Monoclonal antibody inhibition of Neospora caninum tachyzoite invasion into host cells

Monoclonal antibodies were produced against Neospora caninum tachyzoites to identify antigens which may play a role during invasion of host cells. Confocal laser microscopy showed that most antigens recognised by the mAb were located on the surface, but one mAb, 1A5, reacted to the apical end of the parasite. Some mAbs, which recognised 70, 42 and 36kDa parasite proteins, significantly inhibited the invasion of the parasite in vitro. The mAbs which recognised 42 and 36kDa parasite protein, reacted with Nc-p43 and Nc-p36 expressed by vaccinia virus and Escherichia coli, respectively. These results suggest that a 70kDa protein, Nc-p43 and Nc-p36 are involved in the invasion of the parasite into host cells.     

Identification of proteins in Encephalitozoon intestinalis, a microsporidian pathogen of immunocompromised humans: an immunoblotting and immunocytochemical study

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube--the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.     

Trypanosoma cruzi disrupts myofibrillar organization and intracellular calcium levels in mouse neonatal cardiomyocytes

Immunofluorescence studies of normal and Trypanosoma cruzi-infected primary cultures of heart muscle cells were performed to gather information about the arrangement of myofibrillar components during the intracellular life cycle of this parasite. By using a panel of monoclonal antibodies against various myofibrillar proteins, a progressive disruption and loss of contractile proteins (such myosin and actin) of the host cell was detected during infection. The host cell formed a loose network of myofibrillar proteins around the parasites. Breakdown of the myofibrils occurred in regions where the parasites were present, and heavily infected cells showed myofibrillar proteins at their periphery. In parallel, we investigated the effect of T. cruzi infection on intracellular calcium levels by using a Ca²⁺ fluorescent indicator (confocal microscopy). Infected cardiomyocytes displayed a marked impairment in contractility, and calcium influxes became irregular and less intense when compared with those of non-infected cells. Our results demonstrate that T. cruzi infection dramatically affects calcium fluxes and causes myofibrillar breakdown disturbing cardiomyocyte contractility.Financial support through grants and scholarships from the Brazilian funding agencies FAPESP, CNPq, and CAPES is gratefully acknowledged.     

Trichinella spiralis shares epitopes with human autoantigens

Like other helminths, Trichinella spiralis has evolved strategies to allow it to survive in the host organism, including the expression of epitopes similar to those present in either expressed or hidden host antigens. To identify T. spiralis-derived antigens that are evolutionarily conserved in the parasite and its host and that could be responsible for its evasion of the host immune response, we examined the reactivity of six different types of autoantibodies to T. spiralis larvae from muscle. T. spiralis antigens that share epitopes with human autoantigens were identified by assessing the cross-reactivity of autoantibody-containing serum samples with T. spiralis antigens in the absence of specific anti-parasite antibodies. Of the 55 autoantibody-containing human serum samples that we analysed by immunohistological screening, 24 (43.6%) recognised T. spiralis muscle larvae structures such as the subcuticular region, the genital primordium or the midgut. Using Western blots, we demonstrated that the same sera reacted with 24 protein components of T. spiralis muscle larvae excretory-secretory L1 antigens. We found that the human autoantibodies predominantly bound antigens belonging to the TSL1 group; more specifically, the autoantibody-containing sera reacted most frequently with the 53-kDa component. Thus, this protein is a good candidate for further studies of the mechanisms of T. spiralis-mediated immunomodulation.     

The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm

The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM- staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions.     

Identification of proteins specific for human herpesvirus 6-infected human T cells.

Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [35S]methionine- and [3H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [35S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 135,000-Mr polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105k and gp82k, gp116k, gp64k, and gp54k, and gp102k.     

A role for protease activity and host-cell permeability during the process of Trypanosoma cruzi egress from infected cells

The mechanism by which Trypanosoma cruzi egresses from infected cells at the end of the intracellular replication cycle is not understood. This study explored the role of T. cruzi-derived proteases and host-cell membrane permeability during the parasite's egress process. Treatment with a fluoromethyl ketone, known to inhibit the parasite's major protease, significantly reduced parasite egress. In addition, in the late stages of intracellular infection, cells infected with T. cruzi showed increased permeability as evidenced by dye exclusion tests. Furthermore, parasites could be antibody stained inside host cells without chemical permeabilization of the plasma membrane. These results suggest that in advanced stages of the intracellular cycle of T. cruzi, the host cells lose membrane integrity. Previous studies in our laboratory have found that antibodies present in sera of mice chronically infected with T. cruzi (antiegressin) bind the surface of infected cells and reduce parasite egress. In agreement with these reports, western blot analysis showed that several proteins in infected cell membrane extracts reacted with antibodies from infected mouse serum. The findings reported herein might have implications in the process of T. cruzi egress, as well as in the mechanism of action of antiegressin.     

Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s).

Antibodies were raised against the sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met -Glu, which represents a part of the middle T antigen of polyomavirus that is considered to be important in inducing the phenotype of transformed cells. The antibodies reacted with native as well as denatured middle T antigens. In addition, the antibodies immunoprecipitated a cellular protein with an apparent molecular weight of 130,000 (130K) from mouse and rat cells. In some cases, a 33K protein was also immunoprecipitated. Immunoprecipitation of middle T antigen as well as 130K and 33K proteins was blocked by the peptide. The antibodies labeled microfilaments of untransformed mouse, rat, human, and chicken cells by immunofluorescence. This labeling was also blocked by the peptide. The labeling pattern and distribution under a variety of conditions were indistinguishable from those of anti-actin antibodies, although no evidence has been obtained to indicate that the anti-peptide antibodies react with actin. The 130K protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than chicken gizzard vinculin (130K) and slightly faster than myosin light-chain kinase of chicken smooth muscle (130K). Neither of these proteins absorbed the anti-peptide antibodies. The 33K protein does not seem to be tropomyosin (32K to 40K).     

Biogenesis of transverse tubules in skeletal muscle in vitro

The transverse (T) tubules of skeletal muscle are membrane tubules that are continuous with the plasma membrane and penetrate the mature muscle fiber radially to carry surface membrane depolarization to the sites of excitation-contraction coupling. We have studied the development of the T-tubule system in cultured amphibian and mammalian muscle cells using a fluorescent lipid probe and antibodies against T-tubules and plasma membranes. Both the lipid probe and the T-tubule antibody recognized an extensive tubular membrane system which subsequently differentiated into the T-system. At all developmental stages, the molecular composition of the T-system was distinct from that of the plasma membrane, suggesting that during myogenesis T-tubules and the plasma membrane form independently from each other and that exchange of membrane proteins between the two continuous compartments is restricted. In rat muscle cultures, T-tubule-specific antigens were first expressed in terminally differentiated myoblasts. Prior to myoblast fusion the antigens appeared as punctate label throughout the cytoplasm. Shortly after fusion the T-tubule-specific antibody labeled a tubular membrane system that extended from the perinuclear region and penetrated most parts of the cells. In contrast, the lipid probe, which labels the T-tubules by virtue of their direct continuity with the plasma membrane, only labeled short tubules extending from the plasma membrane into the periphery of the myotubes at the early stage in development. Thus, the assembly of the T-tubules appears to begin before their connections with the plasma membrane are established.     

SARCOPLASMIC RETICULUM AND T TUBULES IN DIFFERENTIATING RAT SKELETAL MUSCLE

An electron microscope study has been made of the distribution of membrane couplings between the sarcoplasmic reticulum (SR) and either the plasmalemma or the T tubules in fetal and neonatal rat intercostal muscle. Within primitive muscle cells at 12 days of gestation, the SR forms both simple and specialized membrane junctions with the plasmalemma; caveolae are very few, and T tubules are not detected. Undifferentiated cells neighbor muscle cells. Occasionally these cells contain subsurface couplings between the endoplasmic reticulum and plasmalemmae. Possible relationships between these couplings and the peripheral couplings of muscle cells are discussed. By 15–18 days of gestation, caveolae and beaded T tubules, comparable to those of cultured muscle, develop; T tubules lie along-side myofibrils and are rarely transverse. SR couples both to T tubules and to plasmalemmae during this period. T tubules with lineal profiles appear after further development and their orientation transverse to A–I junctions becomes increasingly evident. Membrane couplings between SR and T tubules also increase in number, whereas the incidence of peripheral coupling declines rapidly Evidence suggests that peripheral couplings are swept into myotubes as caveolae proliferate and T tubules form. SR thus appears to initially couple with the plasmalemma and then to await T tubular growth. This contrasts with the developmental pattern described in cultured chick muscle in which peripheral couplings are not reported and T tubules with diads and triads occur at very primitive stages of muscle differentiation.     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Detection of surface-associated and intracellular glycoconjugates and glycoproteins in Neospora caninum tachyzoites

The surface-associated molecules of the invasive stages of apicomplexan parasites such as Neospora caninum and Toxoplasma gondii are most likely crucially involved in mediating the interaction between the parasite and its host cell. In N. caninum, several antigens have recently been identified which could participate in host cell adhesion and/or invasion. These are antigens which are either constitutively expressed on the outer plasma membrane, or antigens which are only transiently localised on the surface as they are expulsed from the secretory vesicles either prior, or after host cell invasion. Some of these proteins have been characterised at the molecular level, and it has been shown that they are, with respect to protein sequences, closely related to homologous counterparts in T. gondii. Nevertheless, there is only a low degree of cross-antigenicity between the two species. In microbial interactions it has been shown that carbohydrates could also play a crucial role in host cell recognition and immunological host parasite interactions. In this study we present data which strongly suggest that the surface of N. caninum tachyzoites is glycosylated. In SDS-PAGE, glycoproteins comigrated largely with glycosylphosphatidylinositol-anchored proteins which were identified using in vivo [3H]ethanolamine labelling followed by autoradiography. The lectin Con A reacted strongly with the surface of these parasites, binding of which is indicative for the presence of N-glycans. Additional surface binding was observed, although only in a subpopulation of all tachyzoites, for wheat germ agglutinin and Jacalin. Intracellular binding sites for Con A were mainly associated with the parasite dense granules. By lectin labelling of Western blots of N. caninum protein extracts, glycoproteins were identified which reacted specifically with the lectins Con A, wheat germ agglutinin, Jacalin and soy bean agglutinin.     

Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell''s dissemination capabilities.     

Structure and composition of tubular aggregates of skeletal muscle fibres

Unusual regions of densely packed membranous tubules known as tubular aggregates (TAs) have been observed in skeletal muscle fibres of mammals under numerous pathological conditions but also in health. Their causality is unclear. It is neither known whether TAs are destructive and should be treated or whether they have a compensating function in an endangered muscle. In spite of many similarities, the histochemical, immunocytochemical and ultrastructural characteristics of tubular aggregates do vary. Histochemistry provided an overall characteristic of TAs as membranous inclusions with a variety of enzymatic activities. Immunocytochemical evidence revealed that tubular aggregates contain miscellaneous proteins and that derive from membranes of sarcoplasmic reticulum and mitochondria. No evidence for the presence of contractile and cytoskeletal proteins in TAs was found. Ultrastructurally, TAs are characterized as more or less densely packed aggregates of vesicular or tubular membranes of variable forms and sizes that may contain amorphous material, filaments or inner tubules. Various reported types of tubular aggregates, namely, proliferating terminal cisterns, vesicular membrane collections, TAs with double-walled tubules, TAs with single-walled tubules, aggregates of dilated tubules with inner tubules, aggregates of tubulo-filamentous structures, filamentous tubules, riesentubuli, and related membranous structures including cylindrical spirals are sumarized and analyzed here in detail.     

Host and pathogen interaction during vaginal infection by Trichomonas vaginalis and Mycoplasma hominis or Ureaplasma urealyticum

Vaginal infections by Trichomonas vaginalis and Mycoplasma hominis have been shown to be associated. Since M. hominis and Ureaplasma urealyticum are similar pathogens, both belonging to the class of the mycoplasmata, we describe here a molecular study into the interdependence of U. urealyticum and T. vaginalis during infection. Susceptibility towards infection by U. urealyticum depends on genetic polymorphism in the interleukin-1 receptor antagonist (IL-1RA) gene. Now, we defined the relation between IL-1RA genotypes and infection by M. hominis and T. vaginalis. Finally, we also developed a restriction fragment length polymorphism (RFLP) tool for mapping variation in the T. vaginalis AP33 adhesin in order to define putative associations between parasite subtype and mycoplasmata or host. Studies using crudepellets from T. vaginalis culture broth clearly confirm the association between T. vaginalis and M. hominis infection. The association between IL-1RA genotype 2,2 and lack of U. urealyticum infection is corroborated as well. U. urealyticum infection and infection by T. vaginalis are independent. Furthermore, T. vaginalis and M. hominis infection are not depending on IL-1RA genotypes. Interestingly, one of the three AP33 RFLP types identified appeared to be associated with the absence of U. urealyticum infection. In conclusion, the complex interaction between bacterial and parasitic pathogens and the infected host is determined by genetic characteristics of host and microorganisms involved.     

Trypanosoma cruzi: antibodies to a MAP-like protein in chronic Chagas' disease cross-react with mammalian cytoskeleton

Trypanosoma cruzi lambda gt 11 library from epimastogote derived mRNA was screened with human chagasic sera or sera from chronically infected mice. Strong reactive recombinants were detected with both sera. Two recombinant clones were studied in more detail and shown to be composed of the same 114-bp repetitive sequence coding for a 38 amino acid repetition. This repetition is the same size and shares greater than 60% homology with the reported T. brucei microtubule associated protein (MAP) p320. The insert of one of these clones, K1-7 (228 bp), was subcloned into pMSgt11 and the soluble recombinant polypeptide expressed. Antibodies against the K1-7 fusion polypeptide recognized a major 110-kDa band from cytoskeleton. Anti K1-7 monospecific antibodies detected several cytoskeletal proteins from 3T3 fibroblasts and bovine brain microtubule preparations. Reciprocally, anti-MAP1b monoclonal antibodies raised against bovine brain microtubule reacted with the K1-7 polypeptide on Western blots. The protein identified by K1-7 antibodies may be one of the parasite molecules associated to molecular mimicry.     

Sarcotubular development in dystrophic skeletal muscle cells

Summary Dilations of the sarcotubular system and misaligned myofilaments have been reported as early indicators of muscular dystrophy in skeletal muscle. Since the developing tubular component is believed instrumental in initial myofilament alignment during myogenesis, tubular development is evaluated using normal and dystrophic chick embryo skeletal muscle and cultures of normal and dystrophic embryonic pectoral muscle incubated in the presence of horse spleen ferritin. Comparisons of the findings show that periodic tubules are absent from dystrophic somitic muscle and that invaginating tubules from the sarcolemma are found in fewer, randomly located areas of dystrophic pectoral muscle cells. The results indicate that the tubular component is not involved in the bizarre vesiculations seen in mature dystrophic muscle, however, the malalignment of dystrophic myofilaments is probably the result of the poorer development of the T system in this muscle.     

Specific human antibodies do not inhibit Trypanosoma cruzi oligopeptidase B and cathepsin B, and immunoglobulin G enhances the activity of trypomastigote-secreted oligopeptidase B

Trypanosoma cruzi expresses oligopeptidase B and cathepsin B that have important functions in the interaction with mammalian host cells. In this study, we demonstrated that sera from both chagasic rabbits and humans have specific antibodies to highly purified native oligopeptidase B and cathepsin B. Levels of antibodies to cathepsin B were higher than those observed to oligopeptidase B by absorbance values recorded upon ELISA. We next showed that 90% and 30% of sera from individuals with mucocutaneous leishmaniasis have antibodies that recognize oligopeptidase B and cathepsin B as antigens, respectively. In addition, 55% and 40% of sera from kala-azar patients have antibodies to oligopeptidase B and cathepsin B, respectively. Sera from malaria patients did not recognize the proteases as antigens. Despite high levels of specific antibodies, sera from T. cruzi-infected patients did not inhibit the activities of either oligopeptidase B or cathepsin B. Furthermore, sera or IgG purified from either infected or non-infected individuals enhanced the enzymatic activity of the secreted oligopeptidase B. Oligopeptidase B secreted by trypomastigotes and cathepsin B released upon parasite lysis retain their enzymatic activities and may be associated with Chagas' disease pathogenesis by hydrolyzing host proteins and inducing host immune responses.     

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.