Robert E. Lanford

Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.

Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal

A system was developed for the analysis of protein transport to the nucleus. Carrier proteins cross-linked to synthetic peptides were microinjected into the cytoplasm of mammalian cells, and protein transport was evaluated by immunofluorescence staining of fixed cells. A 13-mer synthetic peptide containing seven amino acids homologous to SV40 T antigen was capable of inducing nuclear transport, but no transport was observed when proteins were coupled with a synthetic peptide homologous to a nuclear-transport-defective T antigen. The largest protein-peptide conjugate efficiently transported was ferritin (Mr 465,000). The rate of transport was influenced by the number of peptides per molecule of carrier protein and, to some degree, by the size of the carrier protein. Transport of some conjugates was almost complete in 15 min at room temperature.

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.

Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium

SummaryThe analysis of lipoprotein synthesis and secretion in primary hepatocytes has been restricted by the short-term viability and low proliferative response of hepatocytes in vitro. During this investigation a serum-free medium formulation was developed that supports long-term maintenance (>70 d) and active proliferation of primary baboon hepatocytes. Examination of proliferating cells by electron microscopy revealed a distinctive hepatocyte ultrastructure including intercellular bile canaliculi and numerous surface microvilli. High levels of secreted apolipoproteins A-I and E were detected in the tissue culture medium by gel electrophoresis and immunoblot analysis. Immunoprecipitation of proteins from [35S]-methionine labeled tissue culture medium revealed the synthesis and secretion of numerous plasma proteins. Metabolic labeling of cells with [35S]-methionine followed by single-spin density gradient flotation of the media demonstrated that apolipoproteins were being secreted in the form of lipoprotein particles with buoyant densities corresponding to the very low density lipoprotein and low density lipoprotein range, and to the high density lipoprotein range. The labeled apolipoproteins included Bh, E, and A-I. This system for primary hepatocyte culture should prove very useful in future investigations on the regulation of lipoprotein production by hepatocytes.

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.

Antigenic relationship of SV40 early proteins to purified large T polypeptide.

Abstract Rabbit antiserum was produced against SV40 large T antigen purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. This antiserum immunoprecipitated both large T and small t antigens, and reacted with SV40 T, U, and S antigens by immunofluorescence. these data establish the antigenic relatedness of all the known SV40 early gene products, with the exception of transplantation antigen activity, and confirm the virus-specific nature of each. The reactivity of the anti-T polypeptide serum was compared with the specificities of T-reactive antisera produced by different methods, including conventional tumor-bearing hamster sera, rabbit antiserum directed against whole-cell SDS-lysates of SV40-transformed rabbit kidney cells, and high-titer ascites fluid from hamsters in which ascites was induced by injection of SV40-transformed hamster ascites cells. Each of the antisera was reactive in all of the tests for SV40-induced early antigens, but the relative reactivity toward each protein varied considerably. It is postulated that the differences in reactivity to small t antigen and U antigen represent differences in the immune response of individual animals to the amino and carboxyl termini of the large T antigen polypeptide, respectively. Antiserum produced against the SDS-denatured large T polypeptide exhibited the highest reactivity to both small t and U antigenic sites relative to its reactivity against intranuclear large T antigen.

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.

Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity.

The hepatitis B virus core open reading frame with and without the precore domain was expressed in insect cells using a baculovirus expression system. Precore antigen was not properly processed in insect cells and was present in highly insoluble cytoplasmic aggregates. Core antigen without the precore domain formed core particles with a diameter of 28 nm that were secreted into the medium. Both core and precore antigens were phosphorylated in insect cells. The immune response in mice to both antigens yielded antibodies with a high degree of preferential reactivity for the homologous immunizing polypeptide. A kinase activity that phosphorylated core antigen was associated with highly purified core particles. The kinase activity resembled that previously demonstrated for core particles purified from the cytoplasm of infected hepatocytes and detergent-treated Dane particles. Partial resistance of the phosphate-label to phosphatase treatment suggested that some of the phosphorylated sites are in the interior of the particle. The presence of kinase activity in recombinant core particles demonstrated that this activity is not derived from another hepatitis B virus-encoded polypeptide, and the lack of a kinase consensus sequence in the core open reading frame suggests that the kinase is of cellular origin.

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.

Comparison of diverse transport signals in synthetic peptide-induced nuclear transport

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.