Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Synthesis of surfactant-associated protein, 35,000 daltons, in fetal lung

The major human pulmonary surfactant-associated protein of 35,000 daltons (Da) (SAP-35), consists of a group of related proteins of 27,000-36,000 Da, with isoelectric points ranging from pH 4.6 to 5.2. SAP-35 precursors were identified by immunoprecipitation of protein products of in vitro translation of normal adult human poly(A)+ mRNA with human SAP-35 antiserum. The translation products nearly comigrated with the most basic components of alveolar SAP-35 (mol mass = 24,500-27,000 Da). Processing of the primary translation products by canine pancreatic microsomal membranes increased their apparent molecular weight to 29,000-30,000-Da forms, which were sensitive to endoglycosidase F, suggesting the addition of asparagine-linked oligosaccharides to the molecules. A smaller protein of 24,500 Da was generated during treatment with canine microsomal membranes likely representing cleavage of a signal peptide. SAP-35 was not detected in explants of [35S]methionine-labeled fetal lung (20-24 wk gestation) after 1 day of culture or immunoprecipitates of in vitro translated poly(A)+ mRNA isolated from fetal human lung. However, after 3-5 days of organ culture, synthesis of SAP-35 was readily detected by immunoprecipitation of [35S] methionine-labeled tissue. Fully sialylated (neuraminidase-sensitive forms) comigrated with fully glycosylated SAP-35 isolated from human surfactant. High mannose (endoglycosidase H-sensitive precursors) were also synthesized by the organ cultures and were distinct from the secreted form in surfactant. Synthesis of surfactant-associated SAP-35 and its precursors was induced in association with morphological maturation of the type II epithelial cell during organ culture of human fetal lung.     

Primary translation products, biosynthesis, and tissue specificity of the major surfactant protein in rat

Rat lung tissue was labeled with [35S]methionine and the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (b) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S]methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.     

Synthesis of surfactant-associated glycoprotein A by rat type II epithelial cells. Primary translation products and post-translational modification

Surfactant-associated glycoproteins A, 38 (A3), 32 (A2) and 26 (A1) kDa, pI (4.2-4.8), were identified as related proteins present in surfactant isolated from rat lung lavage fluid. Differences in size and charge among surfactant-associated glycoproteins A were related to differences in glycosylation as determined by reduction of the larger forms (38 and 32 kDa) to 26 kDa by endoglycosidase F and by increased isoelectric points of the glycosylated forms after treatment with neuraminidase. Synthesis and secretion of surfactant-associated glycoproteins A and precursors were demonstrated in purified rat Type II epithelial cells by immunoprecipitation of [35S]methionine-labelled proteins with anti-surfactant-associated glycoprotein A antisera. In pulse-chase experiments, labelled proteins 26-34 kDa, appeared within 10 min and smaller forms co-migrated with surfactant-associated glycoprotein A from alveolar lavage. The relative abundance of the larger molecular mass forms (30-34 kDa, pI 4.8) increased at later times up to 3 h. More acidic mature forms, which co-migrated with surfactant-associated glycoproteins A2 and A3 in surfactant (38 and 32 kDa), were readily detectable in the media, but were not abundant forms in lysates of labelled Type II cells after 1-3 h of incubation. Primary translation products of surfactant-associated glycoprotein A were immunoprecipitated with monospecific anti-surfactant-associated glycoprotein A antiserum after in vitro translation of poly(A)+ mRNA isolated from adult rat lung. The immunoprecipitated translation product migrated at 26 kDa, pI 4.8, and migrated slightly faster than surfactant-associated glycoprotein A1 from surfactant. Treatment of surfactant-associated glycoprotein A with bacterial collagenase resulted in proteolytic fragments 23-20 kDa, pI 4.2-4.8, which no longer underwent sulfhydryl-dependent cross-linking, suggesting that the collagen-like domain was required for the sulfhydryl-dependent oligomerization. Surfactant-associated glycoproteins A are synthesized by rat Type II epithelial cells as pre-proteins, 26-34 kDa. Larger forms result primarily from N-linked glycosylation of the 26 kDa primary translation product. Mature, more acidic forms result from further addition of sialic acid.     

In vitro sulfation of pulmonary surfactant-associated protein-35

Surfactant-associated protein-35 consists of a group of phospholipid-associated proteins of 26-36 kDa isolated from pulmonary alveolar surfactant. In the rat, surfactant-associated protein-35 is synthesized from 26-kDa primary translation products which are cotranslationally acetylated and glycosylated to heterogeneous 30 and 34 kDa forms. High-mannose oligosaccharide-containing precursors of surfactant-associated protein-35 are processed in the rough endoplasmic reticulum and Golgi to complex-type oligosaccharides, resulting in a mature glycoprotein which exhibits extensive charge heterogeneity in two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis. Much of this charge heterogeneity is related to terminal sialylation of the two asparagine-linked oligosaccharides. In the present study, we report that surfactant-associated protein-35 is also sulfated. Sulfation of the 30 and 34 kDa forms of surfactant-associated protein-35 was clearly detected in primary cultures of rat Type II epithelial cells. These sulfated isoforms were sensitive to endoglycosidase F digestion, but resistant to neuraminidase, suggesting that sulfation occurred at oligosaccharide residues other than sialic acid. The lack of sulfation of the 26 kDa forms of surfactant-associated protein-35 and the resistance of the sulfated isoforms to endoglycosidase H digestion are consistent with Golgi-associated sulfation of the complex type oligosaccharides of surfactant-associated protein-35. Thus, sulfation is another component of the complex post-translational processing of surfactant-associated protein-35, which includes acetylation, hydroxylation, glycosylation, sialylation, sulfhydryl-dependent oligomerization and sulfation.     

A comparison of the major surfactant-associated proteins in different species

We examined the major surfactant-associated proteins in a number of species by using two-dimensional gel electrophoresis, protein blotting and immunostaining. All species have a 30,000 to 35,000 mol. wt protein group consisting of multiple isoforms with isoelectric points ranging from pH 4.4 to 5.6. The techniques used in this study have resolved three component subgroups within the 35 K group. A group of proteins at 60,000-65,000 mol. wt has also been consistently identified. We conclude that remarkable similarity exists among the major surfactant-associated proteins from various mammals with regard to isoelectric points, molecular weights and antigenic sites.     

An electrophoretic and immunochemical characterization of human surfactant-associated proteins

We have prepared an antiserum against a serum-free extract of alveolar proteinosis lavage that recognizes the same proteins as an antiserum to human surfactant. Using one and two-dimensional gel electrophoresis, protein blotting and immunostaining we have found proteins with Mr of approx. 35 and 60 kDa to be present in every source of human surfactant we have examined. These proteins are immunologically related to those found in the lavage from alveolar proteinosis patients, have the same electrophoretic characteristics and are not found in serum. The 35 kDa protein is a group of at least eight isoforms ranging in relative molecular mass Mr from 32 to 36 kDa with isoelectric points between 4.8 and 5.5. Neuraminidase digestion studies have shown that at least part of this charge heterogeneity may be due to sialic acid residues. The less abundant form, with a Mr of about 60 kDa is also a sialoglycoprotein with similar isoelectric points.     

Glycosylation and secretion of surfactant-associated glycoprotein A

Synthesis of glycoprotein A, the major surfactant-associated protein, was demonstrated in Type II epithelial cells isolated from rat lung. Predominant, secreted forms migrated as glycoproteins with asparagine-linked, complex-type oligosaccharides (32,000-36,000 daltons, pI 4.2-4.8). Primary in vitro translation products of the glycoprotein migrated as five distinct proteins of approximately 26,000 daltons which were processed by pancreatic microsomal membranes in vitro to 30,000-34,000-dalton, endoglycosidase F-sensitive forms. These in vitro processed forms of glycoprotein A co-migrated with intracellular forms immunoprecipitated from [35S]methionine-labeled, Type II cells. Pulse-chase experiments with [35S]methionine-labeled cells demonstrated rapid synthesis of endoglycosidase H-sensitive precursors of 34,000 daltons, pI 4.7-4.8, which were neither secreted from Type II cells nor detected in surfactant from alveolar lavage. These high-mannose forms were slowly processed to more acidic, endoglycosidase H-resistant, neuraminidase-sensitive forms. At between 10 and 180 min, fully sialylated or other endoglycosidase H-resistant forms were a minor fraction of intracellular glycoprotein A. After 16 h, intracellular glycoproteins A were primarily present as endoglycosidase H-resistant forms. Secretion of mature, sialylated, glycoprotein A was first detected 1 h after labeling, and was also readily detected after 16-24 h chase period. Tunicamycin, which blocks N-linked protein glycosylation, resulted in synthesis of three major 26,000-dalton proteins which co-migrated with the nonglycosylated, surfactant-associated proteins A1 present in surfactant from alveolar lavage and with the major in vitro translation products of rat lung poly(A+) mRNA. Tunicamycin inhibited secretion of glycoprotein A. Swainsonine, which inhibits Golgi alpha-mannosidase II, completely inhibited synthesis of the fully sialylated molecule. Swainsonine produced forms of glycoprotein A which were both neuraminidase- and endoglycosidase H-sensitive and were readily secreted. Monensin, an ionophore that alters protein transport, markedly inhibited intracellular sialylation and secretion. These studies demonstrate that pulmonary Type II cells rapidly synthesize and process surfactant-associated glycoprotein A precursors to endoglycosidase H-sensitive forms, which are slowly sialylated prior to secretion.     

Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages

Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M. tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M. tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ubiquitin or LAMP1 increased in Coro1a KD macrophages during infection. These results demonstrate the formation of autophagosomes around M. tuberculosis in Coro1a KD macrophages. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced in response to M. tuberculosis infection in Coro1a KD macrophages, suggesting that Coro1a blocks the activation of the p38 MAPK pathway involved in autophagosome formation. LC3 recruitment to M. tuberculosis-containing phagosomes was also observed in Coro1a KD alveolar or bone marrow-derived macrophages. These results suggest that Coro1a inhibits autophagosome formation in alveolar macrophages, thereby facilitating M. tuberculosis survival within the lung.     

Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoclonal antibodies

Using a monoclonal antibody and affinity-purified polyclonal antiserum against a 10 KD protein isolated from rat pulmonary lavage, we have localized the protein within Clara cells by a post-embedment protein A-gold technique. The gold particles were localized over the secretory granules of rat Clara cells. Ultrastructural immunolocalization was abolished when the primary antibodies were previously absorbed with purified 10 KD protein. Other pulmonary cells, including type II pneumocytes and ciliated cells, were negative with this technique. These results demonstrate the presence of the 10 KD protein in the secretory granules of the Clara cell and support the concept that this protein constitutes a specific and unique secretory product of Clara cells.     

Structural relationships of the major glycoproteins from human alveolar proteinosis surfactant

Alveolar proteinosis is a disease characterized by accumulation of proteinaceous material in the alveolar space of the lung. Two major collagenase-sensitive polypeptides, alveolar proteinosis peptides of 34 kDa kilodaltons (APP-34) and of 62 kDa (APP-62), were isolated from bronchioalveolar lavage of patients with alveolar proteinosis. These proteins co-purified during fast-performance liquid chromatography (FPLC) chromatofocusing and were separated from each other by electroelution following SDS-polyacrylamide gel electrophoresis. Immunoblot analysis of these proteins demonstrated that both shared antigenic sites with the normal human surfactant-associated protein of Mr 34,000 (SAP-34) using both polyclonal and monoclonal antibodies generated against SAP-34. Removal of asparagine-linked oligosaccharides from the 34 kDa and 62 kDa alveolar proteinosis proteins with endoglycosidase F resulted in polypeptides of 28 kDa from APP-34 and 56 kDa from APP-62. Amino acid analysis and tryptic peptide maps of the electroeluted APP-34 and APP-62 proteins were essentially identical and similar to that previously reported for human SAP-34, supporting the likely relationship of APP-34 and APP-62 as monomer and dimer of the normal SAP-34. APP-34 and APP-62 were both sensitive to bacterial collagenase, yielding collagenase-resistant fragments of 21 kDa, similar in migration and amino acid composition to the fragment generated by collagenase digestion of normal human SAP-34. High molecular weight aggregates of APP-34 and APP-62 were the result of sulfhydryl-dependent and non-sulfhydryl-dependent cross-linking. A domain in the C-terminal non-collagenous portion of the molecules which forms sulfhydryl-dependent oligomers was identified. The two major polypeptides accumulating in the airway of patients with alveolar proteinosis are monomeric (34 kDa) and dimeric (62 kDa) forms of the major surfactant-associated glycoprotein, SAP-34.     

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

Role of alveolar type II cells and of surfactant-associated protein C mRNA levels in the pathogenesis of respiratory distress in mink kits infected with Aleutian mink disease parvovirus.

Neonatal mink kits infected with Aleutian mink disease parvovirus (ADV) develop an acute interstitial pneumonia with clinical symptoms and pathological lesions that resemble those seen in preterm human infants with respiratory distress syndrome and in human adults with adult respiratory distress syndrome. We have previously suggested that ADV replicates in the alveolar type II epithelial cells of the lung. By using double in situ hybridization, with the simultaneous use of a probe to detect ADV replication and a probe to demonstrate alveolar type II cells, we now confirm this hypothesis. Furthermore, Northern (RNA) blot hybridization showed that the infection caused a significant decrease of surfactant-associated protein C mRNA produced by the alveolar type II cells. We therefore suggest that the severe clinical symptoms and pathological changes characterized by hyaline membrane formation observed in ADV-infected mink kits are caused by a dysfunction of alveolar surfactant similar to that observed in respiratory distress syndrome in preterm infants. However, in the infected mink kits the dysfunction is due to the replication of ADV in the lungs, whereas the dysfunction of surfactant in preterm infants is due to lung immaturity.     

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Summary Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit antiadult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA.Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 dalton proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit anti-adult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA. Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 daltons proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

Primary structure differences of human surfactant-associated proteins isolated from normal and proteinosis lung.

The primary structures of human pulmonary surfactant-associated proteins SP-A, SP-B and SP-C isolated from lung lavage of patients with alveolar proteinosis exhibit significant differences from lung surfactant proteins isolated from lungs of healthy individuals. In contrast to SP-A from normal lungs, proteinosis SP-A was shown by SDS gel electrophoresis to contain large amounts of unreducibly cross-linked beta chains. Specific primary structure modifications of SP-C and SP-B proteins were established by direct molecular weight and structural analysis, using [252Cf]plasma desorption mass spectrometry (PD/MS) as the principal method. In comparison to normal lung surfactant SP-B, proteinosis SP-B showed a significantly increased molecular weight by approx. 500 Da for the unreduced protein dimer. SP-C proteins from normal lungs were identified to possess a bis-cysteinyl-5,6-(thioester)palmitoylated structure, and to contain a frayed N-terminus resulting in two sequences of 34 and 35 amino acid residues. In contrast, SP-C from proteinosis patients was modified by (i) partial or even complete removal of palmitate residues and (ii) additional N-terminal proteolytic degradation. These results indicate the presence of pathophysiological structure modifications, which are likely to occur in the alveolar space, and may lead to a reduced surfactant function.     

Identification and characterization of the herpes simplex virus type 1 virion protein encoded by the UL35 open reading frame.

The UL35 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) has been predicted from DNA sequence analysis to encode a small polypeptide with a molecular weight of 12,095. We have investigated the protein product of the UL35 ORF by using a trpE-UL35 gene fusion to produce a corresponding fusion protein in Escherichia coli. The TrpE-UL35 chimeric protein was subsequently isolated and used as a source of immunogen for the production of rabbit polyclonal antiserum directed against the UL35 gene product. The TrpE-UL35 antiserum was found to recognize a 12-kDa protein which was specifically present in HSV-1-infected cells. By utilizing the TrpE-UL35 antiserum, the kinetics of synthesis of the UL35 gene product was examined, and these studies indicate that UL35 is expressed as a gamma 2 (true late) gene. The 12-kDa protein recognized by the TrpE-UL35 antiserum was associated with purified HSV-1 virions and type A and B capsids, suggesting that the UL35 ORF may encode the 12-kDa capsid protein variably designated p12, NC7, or VP26. To confirm this assignment, immunoprecipitation and immunoblotting studies were performed to demonstrate that the TrpE-UL35 antiserum reacts with the same polypeptide as an antiserum directed against the purified p12 capsid protein (anti-NC7) (G.H. Cohen, M. Ponce de Leon, H. Diggelmann, W.C. Lawrence, S.K. Vernon, and R.J. Eisenberg, J. Virol. 34:521-531, 1980). Furthermore, the anti-NC7 serum was also found to react with the TrpE-UL35 chimeric protein isolated from E. coli, providing additional evidence that the UL35 gene encodes p12. On the basis of these studies, we conclude that UL35 represents a true late gene which encodes the 12-kDa capsid protein of HSV-1.