Mice, bacteria, and viruses.
Four-week-old C57BL/6 (B6) and perforin-deficient B6 (PKO) (9) mice were obtained from the National Cancer Institute and the Jackson Laboratory, respectively. rLm strains expressing the LCMV CD8+ (H-2Db) epitope gp33-41 (KAVYNFATC) or the (H-2Ld) epitope np118-126 (RPQASGVYM) were engineered as described previously (29); both epitopes are expressed as fusion proteins with dihydrofolate reductase. The strain expressing gp33-41 is called XFL703 and will be referred to here as rLm-gp33 (28). The strain expressing np118-126 is called XFL303 and will be referred to here as rLm-np118 (29). A recombinant MHV strain (SA59REGFP) expressing EGFP was derived from strain MHV-A59 by targeted recombination of the EGFP gene (catalogue no. 6085-1; Clontech) in place of the gene 4 sequence and has been described previously (5); for simplicity we will refer to this strain here as RA59-gfp (4, 8). Selection of the RA59-gfp/gp33, recombinant MHV-A59 expressing the LCMV gp33 epitope as a fusion protein with GFP is described in the next paragraph. fMHV (obtained from Paul Masters, New York State Department of Health, Albany) is a recombinant MHV-A59 which contains the ectodomain of the feline infectious peritonitis virus spike glycoprotein in place of that of the MHV spike (11). MHV stocks were prepared in 17Cl-1 cells and titrated by plaque assay in murine L2 cells (6).
Selection of RA59-gfp/gp33.
Complementary synthetic oligonucleotides (63 bases) encoding the first 9 amino acids of MHV open reading frame 4a (ORF4a) (including the initial AUG codon) followed by the 9 amino acids of gp33 were synthesized such that, when hybridized, the 5′ and 3′ ends of the double-stranded fragment contained sequences complementary to SalI- and BamHI-cleaved DNA ends, respectively (Table (Table1).1). The double-stranded fragment was ligated into SalI/BamHI-cleaved pEGFP (Clontech vector containing the EGFP gene) such that the ORF4a/gp33 fragment was adjacent to and in frame with the EGFP gene. The ORF4a/gp33/EGFP portion of the plasmids was sequenced using primers EGFP 530+ and EGFP 766− (Table (Table1)1) and the Taq dye terminator procedure according to the manufacturer's protocol (Taq DyeDeoxy Terminator Cycle Sequencing kit; Applied Biosystems). The fragment encoding ORF4a/gp33/EGFP was cleaved from this plasmid with SalI and NotI and inserted into pMH54-EGFP (5) to replace most of gene 4. The plasmid pMH54 and its use in targeted recombination have been described previously (11, 27). Briefly, pMH54 contains a T7 RNA polymerase promoter followed by a 9,139-nucleotide sequence (11); pMH54 encodes the 5′ end of the MHV genome fused in frame to codon 28 of the hemagglutinin esterase pseudogene, followed by the spike and the rest of the 3′ end of the MHV-A59 genome and finally a poly(A) tail. In the final pMH54-gp33/EGFPconstruct, the ATG for ORF4a is 50 nucleotides downstream of the gene 3/gene4 intergenic sequence. This is followed by the nine codons of ORF4a, the nine codons of gp33, seven intervening codons followed by the ATG for the EGFP gene, and the rest of the EGFP gene, such that ORF4a, gp33, and the EGFP protein are all in frame (Fig. (Fig.1).1). Recombination was carried out in feline FCWF cells between the donor RNA (transcribed from pMH54-containing the gp33/EGFP sequences) and the recipient virus, fMHV. The recombinant viruses were selected by two rounds of plaque purification on L2 cells (11, 27). Recombinant virus genomes were amplified and sequenced in the regions that were mutagenized, that is, from the 3′ end of the spike gene into the 5′ end of the EGFP coding region. Reverse transcriptase-mediated PCR (RT-PCR) amplification was carried out, using as templates cytoplasmic RNA extracted from virus-infected L2 cells and the primers FIJ81 and RIJ84 (Table (Table11).
FIG. 1.
FIG. 1.
Recombinant viruses encoding EGFP and gp33. A schematic diagram of the genome of MHV-A59 is shown. Targeted recombination was used to replace gene 4 of MHV-A59 with the EGFP gene (RA59-gfp) or with a fragment of ORF4a, followed by gp33 and the EGFP gene ...
TABLE 1.
TABLE 1.
Oligonucleotides used for cloning and sequencing
Inoculation of mice.
For immunization with rLm-gp33 or rLm-np118, mice were inoculated intraperitoneally (i.p.) with 104 CFU of rLm in 0.5 ml of phosphate-buffered saline (PBS). For i.c. inoculation of virus, mice were anesthetized with isoflurane and the amount of virus designated in each experiment was diluted in PBS containing 0.75% bovine serum albumin, and a total volume of 20 μl was injected into the left cerebral hemisphere. For i.p. inoculation, virus in PBS containing 0.75% bovine serum albumin, and a total volume of 500 μl was injected. Mock-infected controls were inoculated similarly but with an uninfected cell lysate at a comparable dilution.
Virus replication in mice.
For the measurement of virus replication in the brain and liver, at 5 days postinfection, mice were sacrificed and perfused with 10 ml of PBS, and brains and livers were removed. The left half of the brain and the liver were placed directly into 2 ml of isotonic saline with 0.167% gelatin (gel saline). The right half of the brain was used for histology and viral antigen staining as described below. All organs were weighed and stored frozen at −80°C until determination of virus titers. Organs were homogenized, and virus titers were determined by plaque assay on L2 cell monolayers (6).
Histology and immunohistochemistry.
For the analysis of inflammation and viral antigen expression, the right half of the brain harvested at 5 days postinfection (see above), was fixed in formalin overnight. Formalin-fixed tissue was embedded in paraffin and sectioned sagittally. Immunohistochemical analysis was performed by the avidin-biotin-immunoperoxidase technique (Vector Laboratories, Burlingame, Calif.) using 3,3′-diaminobenzidine as substrate and a 1:20 dilution of a monoclonal antibody (MAb) directed against the nucleocapsid protein (N) of MHV-JHM (MAb clone 1-16-1, kindly provided by Julian Leibowitz, Texas A & M University). Control slides from mice either uninfected or inoculated with rLm-gp33 but not with virus were incubated in parallel. All slides were read in a blinded manner.
Isolation and analysis of lymphocytes. (i) Isolation of mononuclear cells from the spleen and CNS.
Spleens were harvested from mice either at 5 or 7 days postinfection. Spleen cell suspensions were prepared by gently homogenizing spleens in a nylon bag (mesh opening, 64 μm) with a syringe plunger in RPMI 1640 medium supplemented with 1% fetal calf serum. Then, red blood cells were lysed with 0.83% NH4Cl. Mononuclear cells were prepared from the CNS as previously described by Phillips et al. (25) on day 7 after i.c. inoculation. Three to five brains were pooled per sample. In brief, animals were sacrificed and perfused with 10 ml of PBS, and a single-cell suspension from the brain was obtained by passing cells through a nylon mesh bag (mesh opening, 64 μm). Percoll (Pharmacia) was added to a final concentration of 30%, and the lysate was centrifuged at 1,300 × g for 30 min at 4°C. The cell pellet was resuspended, passed through a cell strainer (pore diameter, 70 μm; Becton Dickinson), and washed. The cells were then layered over 2 ml of Lympholyte-M (Cedarlane Laboratories) and centrifuged at 1,300 × g for 20 min at room temperature. Cells were removed from the interface, washed once, and counted.
(ii) Intracellular IFN-γ staining.
Intracellular IFN-γ expression in response to peptide stimulation was performed as previously described (19, 25). A total of 106 brain-derived monocytes or splenocytes per well were cultured for 5 h at 37°C in 200 μl of RPMI 1640 medium, supplemented with 5% fetal calf serum, 10 U of human recombinant interleukin-2, and brefeldin A (Golgiplug [1 μl/ml]; PharMingen) either with or without peptides. The peptides were used at a concentration of 1 μg/ml. Cells were then stained as described above for surface expression of CD8 and incubated overnight at 4°C. For intracellular IFN-γ staining, cells were then fixed and permeabilized using the Cytofix/Cytosperm kit (PharMingen) and stained with a fluorescein isothiocyanate-conjugated monoclonal rat anti-mouse IFN-γ antibody (clone XMG 1.2; PharMingen). Cells were washed and then suspended in PBS containing 2% paraformaldehyde and analyzed by FACSan flow cytometry (Becton-Dickinson).
Isolation of epitope escape mutants.
Viruses were plaqued from brain homogenates of infected animals sacrificed at 5 days postinfection as described above. Plaques were circled on the six-well culture dishes and then visualized by fluorescent microscopy. The number of green plaques was counted, and the percentage of fluorescent plaques was calculated. A selection of fluorescent and nonfluorescent plaques were picked, and viruses isolated from these plaques were used to infect L2 cells. Intracellular RNA from such infections was isolated. RT-PCR was used to amplify the region encoding gp33/gfp using primers FIJ81 and RIJ84 (Table (Table1).1). These amplified fragments were analyzed by agarose gel electrophoresis and in some cases sequenced using the same primers.
หนู แบคทีเรีย และไวรัสอายุ 4 สัปดาห์ C57BL/6 (B6) และหนูไม่ perforin B6 (PKO) (9) ได้รับจาก สถาบันมะเร็งแห่งชาติและห้องปฏิบัติการ Jackson ตามลำดับ สายพันธุ์ rLm แสดง CD8 LCMV + (H-2Db) epitope gp33-41 (KAVYNFATC) หรือแบบ (H 2Ld) epitope np118-126 (RPQASGVYM) ถูกออกแบบทางวิศวกรรมตามที่อธิบายไว้ก่อนหน้านี้ (29); ทั้ง epitopes ที่แสดงเป็นโปรตีนฟิวชั่นกับ dihydrofolate reductase สายพันธุ์ gp33-41 แสดงเรียกว่า XFL703 และจะถูกเรียกที่นี่ว่า rLm-gp33 (28) สายพันธุ์ที่แสดง np118 126 เรียกว่า XFL303 และจะถูกเรียกที่นี่ว่า rLm-np118 (29) Recombinant MHV ต้องใช้ (SA59REGFP) แสดง EGFP รับมาจากต้องใช้ MHV A59 โดย recombination เป้าหมายของยีน EGFP (หมายเลขแคตตาล็อก 6085-1 Clontech) แทนลำดับยีน 4 และมีการอธิบายไว้ก่อนหน้านี้ (5); ราย เราจะอ้างถึงนี้ต้องใช้ที่นี่เป็น RA59-gfp (4, 8) ตัวเลือก RA59-gfp/gp33, recombinant MHV-A59 แสดง LCMV gp33 epitope เป็นโปรตีนฟิวชั่นมี GFP ได้อธิบายไว้ในย่อหน้าถัดไป fMHV (ได้รับจาก Paul ต้นแบบ นิวยอร์กรัฐกรมสุขภาพ อัลบานี) เป็นแบบ recombinant MHV-A59 ที่ประกอบด้วย ectodomain ของ peritonitis แมวติดเชื้อไวรัสชั่วไกลโคโปรตีนแทนของสไปค์ MHV (11) หุ้น MHV ถูกเตรียมใน 17Cl-1 เซลล์ และ titrated โดยทดสอบหินปูนในเซลล์ L2 murine (6)เลือก RA59-gfp/gp33Complementary synthetic oligonucleotides (63 bases) encoding the first 9 amino acids of MHV open reading frame 4a (ORF4a) (including the initial AUG codon) followed by the 9 amino acids of gp33 were synthesized such that, when hybridized, the 5′ and 3′ ends of the double-stranded fragment contained sequences complementary to SalI- and BamHI-cleaved DNA ends, respectively (Table (Table1).1). The double-stranded fragment was ligated into SalI/BamHI-cleaved pEGFP (Clontech vector containing the EGFP gene) such that the ORF4a/gp33 fragment was adjacent to and in frame with the EGFP gene. The ORF4a/gp33/EGFP portion of the plasmids was sequenced using primers EGFP 530+ and EGFP 766− (Table (Table1)1) and the Taq dye terminator procedure according to the manufacturer's protocol (Taq DyeDeoxy Terminator Cycle Sequencing kit; Applied Biosystems). The fragment encoding ORF4a/gp33/EGFP was cleaved from this plasmid with SalI and NotI and inserted into pMH54-EGFP (5) to replace most of gene 4. The plasmid pMH54 and its use in targeted recombination have been described previously (11, 27). Briefly, pMH54 contains a T7 RNA polymerase promoter followed by a 9,139-nucleotide sequence (11); pMH54 encodes the 5′ end of the MHV genome fused in frame to codon 28 of the hemagglutinin esterase pseudogene, followed by the spike and the rest of the 3′ end of the MHV-A59 genome and finally a poly(A) tail. In the final pMH54-gp33/EGFPconstruct, the ATG for ORF4a is 50 nucleotides downstream of the gene 3/gene4 intergenic sequence. This is followed by the nine codons of ORF4a, the nine codons of gp33, seven intervening codons followed by the ATG for the EGFP gene, and the rest of the EGFP gene, such that ORF4a, gp33, and the EGFP protein are all in frame (Fig. (Fig.1).1). Recombination was carried out in feline FCWF cells between the donor RNA (transcribed from pMH54-containing the gp33/EGFP sequences) and the recipient virus, fMHV. The recombinant viruses were selected by two rounds of plaque purification on L2 cells (11, 27). Recombinant virus genomes were amplified and sequenced in the regions that were mutagenized, that is, from the 3′ end of the spike gene into the 5′ end of the EGFP coding region. Reverse transcriptase-mediated PCR (RT-PCR) amplification was carried out, using as templates cytoplasmic RNA extracted from virus-infected L2 cells and the primers FIJ81 and RIJ84 (Table (Table11).FIG. 1FIG. 1ไวรัส recombinant รหัส EGFP และ gp33 เป็นแสดงไดอะแกรมแผนผังวงจรของจีโนมของ MHV A59 Recombination เป้าหมายใช้การแทนยีน 4 ของ MHV A59 กับยีน EGFP (RA59-gfp) หรือชิ้นส่วนของ ORF4a ตาม ด้วย gp33 และยีน EGFP ...ตารางที่ 1ตารางที่ 1Oligonucleotides ที่ใช้สำหรับการโคลนและการจัดลำดับInoculation ของหนูการรับวัคซีน rLm-gp33 หรือ rLm np118 หนูได้ inoculated intraperitoneally (i.p.) กับ 104 CFU ของ rLm ใน 0.5 ml ของ buffered ฟอสเฟตน้ำเกลือ (PBS) สำหรับซี inoculation ไวรัส หนูถูกด้วยกับ isoflurane และจำนวนไวรัสที่กำหนดในแต่ละการทดลองถูกทำให้เจือจางใน PBS ประกอบด้วย 0.75% วัว serum albumin และปริมาตรรวมของ 20 μl ถูกฉีดเข้าไปในซีกโลก cerebral ซ้าย สำหรับ i.p. inoculation ถูกฉีดไวรัสใน PBS 0.75% วัว serum albumin และปริมาตรรวมของ 500 μl ควบคุมการติดเชื้อ mock ถูก inoculated ในทำนองเดียวกัน แต่ มีเซลล์ที่เชื้อ lysate ที่เจือจางสามารถเปรียบเทียบจำลองแบบไวรัสในหนูFor the measurement of virus replication in the brain and liver, at 5 days postinfection, mice were sacrificed and perfused with 10 ml of PBS, and brains and livers were removed. The left half of the brain and the liver were placed directly into 2 ml of isotonic saline with 0.167% gelatin (gel saline). The right half of the brain was used for histology and viral antigen staining as described below. All organs were weighed and stored frozen at −80°C until determination of virus titers. Organs were homogenized, and virus titers were determined by plaque assay on L2 cell monolayers (6).Histology and immunohistochemistry.For the analysis of inflammation and viral antigen expression, the right half of the brain harvested at 5 days postinfection (see above), was fixed in formalin overnight. Formalin-fixed tissue was embedded in paraffin and sectioned sagittally. Immunohistochemical analysis was performed by the avidin-biotin-immunoperoxidase technique (Vector Laboratories, Burlingame, Calif.) using 3,3′-diaminobenzidine as substrate and a 1:20 dilution of a monoclonal antibody (MAb) directed against the nucleocapsid protein (N) of MHV-JHM (MAb clone 1-16-1, kindly provided by Julian Leibowitz, Texas A & M University). Control slides from mice either uninfected or inoculated with rLm-gp33 but not with virus were incubated in parallel. All slides were read in a blinded manner.แยกและวิเคราะห์ของ lymphocytes (i) แยกเซลล์ mononuclear จากม้ามและ CNSSpleens ถูกเก็บเกี่ยวจากหนูทั้ง 5 หรือ 7 วัน postinfection บริการเซลล์ม้ามถูกเตรียม โดยเบา ๆ homogenizing spleens ในถุงไนลอน (ตาข่ายเปิด 64 μm) กับจมูกเข็มใน RPMI 1640 เสริม ด้วยเซรั่มลูกครรภ์ 1% แล้ว เซลล์เม็ดเลือดแดงได้ lysed 0.83% NH4Cl เซลล์ Mononuclear ถูกเตรียมจาก CNS ก่อนหน้านี้อธิบายไว้โดยไขควง et al. (25) ในวันที่ 7 หลังจาก inoculation ซี สมองสามถึงห้าถูกทางถูกพูต่อตัวอย่าง สังเขป สัตว์เสียสละ และ perfused กับ 10 ml ของ PBS และระงับเซลล์เดียวจากสมองได้รับ โดยผ่านเซลล์ถุงตาข่ายไนล่อน (ตาข่ายเปิด 64 μm) Percoll (Pharmacia) ถูกเพิ่มความเข้มข้นสุดท้าย 30% และที่ lysate ถูก centrifuged ที่ 1300 × g ใน 30 นาทีที่ 4 องศาเซลเซียส เม็ดเซลล์ถูก resuspended ผ่านเครื่องร่อนเซลล์ (เส้นผ่าศูนย์กลางของรูขุมขน 70 μm Becton สัน), และเครื่องซักผ้า เซลล์ได้แล้วชั้น Lympholyte-M (ห้อง Cedarlane) กว่า 2 ml และ centrifuged ที่ 1300 × g สำหรับ 20 นาทีที่อุณหภูมิห้อง เซลล์ถูกเอาออกจากอินเทอร์เฟซ ล้างครั้งเดียว และนับ(ii) intracellular IFN-γย้อมสีIntracellular IFN-γ expression in response to peptide stimulation was performed as previously described (19, 25). A total of 106 brain-derived monocytes or splenocytes per well were cultured for 5 h at 37°C in 200 μl of RPMI 1640 medium, supplemented with 5% fetal calf serum, 10 U of human recombinant interleukin-2, and brefeldin A (Golgiplug [1 μl/ml]; PharMingen) either with or without peptides. The peptides were used at a concentration of 1 μg/ml. Cells were then stained as described above for surface expression of CD8 and incubated overnight at 4°C. For intracellular IFN-γ staining, cells were then fixed and permeabilized using the Cytofix/Cytosperm kit (PharMingen) and stained with a fluorescein isothiocyanate-conjugated monoclonal rat anti-mouse IFN-γ antibody (clone XMG 1.2; PharMingen). Cells were washed and then suspended in PBS containing 2% paraformaldehyde and analyzed by FACSan flow cytometry (Becton-Dickinson).Isolation of epitope escape mutants.Viruses were plaqued from brain homogenates of infected animals sacrificed at 5 days postinfection as described above. Plaques were circled on the six-well culture dishes and then visualized by fluorescent microscopy. The number of green plaques was counted, and the percentage of fluorescent plaques was calculated. A selection of fluorescent and nonfluorescent plaques were picked, and viruses isolated from these plaques were used to infect L2 cells. Intracellular RNA from such infections was isolated. RT-PCR was used to amplify the region encoding gp33/gfp using primers FIJ81 and RIJ84 (Table (Table1).1). These amplified fragments were analyzed by agarose gel electrophoresis and in some cases sequenced using the same primers.
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