Suppression of in vivo tumor formation induced by simian virus 40-transformed cells in mice receiving antiidiotypic antibodies.

This study characterizes four private idiotypes (Id) associated with monoclonal antibodies (mAb) to simian virus 40 (SV40) tumor antigen (T-Ag), and to a cellular protein, p53. Anti-Id recognized Id determinants associated with the antibody-combining site. BALB/c mice receiving a pool of anti-Id directed against mAb recognizing distinct amino and carboxyl terminal epitopes of T-Ag before receiving a tumorigenic dose of SV40-transformed cells showed suppression of tumor formation. Serum obtained from these mice before tumor challenge contained anti-anti-Id that failed to bind T-Ag. These data support the potential role of regulatory idiotopes in tumor immunity.

Nuclear transport: a guide to import receptors.

After synthesis in the cytoplasm, nuclear proteins traverse the nuclear envelope as a result of the specific recognition of nuclear localization signals by import. Various approaches have now uncovered a range of proteins with at least some of the characteristics expected of import receptors. This article focuses on early steps in the nuclear import of proteins and surveys the recently identified candidate import receptors.

The effects of variations in the number and sequence of targeting signals on nuclear uptake.

To determine if the number of targeting signals affects the transport of proteins into the nucleus, Xenopus oocytes were injected with colloidal gold particles, ranging in diameter from 20 to 280 A, that were coated with BSA cross-linked with synthetic peptides containing the SV-40 large T-antigen nuclear transport signal. Three BSA conjugate preparations were used; they had an average of 5, 8, and 11 signals per molecule of carrier protein. In addition, large T-antigen, which contains one signal per monomer, was used as a coating agent. The cells were fixed at various times after injection and subsequently analyzed by electron microscopy. Gold particles coated with proteins containing the SV-40 signal entered the nucleus through central channels located within the nuclear pores. Analysis of the intracellular distribution and size of the tracers that entered the nucleus indicated that the number of signals per molecule affect both the relative uptake of particles and the functional size of the channels available for translocation. In control experiments, gold particles coated with BSA or BSA conjugated with inactive peptides similar to the SV-40 transport signal were virtually excluded from the nucleus. Gold particles coated with nucleoplasmin, an endogenous karyophilic protein that contains five targeting signals per molecule, was transported through the nuclear pores more effectively than any of the BSA-peptide conjugates. Based on a correlation between the peri-envelope density of gold particles and their relative uptake, it is suggested that the differences in the activity of the two targeting signals is related to their binding affinity for envelope receptors. It was also determined, by performing coinjection experiments, that individual pores are capable of recognizing and transporting proteins that contain different nuclear targeting signals.

Anti-idiotypic antibody vaccine for type B viral hepatitis in chimpanzees.

Anti-idiotypic antibodies (anti-Id) that contain an internal image component that mimics the surface antigen of hepatitis B virus (HBsAg) were used to immunize chimpanzees. Four injections of the rabbit anti-Id preparation elicited an antibody response to HBsAg (anti-HBs). The antibody specificity appeared to be against the anti-Id, since the anti-Id immunogen was shown to bind the chimpanzee anti-HBs. Two chimpanzees immunized with the anti-Id, along with two control animals that were either untreated or received a nonimmune rabbit immunoglobulin G preparation, were challenged with infectious hepatitis B virus. Both control chimpanzees developed clinical and serological characteristics consistent with an active hepatitis B virus infection, whereas the two anti-Id treated chimpanzees were protected from infection. Since chimpanzees provide a relevant model of a human response to hepatitis B virus immunization and infection, these results indicate that anti-Id preparations such as that described here might be candidates for vaccines against human diseases.

Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.

Differential ability of a T-antigen transport-defective mutant of simian virus 40 to transform primary and established rodent cells.

The transforming potential and oncogenicity of a simian virus 40 (SV40) mutant affecting T-antigen (T-ag), SV40(cT)-3, was examined in an effort to dissect T-ag functions in transformation. SV40(cT)-3 has a point mutation at nucleotide 4434 that abolishes the transport of T-ag to the nucleus but does not affect its association with the cell surface. Transfection-transformation assays were performed with primary cells and established cell lines of mouse and rat origin. The efficiency of transformation for established cell lines by SV40(cT)-3 was comparable to that of wild-type SV40, indicating that transformation of established cell lines can occur in the absence of detectable amounts of nuclear T-ag. Transformation of primary mouse embryo fibroblasts by SV40(cT)-3 was markedly influenced by culture conditions; the relative transforming frequency was dramatically reduced in assays involving focus formation in low serum concentrations or anchorage-independent growth. Immunofluorescence tests revealed that the transformed mouse embryo fibroblasts partially transport the mutant cT-ag to the cell nucleus. Transformed cell lines induced by SV40(cT)-3 did not differ in growth properties from wild-type transformants. SV40(cT)-3 was completely defective for the transformation of primary baby rat kidney cells, a primary cell type unable to transport the mutant T-ag to the nucleus. The intracellular localization of cellular protein p53 was found to mimic T-ag distribution in all the transformants analyzed. The mutant virus was weakly oncogenic in vivo: the induction of tumors in newborn hamsters by SV40(cT)-3 was reduced in incidence and delayed in appearance in comparison to wild-type SV40. These observations suggest that cellular transformation is regulated by both nuclear and surface-associated forms of SV40 T-ag.

Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.

A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and 129, since substitutions of neutral asparagines at these positions abolished transport. However, partial transport was observed with the peptide having an asparagine at position 128 when a high number of peptides were conjugated to the carrier protein.

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.

Expression of mammary tumor virus proteins in preneoplastic outgrowth lines and mammary tumors of BALB/cV mice.

A subline of BALB/c mice, designated BALB/cV, has been segregated which exhibits an intermediate mammary tumor incidence and which harbors a unique milk-transmitted virus. Six stable hyperplastic alveolar nodule outgrowth lines were established from chemical carcinogen-treated and hormonally stimulated mice. Tumor incidences exhibited by the individual preneoplastic lines ranged from 22 to 95%; no differences in tumor-producing capability were observed when lines were transplanted in virus-negative (BALB/c) or virus-positive (BALB/cV) animals. Viral antigen expression was monitored using antisera prepared against C3H mouse mammary tumor virus (MMTV) proteins. The preneoplastic lines exhibited more virus antigenpositive cells in a peroxidase-antiperoxidase immunocytochemical assay than did primary tumors which arose from the hyperplastic alveolar nodule transplants. Analysis of BALB/cV preneoplastic and tumor tissue by metabolic labeling and by protein electroblotting methodologies revealed that viral precursor and structural proteins were expressed in both types of tissue; the polypeptides were similar in molecular weight to those encoded by exogenous MMTVs. These studies demonstrate that the coding capacity of the BALB/cV isolate of MMTV is similar to that of known MMTV isolates and that each of the BALB/cV structural polypeptides shares group-specific antigenic determinants with the analogous protein encoded by MMTV from the C3H mouse. The hyperplastic outgrowth lines established in the BALB/cV subline provide an additional system for the study of mammary tumorigenesis.

Production of lipoprotein(a) by primary baboon hepatocytes.

Primary baboon hepatocytes were cultured in a serum-free medium formulation that permitted the analysis of lipoprotein(a) (Lp(a] production by the cells. The hepatocytes were determined to synthesize Lp(a) on the basis of the following observations: (1) the culture medium reacted in an ELISA designed for detection of baboon Lp(a) in serum samples; (2) the Lp(a)-specific protein, apo(a), was detected in the culture medium by immunoblotting techniques; (3) the unique protein structure of Lp(a) was demonstrated (i.e., association of apo(a) with apoB via interchain disulfide bonds to form apoLp(a]; and (4) the Lp(a) proteins occurred in the medium at a density of about 1.05 g/ml when subjected to density gradient ultracentrifugation. De novo synthesis of Lp(a) by cultured hepatocytes was demonstrated by incorporation of [35S]cysteine. Lp(a) was produced by the hepatocytes throughout a 20 day culture period. Finally, apo(a) isoform patterns in the hepatocyte culture medium and the hepatocyte donors' serum were indistinguishable.

Polyhedrin initiator codon altered to AUU yields unexpected fusion protein from a baculovirus vector.

A recombinant baculovirus expression vector was constructed to express the core (capsid) protein of the hepatitis B virus. Along with the expected 21-kDa polypeptide, a second 24-kDa protein was observed. Immunoprecipitation and immunoblotting using a rabbit polyclonal anticore antiserum demonstrated that the two proteins were related. The core gene originally was cloned in-frame with the polyhedrin initiator codon that had been altered to AUU as a means of preventing fusion protein formation. A transient expression assay revealed expression of the 24-kDa protein was prevented if a frame-shift mutation was created upstream of the HBV core translation start site. These results suggest that the 24-kDa protein was the result of an unexpectedly high level of translation initiation at the AUU codon that gave rise to a polyhedrin-HBV core fusion protein. The 24-kDa core protein was shown to be a polyhedrin fusion protein by immunoblotting with an antipolyhedrin antiserum, and initiation at the AUU was demonstrated by amino terminal protein sequencing. Methods to prevent undesired fusion protein expression using this or similar vectors are discussed.

Expression and characterization of hepatitis B virus precore-core antigen in E. coli.

The hepatitis B virus core antigen, including the precore sequence (HBcAg-p25), was expressed at very high levels in bacteria. Three expression vectors were constructed in which the synthesis of HBcAg-p25 was controlled by the tac promoter, and the number of nucleotides between the bacterial ribosome binding site and the precore initiation codon was varied in order to maximize HBcAg-p25 synthesis. The relative amount of HBcAg-p25 polypeptide expressed by the different vectors was estimated by SDS-polyacrylamide gel electrophoresis and immunoblot. HBcAg-p25 was associated with an insoluble fraction of bacterial extracts and required ionic detergents for solubilization. Comparison by ELISA of the immunoreactivity of HBcAg with and without the precore sequence suggested that human anti-HBcAg IgG preferentially recognizes HBcAg lacking the precore sequence.

Amplification of simian retroviral sequences from human recipients of baboon liver transplants.

Investigations into the use of baboons as organ donors for human transplant recipients, a procedure called xenotransplantation, have raised the specter of transmitting baboon viruses to humans and possibly establishing new human infectious diseases. Retrospective analysis of tissues from two human transplant recipients with end-stage hepatic disease who died 70 and 27 days after the transplantation of baboon livers revealed the presence of two simian retroviruses of baboon origin, simian foamy virus (SFV) and baboon endogenous virus (BaEV), in multiple tissue compartments. The presence of baboon mitochondrial DNA was also detected in these same tissues, suggesting that xenogeneic "passenger leukocytes" harboring latent or active viral infections had migrated from the xenografts to distant sites within the human recipients. The persistence of SFV and BaEV in human recipients throughout the posttransplant period underscores the potential infectious risks associated with xenotransplantation.

Intracellular processing of apo(a) in primary baboon hepatocytes.

We have developed a serum-free medium for the long-term culture of highly differentiated primary baboon hepatocytes. Hepatocytes isolated from animals with defined plasma Lp(a) levels and apo(a) glycoprotein phenotypes were used to study the assembly of Lp(a). A combination of steady-state and pulse-chase labeling studies and endoglycosidase digests demonstrated that apo(a) was synthesized as a lower molecular weight precursor. After a prolonged period of time in the endoplasmic reticulum, apo(a) was converted to a mature form and secreted. A proportion of mature apo(a) also had a prolonged residence time in the trans Golgi apparatus. In all experiments, apoB co-immunoprecipitated with apo(a) from the culture medium but not from the cell lysates, supporting an extracellular association of the proteins for the formation of Lp(a). Analysis of hepatic RNA from 29 'null' Lp(a) phenotype baboons revealed that one-third of the animals had detectable apo(a) transcripts, whereas the remainder had no detectable apo(a) mRNA. The baboon hepatocyte system therefore represents a valuable model to examine the effect of allelic variation at the apo(a) locus on Lp(a) assembly.

Strand-Specific rTth RT-PCR for the Analysis of HCV Replication.

Because of the very low level of HCV present in the serum of infected individuals, as well as the low level of replication in the host, reverse transcription-polymerase chain reaction (RT-PCR) assays are the only method suitable for the routine detection of HCV RNA. The use of RT-PCR to monitor HCV replication in vivo as well as the in vitro inoculation of cultured cells presents unique problems. The extreme sensitivity of PCR permits the detection of HCV RNA in tissues not permissive for replication of the virus, and following in vitro infections, the residual inoculum can be detected for extended time periods depending on the sensitivity of the PCR procedure. Since HCV is a positive-stranded RNA virus, the detection of negative-strand RNA should be indicative of active viral RNA replication, assuming that the inoculum contains primarily positive-strand RNA. Early attempts to detect negative-strand RNA employed a strand-specific PCR technique that utilized only one primer during cDNA synthesis, followed by inactivation of the RT and amplification of the cDNA by PCR. During the course of our studies, we found this technique to lack significant strand specificity using synthetic RNA. A series of experiments suggested that the lack of specificity was probably owing to a combination of factors, including false priming of the incorrect strand (e.g., the positive strand in a negative-strand assay) by the cDNA primer, self-priming of the RNA, and random priming by extraneous nucleic acids (illustrated in Fig. 1 A). Fig. 1 Schematic diagram of conventional strand-specific RT-PCR and rTth RT-PCR procedures. (A) The amplification of HCV negative-strand RNA using conventional strand-specific RT-PCR and the false amplification of the HCV positive-strand RNA by the same procedure A cDNA copy of the RNA is made with primer complementary to negative-strand RNA. At the reduced temperatures used for cDNA synthesis, this primer can misanneal at sites on the positive-strand RNA with partial homology to the primer. cDNA synthesis can also be primed by contaminating nucleic acids and by terminal hairpin structures that can occur anywhere in genome if the RNA is partially degraded Following cDNA synthesis, the RT activity is inactivated and PCR amplification is performed with Taq. False-primed positive-strand RNA yields the same product as negative-strand RNA provided that the cDNA produce from false priming spans the sequence encompassed by the PCR primers. (B) The amplification of HCV negative-strand RNA by rTth RT-PCR is depicted A cDNA copy of the RNA is made with a primer complementary to the negative strand of RNA using the rTth thermostable reverse transcriptase at 70°C The RT activity of rTth is inactivated by chelation of Mn(2+) with EGTA. Following the addition of Mg(2+) and the reverse primer, PCR is conducted using the thermostable DNA polymerase activity of rTth.

A cultivation method for highly differentiated primary chimpanzee hepatocytes permissive for hepatitis C virus replication.

The liver performs a wide array of functions, a few of which include the synthesis and secretion of most of the plasma proteins, including the lipoproteins, cholesterol, and bile acid metabolism, and detoxification of the blood. In vitro analysis of most liver functions has been hampered by the difficulties encountered in isolating and maintaining functional cultures of primary hepatocytes. Although in vivo the liver has an amazing capacity for regeneration, hepatocytes in culture have limited proliferation capacity and are normally short-lived. We have developed methods for the isolation and cultivation of highly differentiated primate hepatocyte cultures that can be maintained for over 100 d without significant loss of differentiated function. This system has been used in our lab for the analysis of lipoprotein synthesis and hepatotropic virus replication. This chapter is designed to provide a detailed methodology of our approach. Numerous alternative hepatocyte cultivation systems have been described, but owing to space limitations, these systems will not be described here. A number of excellent reviews are dedicated to this subject, one of which is in a previous volume of this series (1), and another that is an entire book dedicated to the subject (2).

GB virus B as a model for hepatitis C virus.

GB viruses A and B (GBV-A and GBV-B) are members of the Flaviviridae family and are isolated from tamarins injected with serum from a human hepatitis patient. Along with a related human virus, GB virus C, or alternatively, hepatitis G virus (GBV-C/HGV), the three viruses represent the GB agents. Of the three viruses, GBV-B has been proposed as a potential surrogate model for the study of hepatitis C virus (HCV) infections of humans. GBV-B is phylogenetically most closely related to HCV and causes an acute, self-resolving hepatitis in tamarins as indicated by an increase in alanine aminotransferase and changes in liver histology. Similarities between GBV-B and HCV are found at the nucleotide sequence level with the two viruses sharing 28% amino acid homology over the lengths of their open reading frames. Short regions have even higher levels of homology that are functionally significant as shown by the ability of the GBV-B NS3 protease to cleave recombinant HCV polyprotein substrates. The shared protease substrate specificities suggest that GBV-B may be useful in testing antiviral compounds for activity against HCV. Although there are numerous similarities between GBV-B and HCV, there are important differences in that HCV frequently causes chronic infections in people, whereas GBV-B appears to cause only acute infections. The acute versus chronic course of infection may point to important differences between the two viruses that, along with the numerous similarities, will make GBV-B in tamarins a good surrogate model for HCV.

The chimpanzee model of hepatitis C virus infections.

The chimpanzee (Pan troglodytes) is the only experimental animal susceptible to infection with hepatitis C virus (HCV). The chimpanzee model of HCV infection was instrumental in the initial studies on non-A, non-B hepatitis, including observations on the clinical course of infection, determination of the physical properties of the virus, and eventual cloning of the HCV nucleic acid. This review focuses on more recent aspects of the use of the chimpanzee in HCV research. The chimpanzee model has been critical for the analysis of early events in HCV infection because it represents a population for which samples are available from the time of exposure and all exposed animals are examined. For this reason, the chimpanzee represents a truly nonselected population. In contrast, human cohorts are often selected for disease status or antibody reactivity and typically include individuals that have been infected for decades. The chimpanzee model is essential to an improved understanding of the factors involved in viral clearance, analysis of the immune response to infection, and the development of vaccines. The development of infectious cDNA clones of HCV was dependent on the use of chimpanzees, and they will continue to be needed in the use of reverse genetics to evaluate critical sequences for viral replication. In addition, chimpanzees have been used in conjunction with DNA microarray technology to probe the entire spectrum of changes in liver gene expression during the course of HCV infection. The chimpanzee will continue to provide a critical aspect to the understanding of HCV disease and the development of therapeutic modalities.

Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector.

Simian virus 40 (SV40) large T and small t antigens were synthesized in insect cells using the baculovirus Autographa californica as an expression vector. A recombinant virus containing a genomic copy of the SV40 early region expressed high levels of small t antigen but only low levels of large T antigen. However, very high levels of T antigen synthesis were observed when viruses were constructed with a cDNA copy of the large T antigen mRNA. Insect cells were capable of modifying T antigen by phosphorylation, palmitylation, glycosylation, and oligomerization. Functional assays demonstrated that the origin-specific DNA binding, ATPase, and helicase activities of insect cell-derived T antigen were comparable to T antigen synthesized in mammalian cells. Use of the baculovirus vector system to produce T antigen should facilitate future investigations requiring large quantities of T antigen.