A small region of the ecotropic murine leukemia virus (MuLV) gag gene profoundly influences the types of polytropic MuLVs generated in mice

The vast majority of recombinant polytropic murine leukemia viruses (MuLVs) generated in mice after infection by ecotropic MuLVs can be classified into two major antigenic groups based on their reactivities to two monoclonal antibodies (MAbs) termed Hy 7 and 516. These groups very likely correspond to viruses formed by recombination of the ecotropic MuLV with two distinct sets of polytropic env genes present in the genomes of inbred mouse strains. We have found that nearly all polytropic MuLVs identified in mice infected with a substrain of Friend MuLV (F-MuLV57) are reactive with Hy 7, whereas mice infected with Moloney MuLV (Mo-MuLV) generate major populations of both Hy 7- and 516-reactive polytropic MuLVs. We examined polytropic MuLVs generated in NFS/N mice after inoculation with Mo-MuLV-F-MuLV57 chimeras to determine which regions of the viral genome influence this difference between the two ecotropic MuLVs. These studies identified a region of the MuLV genome which encodes the nucleocapsid protein and a portion of the viral protease as the only region that influenced the difference in polytropic-MuLV generation by Mo-MuLV and F-MuLV57.     

A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes

The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential beta-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.     

Deletions in one domain of the Friend virus-encoded membrane glycoprotein overcome host range restrictions for erythroleukemia

Although the Friend virus-encoded membrane glycoprotein (gp55) activates erythropoietin receptors (EpoR) to cause erythroblastosis only in certain inbred strains of mice but not in other species, mutant viruses can overcome aspects of mouse resistance. Thus, mice homozygous for the resistance allele of the Fv-2 gene are unaffected by gp55 but are susceptible to mutant glycoproteins that have partial deletions in their ecotropic domains. These and other results have suggested that proteins coded for by polymorphic Fv-2 alleles might directly or indirectly interact with EpoR and that changes in gp55 can overcome this defense. A new viral mutant with an exceptionally large deletion in its ecotropic domain is now also shown to overcome Fv-2rr resistance. In all cases, the glycoproteins that activate EpoR are processed to cell surfaces as disulfide-bonded dimers. To initiate analysis of nonmurine resistances, we expressed human EpoR and mouse EpoR in the interleukin 3-dependent mouse cell line BaF3 and compared the abilities of Friend virus-encoded glycoproteins to convert these cells to growth factor independence. Human EpoR was activated in these cells by erythropoietin but was resistant to gp55. However, human EpoR was efficiently activated in these cells by the same viral mutants that overcome Fv-2rr resistance in mice. By construction and analysis of human-mouse EpoR chimeras, we obtained evidence that the cytosolic domain of human EpoR contributes to its resistance to gp55 and that this resistance is mediated by accessory cellular factors. Aspects of host resistance in both murine and nonmurine species are targeted specifically against the ecotropic domain of gp55.     

The mouse H-2A region influences the envelope gene structure of tumor-associated murine leukemia viruses

C57BL/10 (B10) strains congenic at the mouse major histocompatibility locus (H-2) were injected with a modified ecotropic SL3-3 murine leukemia virus (MuLV) to determine the effect of the H-2 genes on the envelope gene structure of recombinant MuLVs. All tested strains rapidly developed T-cell lymphomas, and recombinant proviruses were detected in the tumor DNAs by Southern blot. The B10.D2 (H-2d), B10.Br (H-2k), B10.Q (H-2q), and B10.RIII (H-2r) strains exhibited a TI phenotype in which almost all tumors contained type I recombinants. These recombinants characteristically acquire envelope gene sequences from the endogenous polytropic viruses but retain the 5' p15E (TM) gene sequences from the ecotropic virus. The parental B10 (H-2b) strain, however, had a novel phenotype that was designated NS for nonselective. Only 30% of the B10 tumors had detectable type I recombinants, whereas a proportion of the others appeared to contain type II recombinants that lacked the type I-specific ecotropic p15E gene sequences. Studies of other B10 congenic strains with hybrid H-2 loci and selected F1 animals revealed that the NS phenotype was regulated by a dominant gene(s) that mapped to the A region of H-2b. These results demonstrate that a host gene within the major histocompatibility complex can influence the genetic evolution of pathogenic retroviruses in vivo.     

The entire nucleotide sequence of friend-related and paralysis-inducing PVC-441 murine leukemia virus (MuLV) and its comparison with those of PVC-211 MuLV and Friend MuLV

PVC-441 murine leukemia virus (MuLV) is a member of the PVC group of Friend MuLV (F-MuLV)-derived neuropathogenic retroviruses. In order to determine the molecular basis for the difference in neuropathogenicity between PVC-441 and the previously characterized PVC-211 MuLVs, the entire nucleotide sequence of PVC-441 MuLV was determined and compared with those of PVC-211 and F-MuLV. The results suggest that PVC-441 and PVC-211 MuLVs were formed as a result of random mutations of F-MuLV and developed differently. The distinct pathogenicities of PVC-441 and PVC-211 MuLVs were maintained in the viruses regenerated from their molecular clones, and the sequences responsible for the pathological differences observed can be localized to the env gene. The amino acid sequence of PVC-441 deduced from its nucleotide sequence revealed a number of differences from PVC-211, the most striking of which was a difference at position 129 of the SU proteins in the two viruses. Host range studies with a brain capillary endothelial cell line (RTEC-6) and Chinese hamster ovary cells (CHO-K1) revealed that PVC-441, like PVC-211, could infect these cells but its efficiency of infection was lower than that of PVC-211. These results may account for the difference in neuropathogenicity between PVC-441 and PVC-211.     

Apoptosis induction by the binding of the carboxyl terminus of human immunodeficiency virus type 1 gp160 to calmodulin

The role of calmodulin (CaM) in apoptosis induced by gp160 of human immunodeficiency virus type 1 was investigated with cells undergoing single-cell killing. These cells were found to express, under the control of an inducible promoter, wild-type gp160 or mutant gp160 devoid of various lengths of the carboxyl terminus. Immunoprecipitation accompanied by immunoblotting revealed binding of CaM to wild-type gp160 but not to mutant gp160 bearing a carboxyl terminus with a deletion spanning more than five amino acid residues. A significant coenzyme activity was detected in the CaM bound to gp160 even in the presence of a Ca2+ chelater, EGTA. The cells forming this gp160-CaM complex exhibited an elevated intracellular Ca2+ level followed by DNA fragmentation, which is a hallmark of apoptosis, and finally cell killing, while the cells not forming this complex did not show any significant elevation in Ca2+ level or DNA fragmentation. These results thus indicated that CaM plays a key role in gp160-induced apoptosis.     

Xenopus laevis sperm receptor gp69/64 glycoprotein is a homolog of the mammalian sperm receptor ZP2

Little is known about sperm-binding proteins in the egg envelope of nonmammalian vertebrate species. We report here the molecular cloning and characterization of a recently identified sperm receptor (gp69/64) in the Xenopus laevis egg vitelline envelope. Our data indicate that the gp69 and gp64 glycoproteins are two glycoforms of the receptor and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation. The amino acid sequence of the receptor is closely related to that of the mouse zona pellucida protein ZP2. Most of the sequence conservation, including a ZP domain, a potential furin cleavage site, and a putative transmembrane domain are located in the C-terminal half of the receptor. Proteolytic cleavage of the gp69/64 protein by a cortical granule protease during fertilization removes 27 amino acid residues from the N terminus of gp69/64 and results in loss of sperm binding to the activated eggs. Similarly, we find that treatment of eggs with type I collagenase removes 31 residues from the N terminus of gp69/64 and has the same effect on sperm binding. The isolated and purified N terminus-truncated receptor protein is inactive as an inhibitor of sperm-egg binding. Earlier studies on the effect of Pronase digestion on receptor activity suggest that this N-terminal peptide may contain an O-linked glycan that is involved in the binding process. Based on these results and the findings on the primary structure of the receptor, a pathway for the maturation and secretion of gp69/64, as well as its inactivation following fertilization, is proposed.     

Adenovirus facilitated infection of human cells with ecotropic retrovirus

Retroviral infection is restricted by the expression of a viral receptor on the surface of the target cell. Retrovirus-mediated gene transfer is therefore not possible in cells that fail to express sufficient levels of the appropriate receptor, representing one major obstacle to the use of recombinant retroviruses in experimental and clinical applications. In this study, we utilized an adenoviral vector to express transiently the receptor for the ecotropic murine leukemia virus in a panel of human cell lines. Following adenoviral infection, the susceptibility to ecotropic retroviral particles of A549, HeLa, RC39 and Meso 33 cells, derived from human lung epithelium, cervical epithelium, kidney and mesothelium, respectively, was measured on a single-cell basis by the detection of a cell surface marker encoded by the recombinant retrovirus. The marker, termed NTP, was found in 10-30%, 25-50% and 50-90% of cells infected at 5, 50 and 250 adenovirus multiplicity of infection, respectively. Southern blot analysis demonstrated the integration of intact retroviral DNA. The integrated vector copy number increased with the adenoviral multiplicity of infection, suggesting that retrovirus infection is proportional to receptor expression by the target cell, albeit not in a linear fashion. Susceptibility to ecotropic retroviral infection was maintained undiminished for at least 3 days, indicating the persistent expression of ecotropic receptor by the adenovirus-transduced cells in that time period and the lack of a major cellular defense triggered by adenovirus infection against the subsequent retroviral infection. Thus, the infection of human cells of various tissues with a recombinant adenovirus expressing the ecotropic murine leukemia virus receptor generates a window of susceptibility where a high retroviral infection rate can be achieved. Increased efficiency of retroviral infection obtained in this fashion is amenable to specific regulation via the controlled expression of the adenovirus-encoded retroviral receptor.     

Amplicons of maize zein genes are conserved within genic but expanded and constricted in intergenic regions

The properties are discussed of system y+L, a broad scope amino acid transporter which was first identified in human erythrocytes. System y+L exhibits two distinctive properties: (a) it can bind and translocate cationic and neutral amino acids, and (b) its specificity varies depending on the ionic composition of the medium. In Na+ medium, the half-saturation constant for L-lysine influx was 9.5 +/- 0.67 microM and the inhibition constant (Ki) for L-leucine was 10.7 +/- 0.72 microM. L-Leucine is the neutral amino acid that binds more powerfully, whereas smaller analogues, such as L-alanine and L-serine interact less strongly (the corresponding inhibition constants were Ki,Ala, 0.62 +/- 0.11 mM; Ki,Ser, 0.49 +/- 0.08 mM). In the presence of K+, the carrier functions as a cationic amino acid specific carrier, but Li+ is able to substitute for Na+ facilitating neutral amino acid binding. The effect of the inorganic cations is restricted to the recognition of neutral amino acids; translocation occurs at similar rates in the presence of Na+, K+ and Li+. The only structural feature that appears to impair translocation is bulkiness and substrates with half-saturation constants differing by more than 100-fold translocate at the same rate. This suggests that translocation is largely independent of the forces of interaction between the substrate and the carrier site. System y+L activity has been observed in Xenopus laevis oocytes injected with the cRNA for the heavy chain of the 4F2 human surface antigen. 4F2hc is an integral membrane protein with a single putative membrane-spanning domain and it remains to be clarified whether it is part of the transporter or an activator protein.     

Host range and interference studies of three classes of pig endogenous retrovirus

Recent interest in the use of porcine organs, tissues, and cells for xenotransplantation to humans has highlighted the need to characterize the properties of pig endogenous retroviruses (PERVs). Analysis of a variety of pig cells allowed us to isolate and identify three classes of infectious type C endogenous retrovirus (PERV-A, PERV-B, and PERV-C) which have distinct env genes but have highly homologous sequences in the rest of the genome. To study the properties of these env genes, expression plasmids for the three env genes were constructed and used to generate retrovirus vectors bearing corresponding Env proteins. Host range analyses by the vector transduction assay showed that PERV-A and PERV-B Envs have wider host ranges, including several human cell lines, compared with PERV-C Env, which infected only two pig cell lines and one human cell line. All PERVs could infect pig cells, indicating that the PERVs have a potential to replicate in pig transplants in immunosuppressed patients. Receptors for PERV-A and PERV-B were present on cells of some other species, including mink, rat, mouse, and dog, suggesting that such species may provide useful model systems to study infection and pathogenicity of PERV. In contrast, no vector transduction was observed on nonhuman primate cell lines, casting doubt on the utility of nonhuman primates as models for PERV zoonosis. Interference studies showed that the three PERV strains use receptors distinct from each other and from a number of other type C mammalian retroviruses.     

Genetic variation of envelope gp90 gene of equine infectious anemia virus isolated from an experimentally infected horse

Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the principal neutralizing domain (PND) region. The insertions were arranged regularly with smaller segments. The nucleotide sequence of the P337-V26 gp90 gene was found to contain a six-nucleotides insertion and seven nucleotide substitutions outside the PND region, when compared with that of the P337-V70 strain.     

71-kilodalton heat shock cognate protein acts as a cellular receptor for syncytium formation induced by human T-cell lymphotropic virus type 1

We previously reported that the region corresponding to amino acids 197 to 216 of the gp46 surface glycoprotein (gp46-197) served as a binding domain for the interaction between gp46 and trypsin-sensitive membrane components of the target cell, leading to syncytium formation induced by human T-cell lymphotropic virus type 1 (HTLV-1)-bearing cells. Our new evidence shows that the 71-kDa heat shock cognate protein (HSC70) acts as a cellular receptor for syncytium formation. Using affinity chromatography with the peptide gp46-197, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we isolated three components (bands A, B, and C) from MOLT-4 cell lysate which exhibited specific interactions with gp46 and inhibitory activities for syncytium formation induced by HTLV-1-bearing cells. Band A and B components were identified as HSC70 and beta-actin, respectively, through amino acid sequencing by tandem mass spectrometry and immunostaining with specific monoclonal antibodies. Band C is likely to be a nonprotein component, because full activity for syncytium formation was seen after extensive trypsin digestion. Anti-HSC70 monoclonal antibody clearly blocked syncytium formation in a coculture of HTLV-1-bearing cells and indicator cells, whereas no inhibition was seen with anti-beta-actin monoclonal antibody. Furthermore, flow cytometric analysis indicated that anti-HSC70 antibody reacted with MOLT-4 cells. Thus, we propose that HSC70 expressed on the target cell surface acts as a cellular acceptor to gp46 exposed on the HTLV-1-infected cell for syncytium formation, thereby leading to cell-to-cell transmission of HTLV-1.     

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-beta in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-beta (aa128-134) and HIV-1 gp41 (aa586-595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-beta antibody levels by ELISA. The levels of anti-IFN-beta antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-beta in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-beta recognized rsgp41 (recombinant soluble gp41 Env amino acid 539-684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-beta and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128-134 of human IFN-beta and the immunosuppressive domain (aa583-599) of HIV-1 gp41. The increased levels of antibodies against interferon-beta in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-beta and HIV-1 gp41.     

Host range conversion of murine leukemia virus resulting from recombination with endogenous virus

Ecotropic murine leukemia viruses (MuLVs) are classified into B-N-, or NB-tropic MuLV by their host range determined by the Fv-1 gene product. B-tropic MuLV is restricted in N-type mouse cells (Fv-1 n/n) and N-tropic MuLV is restricted in B-type mouse cells (FV-1 b/b). Although forced passages in a restrictive host grant a wider host range (NB-tropism), we show here a host range conversion from B to N tropism. The conversion was most likely a result of recombination between the exogenously infected B-tropic MuLV and an endogenously expressed N-tropic MuLV in a C57BL/6 mouse cell line, YH-7.     

New concepts in anionic and cationic amino acid transport]

In mammalian cells, amino acids are taken up by different transport systems present in the plasma membrane. The transport systems were originally characterized by kinetic and competition studies. However, it was difficult to assign specific amino acids to specific transport systems. With recent advances in molecular biology, it has been possible to identify the specific transporter proteins for specific amino acids. In this review we describe the anionic and cationic amino acid transport systems reported at the molecular level. The anionic amino acids are movilized mainly by the XaG- and Xc- systems which are important in the inactivation of glutamatergic nervous transmission in the brain and for the synthesis of glutathione, respectively. Four isoforms of the XAG- system in the brain belong to the family of Na+ dependent amino acid transporters. Transport systems for cationic amino acids also recognize zwitterionic substrates, and the better characterized systems at the present time are y+, y+L, bo,+ and Bo,+. The regulation of the entrance of cationic amino acid such as arginine, lysine, and ornithine to the cell is important in the biosynthesis of nitric oxide, creatine, carnitine, and polyamines. An inherited defect associated to bo,+ system is cysteinuria.     

Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.     

Tension relaxation induced by pulse photolysis of caged ATP in partially crosslinked fibers from rabbit psoas muscle

We previously isolated gp17, a human seminal plasma glycoprotein, which specifically interacts with the D1-D2 region of CD4, a T cell surface molecule involved in antigen recognition mediated by helper T cells also acting as a receptor for the human immunodeficiency virus. In this study we report that monoclonal antibodies (mAb) reacting with gp17 are able to inhibit the binding of gp17 to immobilized soluble CD4. An immunohistochemical analysis shows that gp17 is also expressed in mammary tumor cells upon hormone treatment and in biopsies from breast cancer patients. A structural characterization of gp17, including amino acid sequencing, indicates that the protein has an extensive structural similarity with a glycoprotein designated as seminal actin-binding protein (SABP), also secreted by male sexual glands. SABP is in turn identical to gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP), a factor known as a highly specific and sensitive marker of primary and metastatic apocrine breast cancer. To establish further the correspondence of gp17 and GCDFP-15/PIP/SABP, the latter was expressed in bacteria from a cloned cDNA and purified by affinity chromatography to either anti-gp17 mAb-Sepharose or CD4-Sepharose. The purified recombinant protein is shown to inhibit the binding of labeled, pure g17 to immobilized soluble CD4. The finding that breast cancer cells express a protein able to interact with the CD4 domains involved in the recognition of class II major histocompatibility antigens suggests a possible mechanism by which a tumor may affect the activity of tumor-infiltrated CD4+ T cells.