Heterosexual Transmission of a Murine AIDS Virus

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.Human immunodeficiency virus (HIV) infection is now pandemic. In many countries, HIV has been spread mainly by heterosexual transmission (3, 5). For the prevention of HIV infection, as well as for the development of vaccines against HIV, it is of a great importance to understand the mechanisms of the heterosexual transmission of retroviruses. Since it is difficult to investigate the mechanisms of heterosexual transmission of HIV in humans experimentally, an animal model with a retrovirus which induces an acquired immunodeficiency syndrome like human AIDS would be useful. A murine leukemia virus mixture called LP-BM5 MuLV induces a severe acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice (10). The mixture includes a replication-competent ecotropic virus, mink cell focus-inducing virus, and a replication-defective virus which has been considered to be involved in the pathogenesis of MAIDS (4). With many similarities to human AIDS patients, mice infected with the LP-BM5 MuLV mixture develop splenomegaly, systemic lymphadenopathy, and severe immunodeficiency (4, 11). We previously reported that maternal transmission of LP-BM5 MuLV occurs via mother’s milk with high frequency (12). In the present study, we demonstrate that the heterosexual transmission of LP-BM5 MuLV also occurs with high frequency via genital organs.C57BL/10 (B10) mice were purchased from Japan SLC Inc., Shizuoka, Japan. All mice were specific-pathogen free and were housed in an air-conditioned room. They were given autoclaved water and sterilized pelleted feed. An SC-1 clone chronically infected with LP-BM5 MuLV, the G6 cell line, was kindly supplied by H. C. Morse III, National Institutes of Health, Bethesda, Md. Virus was prepared from the supernatant of G6 cells as previously described (12). The virus preparation was stored at −70°C until use. B10 mice were inoculated by the intraperitoneal route with 0.3 ml of the LP-BM5 MuLV preparation. To increase the frequency of sexual contacts and to avoid pregnancy in the female mice, all male mice were sterilized by vasectomy under anesthesia with pentobarbital (Nembutal). The vasectomized male mice were mated with female mice at least 4 weeks postoperation, since sperm are usually kept alive for 2 to 3 weeks in spermiducts. Excised genital organs were crushed with plastic sticks in 1 ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% sodium dodecyl sulfate, and proteinase K (0.5 mg/ml). Spleen cells were lysed after hemolysis with 0.83% NH₄Cl. Lysed samples were incubated at 50°C for 3 h. DNA was extracted three times with phenol-chloroform, precipitated with cold ethanol, treated with RNase and proteinase K, and dissolved in 0.1 ml of H₂O. LP-BM5 MuLV defective virus genome was detected by Southern blot hybridization combined with PCR as described previously (12). In brief, template DNAs (1 μg per tube) were added to a cocktail adjusted to final concentrations of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 200 μM deoxynucleoside triphosphate, 100 pmol of each primer (5′-CCTCTTCCTTTATCGACACT-3′ [sense] and 5′-ATTAGGGGGGGAATAGCTCG-3′ [antisense]), and 2 U of Taq DNA polymerase (Boehringer Mannheim) in a total volume of 100 μl and were subjected to 32 cycles of amplification. In each cycle of PCR, the mixture was denatured at 95°C for 1 min (5 min for the first cycle), annealed at 55°C for 3 min, and extended at 72°C for 1 min. The PCR-amplified products were subjected to gel electrophoresis (1.5% agarose) and transferred to a Hybond N+ membrane (Amersham) by the alkaline blotting method. Hybridization was achieved with a 5′ ³²P-labeled probe (5′-TGTCAAAGGGACCAGTTAAG-3′) at 45°C overnight in 6× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.5% sodium dodecyl sulfate–100 μg of salmon sperm DNA per ml. Hybridized membranes were washed twice in 2× SSC at 37°C for 10 min and then in 0.5× SSC at 45°C for 30 min. DNA derived from uterine cervices of uninfected mice was used as a negative control. The limit of sensitivity was approximately 10 copies per tube, as assessed by Southern blot analysis with plasmid DNAs (1/10 of the PCR product).Concanavalin A (ConA) was obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Responder spleen cells (2 × 10⁵) were cultured with ConA (5 μg/ml) in 96-well flat-bottomed microculture plates in 0.2 ml of culture medium at 37°C in 7.5% CO₂. The culture medium consisted of RPMI 1640 supplemented with 10% fetal calf serum, penicillin (5,000 IU/100 ml), streptomycin (5,000 μg/100 ml), nonessential amino acids, sodium pyruvate (11.0 mg/100 ml), 2-mercaptoethanol (5 × 10⁻⁵ M), and l-glutamine (29.2 mg/100 ml). On day 2, cultures were pulsed with 1 μCi of [³H]thymidine and incubated for an additional 12 to 18 h. Incorporation of [³H]thymidine into responder spleen cells was quantitated by liquid scintillation counting. Determinations were performed in triplicate; standard errors of the means were generally <5% and therefore have not been indicated.As illustrated in Fig. ​Fig.1,1, in order to investigate the heterosexual transmission of LP-BM5 MuLV from male to female mice, normal male mice were inoculated with LP-BM5 MuLV and vasectomized 1 week later. At 5 weeks after virus inoculation, they were mated with uninfected female mice. After 8 weeks of breeding, female mice were sacrificed and their vaginae, cervices uteri, corpora uteri, inguinal lymph nodes, and spleens were removed and stored at −70°C until use. In the opposite direction, to investigate virus transmission from female to male, normal female mice were inoculated with LP-BM5 MuLV and then mated with uninfected, vasectomized male mice as described above. After 8 weeks of breeding, male mice were sacrificed and their penes, prepuces, inguinal lymph nodes and spleens were removed and stored at −70°C until use. Figure ​Figure22 shows the detection by PCR of the LP-BM5 defective virus genome in genital organs and spleens that were taken from mice mated with their virus-infected counterparts. It was demonstrated that although the defective virus genome was detected in both spleens and genital organs in some male mice (2 of 17 [see Table ​Table1]),1]), as shown in Fig. ​Fig.2,2, lanes 3 and 4, the defective virus genome was detected only in the genital organs, not the spleens (Fig. ​(Fig.2,2, lanes 5 and 6), from most of the male mice. In contrast, all of the female mice were positive for defective virus genome only in the genital organs (Fig. ​(Fig.2,2, lanes 1 and 2). None of the mice examined were positive for the virus genome only in the spleens (this issue is discussed below). It should be noted here that the efficacy of PCR amplification, which was measured by experiments using the mixture of genomic DNA and plasmid DNA containing the defective virus, did not differ among the genital organs and spleens. By using the above strategy, the heterosexual transmission of LP-BM5 MuLV was investigated according to the protocol shown in Fig. ​Fig.1.1. Open in a separate windowFIG. 1Experimental design for examination of heterosexual transmission of the MAIDS virus in B10 mice. i.p., intraperitoneal.Open in a separate windowFIG. 2Detection of the LP-BM5 MuLV defective virus genome by PCR in genital organs and spleens. The template DNAs (1 μg) derived from female or male mice which were bred with LP-BM5 MuLV-infected mice were amplified by PCR. Samples were prepared from either female (lanes 1 and 2) or male (lanes 3 to 6) mice. Lanes 1, 3, and 5, spleen; lane 2, uterine cervix; lanes 4 and 6, penis (from two representative male mice). The PCR products (5 μl) were applied to a 1.5% agarose gel and analyzed by Southern blotting with a probe for the defective virus (12).

TABLE 1

Heterosexual transmission of LP-BM5 MuLV

Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice

The structures of the asparagine-linked oligosaccharide chains of IgG from autoimmune arthritic MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice and control MRL/Mp(-)+/+ (MRL(-)+/+) mice were investigated. Two subpopulations of IgG, M1-I and M1-II, were obtained from serum of MRL-lpr/lpr mice by column chromatography on protein A-Sepharose CL-4B. Although M1-I did not bind to the column, its elution was retarded, whereas M1-II was bound and was eluted in acidic buffer. IgG (Mn) from MRL(-)+/+ mice showed the same chromatographic behavior as M1-II. The structures of oligosaccharide chains liberated quantitatively by hydrazinolysis from IgG samples Mn, M1-I, M1-II, and a pooled mixture (M1) of M1-I and M1-II were determined by sequential exoglycosidase digestion, lectin (RCA120) affinity HPLC, and by methylation analysis. Their oligosaccharide structures were the same and shown to be biantennary complex-type chains +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The proportion of each oligosaccharide in Mn and M1-II was the same but differed from that in M1-I where the degree of the galactosylation was significantly decreased which caused the change in the oligosaccharide pattern of total serum IgG (M1) of autoimmune MRL-lpr/lpr mice. This phenomenon, which is also found in total serum IgG of patients with rheumatoid arthritis, suggests that alteration of oligosaccharides in IgG may be a common feature in animals which develop arthritis with the production of rheumatoid factor regardless of species.     

Two tunicamycin-sensitive components involved in agglutination and fusion ofChlamydomonas reinhardtii gametes

To find out glycoproteins involved in the mating reaction ofChlamydomonas reinhardtii, the effect of tunicamycin (TM), a potent inhibitor of glycosylation of proteins, was studied. TM, when present during gametogenesis, blocked the acquisition of agglutinability ofmt ⁺ cells. TM also inhibited the recovery of agglutinability ofmt ⁺ gamete after trypsin treatment. On the contrary, TM blocked neither the acquisition of agglutination during gametogenesis ofmt ^- cells nor the recovery of their agglutinability after trypsinization. It was found, however, that the TM-treatedmt ^- gametes can agglutinate but do not fuse with non treatedmt ⁺ gametes at all. When gametes of gam-1mt ^-, a conditional mutant strain for cell fusion, were induced at non permissive temperature of 35°C and then transferred to 25°C, the ability of cell fusion was acquired after about 5 h incubation. Presence of TM completely blocked this acquisition. Based on these evidence, we conclude that at least two TM-sensitive glycoproteins are included in the mating reaction. The first component is located on the flagellar surface ofmt ⁺ gamete and responsible for agglutination withmt ^- flagella. The second component occurs on the surface ofmt ^- gamete and plays a role in the fusion withmt ⁺ gamete.Abbreviations CHI cycloheximide - mt mating type - TM tunicamycin     

MHC Class I Presentation of an Exogenous Polypeptide Antigen Encoded by the Murine AIDS Defective Virus

Peptides derived from endogenous proteins are presented by MHC class I molecules, whereas those derived from exogenous proteins are presented by MHC class II molecules. This strict segregation has been reconsidered in recent reports in which exogenous antigens are shown to be presented by MHC class I molecules in the phagocytic pathway. In this report, the presentation pathway of an exogenously added highly antigenic polypeptide encoded by the murine AIDS (MAIDS) defective virus gag p12 gene is investigated. A 25-mer polypeptide (P12–25) encoded within the gag p12 region of the MAIDS defective virus was found to be effective in stimulating unprimed B6 (H-2^b) CD8⁺ T cells in vitro. The presentation of P12–25 is sensitive to cytochalasin B and D, brefeldin A and gelonin, a ribosome-inactivating protein synthesis inhibitor, but less sensitive or resistant to lactacystin, a highly specific inhibitor of the proteasome. Interestingly, CA-074, a selective inhibitor of cathepsin B, inhibited presentation of the polypeptide, indicating its involvement in the degradation of the P12–25 polypeptide. In fact, when P12–25 was digested with purified cathepsin B in vitro, a highly antigenic 11-mer peptide containing the class I (H-2D^b)-binding motif was obtained. Our results favor the phagosome/macropinosome-to-cytosol-to-endoplasmic reticulum (ER)-to-cell surface pathway for exogenous antigens presented by MHC class I molecules. These findings may be relevant to exploiting peptide vaccines that specifically elicit CD8⁺ T cell immunity in vivo.     

Effects of hapten epitope structure and hapten-self conjugation pattern on T cell specificity and Ir gene control in hapten-self cytotoxic and helper T cell responses

Mouse strains of H-2b haplotype exhibit much weaker cytotoxic T lymphocyte (CTL) responses to haptens reactive with amino groups of cell surface (NH2-reactive haptens) compared with H-2k strains. However, H-2b strains can generate high CTL responses to haptens reactive with sulfhydryl groups of cell surface (SH-reactive haptens). The present study investigates the role of haptenic structure and hapten-cell surface reaction patterns in influencing the generation of the T cell specificity as well as the H-2-linked genetic control. CTL and helper T cell responses were generated against two structurally related haptens, N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene-diamine (SH-reactive AEDANS; AED-SH) and 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (NH2-reactive form of AEDANS; AED-NH2) by immunizing C57BL/6N (H-2b) mice with these hapten-modified syngeneic spleen cells. Spleen cells from primed C57BL/6N mice generated strong CTL and helper T cell activities upon in vitro restimulation with the respective hapten-modified self. The generation of potent anti-AED-NH2 CTL and helper T cell responses in C57BL/6N mice sharply contrasted with the failure of NH2-reactive haptens studied thus far to generate strong anti-hapten cytotoxic responses in H-2b mice. Antibodies induced against the above two haptens exhibited extensive cross-reactivity detected by hemagglutination, whereas CTL and helper T cells clearly discriminated the structural difference between AED-NH2 and AED-SH haptens. The hapten specificity in T cell recognition was also observed between AED-NH2 and trinitrophenyl (TNP) haptens, which were demonstrated to functionally modify similar cell surface sites. These results indicate that hapten epitope structure and hapten-cell membrane conjugation patterns influence the generation of H-2-linked genetic control and T cell specificity in anti-hapten self cytotoxic as well as helper T cell responses.     

Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides.

Conditions have been established for the rapid and efficient conjugation of reducing oligosaccharides (di- to deca-saccharides) to dipalmitoyl phosphatidylethanolamine. The resulting neoglycolipids derived from several naturally occurring oligosaccharides and a series of N-linked high-mannose-type oligosaccharides released by hydrazinolysis from RNAase B showed specific and potent reactivities, as appropriate, with monoclonal antibodies to blood group Lewis(b), blood group A or a stage-specific embryonic (SSEA-1) antigen, or the lectin concanavalin A.     

Diversity of oligosaccharide structures on the envelope glycoprotein gp 120 of human immunodeficiency virus 1 from the lymphoblastoid cell line H9. Presence of complex-type oligosaccharides with bisecting N-acetylglucosamine residues

The N-linked oligosaccharide structures on the envelope glycoprotein gp120 of human immunodeficiency virus 1 derived from chronically infected lymphoblastoid (H9) cells have been investigated by enzymatic microsequencing after release from protein by hydrazinolysis, labeling with NaB3H4, and chromatography on adsorbent columns of Phaseolus vulgaris erythrophytohemagglutinin and Ricinus communis agglutinin (Mr 120,000) and on Bio-Gel P-4. A substantially greater diversity of oligosaccharide structures was detected than among those released by hydrazinolysis from recombinant gp120 produced in Chinese hamster ovary cells and investigated by similar procedures (Mizuochi, T., Spellman, M.W., Larkin, M., Solomon, J., Basa, L.J., and Feizi, T. (1988) Biochem J. 254, 599-603) and among those released by endoglycosidases from virus-derived gp120 isolated from infected H9 cells after metabolic labeling with D-[2-3H]mannose or D-[6-3H]glucosamine (Geyer, H., Holschbach, L., Hunsmann, G., and Schneider, J. (1988) J. Biol. Chem. 263, 11760-11767). In this study, 16% of the oligosaccharides were identified as complex-type bi-, tri-, and tetraantennary sialo-oligosaccharides with bisecting N-acetylglucosamine residues. Such structures were lacking on recombinant gp120 and could not be detected on the metabolically labeled, virus-derived glycoprotein. As in the earlier investigations, complex-type chains lacking bisecting N-acetylglucosamine residues, hybrid-type chains, and a series of high mannose-type structures with 5-9 mannose residues were identified. In addition, an array of complex-type chains having one or more outer chains with beta-galactosyl residues were detected in this study, but with additional substitutions that require further investigation. The number of potential N-glycosylation sites on gp120 is on the order of 20, but the oligosaccharide structures are far more numerous. Thus, the salient conclusion from this and earlier investigations is that alternative structures occur on at least some of the glycosylation sites and that numerous glycosylation variants of this glycoprotein are produced even within a single cell line. Since the glycosylation is the product of host cell glycosyltransferases, an even greater number of glycosylation variants of gp120 are predicted to arise from the heterogeneous cell populations harboring the virus in in vivo infection.     

Role of lymphokine-secreting CD8+ T cells in cytotoxic T lymphocyte responses against vaccinia virus

The present study has examined the relative role of CD4+ and CD8+ Th cells in the generation and reactivation of antivaccinia virus memory CTL responses. We show that mice primed in vivo to vaccinia virus generate in vitro antivaccinia virus memory CTL responses through both CD4+ and CD8+ Th cell pathways, with the CD4+ Th pathway being the more prominent of the two. In addition, we show that vaccinia virus-specific CD8+ Th cell function is mediated through production of lymphokines, including IL-2, and that the CD8+ Th cell component in the CTL response is labile, decreasing progressively with increasing time after in vivo priming. Thus, this study demonstrates the existence of two phenotypically distinct Th cell pathways in the generation of antivirus CTL responses.     

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β-subunit by disruption of disulfide bonds

The human chorionic gonadotropin β-subunit (hCGβ) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGβ produced in Chinese hamster ovary (CHO) cells: wild-type hCGβ (βWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of βWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled βWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGβ molecule. Abbreviations: hCGβ, human chorionic gonadotropin β-subunit; βWT, wild type hCGβ; CHO, Chinese hamster ovary; Endo-H, endoglycosidase H; Endo-F, endoglycosidase F This revised version was published online in November 2006 with corrections to the Cover Date.     

Beta-subunits of equine chorionic gonadotropin and lutenizing hormone with an identical amino acid sequence have different asparagine-linked oligosaccharide chains.

The glycoprotein hormones, equine chorionic gonadotropin (eCG) and lutenizing hormone (eLH), possess a beta-subunit with an identical amino acid sequence. The Asn-linked oligosaccharide chains of eCG beta and eLH beta were quantitatively liberated as tritium-labeled oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4-reduction. Paper electrophoresis in combination with sialidase digestion and solvolytic desulfation indicated that eCG beta contained neutral and sialylated oligosaccharides, while eLH beta contained neutral, sialylated, sulfated, and both sialylated and sulfated oligosaccharides. In addition, elution profiles on a Bio-Gel P-4 column of the neutralized oligosaccharide mixtures of eCG beta and eLH beta were different, indicating that the molecular masses of oligosaccharides of the two glycoproteins are different. Therefore, this suggests that the structures of the Asn-linked oligosaccharide chains of eCG beta and eLH beta are different although they have an identical amino acid sequence.