1 to 9). To enhance the detection of size differences among the mutant envelope proteins, a smaller fragment of the envelope protein was generated by acid hydrolysis. Labeled proteins were immunoprecipitated from transfected COS-1 cells and treated with dilute acetic acid. This treatment hydrolyzes proteins between the aspartic acid and proline residues at positions 385 and 740 of the SIV gp160, resulting in fragments of 385, 355, and 140 residues. The first 385 amino acids of gp160 contain 20 signals for N-linked carbohydrates, and each glycan adds approximately 2,500 Da to the relative mass of the fragment (16). Thus, this 385-aa fragment of the wild-type protein is predicted to migrate at approximately 91 kDa. Protein fragments representing these amino-terminal 385 residues of the wild-type and mutant envelope proteins are shown in Fig. 3B. The three single mutants showed an increase in mobility of approximately 3 kDa with respect to the wild-type protein which migrated, as expected, between 90 and 92 kDa (Fig. 3B, lanes 2 to 5). The double mutants showed an additional increase in mobility (lanes 6 to 8), while the triple mutant, g456, had a further increase in mobility (lane 9). These results are consistent with utilization of each of the fourth, fifth, and sixth N-linked glycosylation sites.

Identification of a revertant of the triple mutant.

In contrast to the results with the single and double mutants, replication of the triple mutant (g456) was undetectable after transfection of CEMx174 and 221 cells (data not shown). In one CEMx174 culture transfected with g456 viral DNA, detectable virus began to appear beyond 40 days after transfection (Fig. 4A). When virus from day 63 of this transfection was used to infect CEMx174 cells, the virus replicated with only a slight delay compared to the parental virus (Fig. 4B). These findings suggested that reversions or compensatory changes had appeared in the culture to allow wild-type or near-wild-type levels of viral replication.

Sequence analysis of viral DNA derived from CEMx174 cells infected with the g456 revertant revealed a single predominant change of Met to Val at position 144 (Fig. 5). This position is located 2 amino acids upstream of the mutated fifth N-linked site. No changes were observed in the fourth, fifth, and sixth QXS/T sites themselves (Fig. 5). We introduced the Val-to-Met change into the parental SIV239 DNA and into the g456 mutant in the absence of any other changes. Virus containing the M144V change in the 239 background replicated similarly to the parental SIVmac239 upon transfection (data not shown) and infection of both the CEMx174 and 221 cell lines (Fig. 6A and B). Virus containing the M144V change in the g456 background replicated with only a slight delay compared to SIVmac239 upon both transfection (data not shown) and infection of both CEMx174 and 221 cells (Fig. 6A and B). The virus with the M144V mutation in the g456 background replicated with kinetics similar to that of the revertant recovered from the original transfection (Fig. 4B). Thus, the change of Met to Val at position 144 is able to compensate for the loss of the fourth, fifth, and sixth NXS/T sites. However, replication of the g456MV variant was severely impaired after infection of stimulated rhesus monkey PBMC (Fig. 6C).

Nature of the block to replication.

We performed experiments to evaluate both the nature of the block to replication with the g456 mutant and the way in which the M-to-V substitution may relieve this block. Vectors for transient expression of wild-type, g456, and g456MV envelope proteins were transfected into COS-1 cells. Envelope protein expression, processing, and size were evaluated by Western blot analysis of cell extracts on days 2, 3, 4, and 5 after transfection (Fig. 7). The parental clone yielded both the gp160 precursor and the proteolytically processed gp120 external surface subunit as expected during the 2- to 5-day period that was examined (Fig. 7). Both the g456 and g456MV mutants yielded a precursor that migrated faster than the gp160 precursor of the parental virus (Fig. 7). Less processed “gp120” was observed in cells expressing the g456 mutant than in cells expressing the parental envelope, while g456MV expressed intermediate levels (Fig. 7).

We next assessed the ability of the g456 envelope protein to bind CD4 receptor by measuring binding to sCD4. Transfected COS-1 cells were cultured for 3 days and then labeled with [³⁵S] methionine and [³⁵S]cysteine for 24 h. Labeled envelope protein in the cell lysate was tested for its ability to bind to soluble CD4. CD4-env protein complexes were immunoprecipitated with the OKT4 anti-CD4 monoclonal antibody, whose reactivity is not blocked by the CD4-env interaction. Under the conditions used for this assay, both mutant envelope proteins were able to bind to the soluble CD4 (Fig. 8). We have shown previously that the gp160 precursor of SIVmac239 is capable of binding to soluble CD4 (23).

Since only a small amount of g456 gp120 equivalent was detected in cells both by Western blot analysis and immunoprecipitation, loss of “gp120” by release into the medium was evaluated by labeling transfected COS-1 cells with [³⁵S]methionine and [³⁵S]cysteine and immunoprecipitating “gp120” from the cell-free supernatants. “gp120” was detected in the supernatant in all cases (Fig. 9). Upon two repetitions of the assay, the g456 virus consistently released higher levels of gp120 than did the wild-type. Processing of “gp160” precursor and loss of “gp120” by release into the medium was also evaluated by a pulse-chase experiment (Fig. 10). The results of these assays were consistent with those of the other methods, suggesting that there were decreased amounts of processed “gp120” of g456 in cells and increased amounts of “gp120” released into the media.

Replication of additional mutants.

To examine the importance for replication of other sites for N-linked carbohydrates around the V1 and V2 regions, additional asparagine codons were replaced with the codon for glutamine. Mutants were created that lacked various combinations of the 4th through 13th glycosylation sites. Alteration of the 8th or 10th site or both the 12th and 13th sites for N-linked glycosylation had no effect on viral replication in CEMx174 cells (data not shown). Viral replication was not significantly affected when three consecutive or nonconsecutive potential glycosylation sites were altered (Fig. 11A and C). Furthermore, multiple combinations of env mutants lacking four or five glycosylation sites were also replication competent in CEMx174 cells (Fig. 11B) as well as 221 cells (not shown) and stimulated rhesus PBMCs (Fig. 11D). Thus, elimination of multiple combinations of carbohydrate attachment sites between g4 and g13 yielded virus that was still replication competent.