Adeno-associated virus rep protein synthesis during productive infection.

Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. We studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with [35S]methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.     

Protein synthesis during germination of Allomyces macrogynus mitospores.

Protein synthesis in Allomyces marcogynus mitospores began at the time of encystment and was required for further germination. The rate of protein synthesis in germinating spores decreased in the absence of ribonucleic acid synthesis relative to the uninhibited control. There was a concomitant decrease in polysome content, suggesting that some of the messenger ribonucleic acid synthesized during early germination is translation.     

Synthesis and Turnover of Cytoskeletal Proteins in Cultured Astrocytes

Abstract: We previously reported that the cytoskeleton of rat astrocytes in primary culture contains vimentin, glial fibrillary acidic protein (GFAP), and actin. These proteins were found in a fraction insoluble in Triton X-100 and thought to be assembled in filamentous structures. We now used primary astrocyte cultures to study the kinetics of synthesis and turnover of these cytoskeletal proteins. The intermediate filament proteins were among the most actively synthesized by astrocytes. High levels of synthesis were detectable by the third day of culture in the early log phase of growth, and the pattern of labeling at day 3 was similar to that at 14 days when the cultures had reached confluency. In short-term incorporation experiments vimentin, GFAP, and actin in the Triton-insoluble fraction were labeled within 5 min after exposure of the cultures to radioactive leucine. We did not detect any saturation of labeling for up to 6 h of incubation. The turnover of filament proteins studied by following the decay of radioactivity from prelabeled vimentin, GFAP, and cytoskeletal actin displayed biphasic decay kinetics for all three proteins. In the initial phase a fast-decaying pool with a half-life of 12–18 h contributed about 40% of the total activity in each protein. A major portion, about 60%, of each protein, however, decayed much more slowly, exhibiting a half-life of about 8 days.     

Kinetics of utilization of Sendai virus RNA and protein in the process of virion assembly.

The synthesis of the 50S genomic RNA and strucural proteins of Sendai virus was examined with respect to their utilization in virus assembly. It was found that during a single cycle of infection, 50S RNA was synthesized before the structural proteins and that both RNA and protein were synthesized 2 to 4 h before their appearance in released virions. Pulse-chase labeling indicated that the NP and P proteins synthesized early and the M and F proteins synthesized late were preferentially incorporated into virus relative to the other viral proteins. The kinetics of incorporation of pulse-labeled NP protein suggested that it was withdrawn from a relatively large pool whereas the M protein appeared to be present in a relatively small pool in the cytoplasm. Further, it was possible to chase pulse-labeled M protein, but not NP protein, from the cell during an 8-h time period.     

Sequential Protein Synthesis Following Vaccinia Virus Infection

Inhibition of HeLa cell protein synthesis and the sequential synthesis of viral proteins were followed by pulse-labeling infected cells with (14)C-phenylalanine. Proteins were resolved by polyacrylamide gel electrophoresis. The viral origin of native proteins was confirmed by immunodiffusion. The inhibition of host protein synthesis and the synthesis of early viral proteins occur 1 to 3 hr after infection. This early sequence of events also occurs in the presence of 5-fluorodeoxyuridine, an inhibitor of deoxyribonucleic acid synthesis. Other viral proteins are synthesized at a later time. Those proteins which are not made in the absence of viral deoxyribonucleic acid synthesis can be further subdivided into intermediate and late classes. The intermediate protein is synthesized before the late proteins but does not appear to be a precursor of them. Many more viral polypeptides were resolved by polyacrylamide gel electrophoresis after solubilization of the entire cytoplasmic fraction with sodium dodecyl sulfate. Virion and nonvirion proteins were identified. Kinetic experiments suggested that certain structural proteins as well as certain nonstructural proteins are made early, whereas others of both classes are made primarily at later times.     

Detection of G1 proteins in Chinese hamster cells synchronized by isoleucine deprivation or mitotic selection.

Examination of labeling patterns of proteins in Chinese hamster cells(line CHO) revealed the presence of a class of protein(s) that is synthesized during G1 phase of the cell cycle. Cells arrested in G1 by isoleucine (Ile) deprivation were prelabeded with [14-C]Ile, induced to traverse G1 by addition of unlabeled Ile, and labeled with [3-H]Ile at hourly intervals. Cells were fractionated into neclear and cytoplasmic portions, and proteins were separated by sodium dodecyl sulfate-polyacrylamide get electrophoresis. Gel profiles of proteins in the 45,000-160,000 mol wt range from the cytoplasm of cells in G1 were similar to those from cells arrested in G1 except for the presence of a mojor peak of [1-H]Ile incorporated into a protein(s) of approximately 80,000 mol wt. Peaks of net [3-H]Ile incorporation were not detected in neclear preparations. Cellular fractionation by differential centrifugation showed the peak I protein was located in the soluble supernatant fraction of the cytoplasm. Time-course studies showed that synthesis of this protein began 1-2 h after initiation of G1 traverse; the protein reached maximum levels in 4-6 h and was reduced to undetectable levels by 9 h. A cytoplasmic protein with similar electrophoretic mobility was found in G1 phase of cells synchronized by mitotic selection. This class of proteins is synthesized by cells before entry into S phase and may be involved in initiation of DNA synthesis.     

Differential protein synthesis in the induction of thyroid cell proliferation by thyrotropin, epidermal growth factor or serum

Protein synthesis in the G1 period of the cell cycle has been investigated using two-dimensional gel electrophoresis in primary cultures of dog quiescent thyroid cells, incubated in defined medium and induced to proliferate by the combined action of thyrotropin (TSH), epidermal growth factor (EGF) and serum or by each of these agents, acting alone. The analysis of the proteins, pulse-labeled for 3 h with [35S]methionine, in quiescent cells deprived of serum and in cells that had been stimulated for various periods of time by the addition of TSH, EGF and serum showed maximal modifications before entry into S phase: the labeling of at least ten proteins was enhanced while that of at least six proteins was decreased. The synthesis of one of these proteins (protein 1; Mr approximately equal to 81 000) was maximal 9-12 h after stimulation by the proliferative agents but began to decrease at 15-18 h and was still decreased at 29-32 h. The study of the effect of each of the proliferation agents alone on the labeling of these sixteen proteins showed that TSH specifically stimulated the labeling of eight polypeptides (proteins 2-9) and that, in contrast, EGF and serum specifically increased the labeling of two other proteins (proteins 1 and 10). The labeling of one protein was decreased by each of the different agents (protein 6') while TSH specifically decreased the labeling of four polypeptides (proteins 1'-4') and increased the labeling of one polypeptide (protein 5') whose synthesis was decreased by EGF and serum. The specific effect of TSH on one protein labeling (protein 7; Mr approximately equal to 39 000) was potentiated by EGF and serum while the specific effect of EGF and serum on another protein labeling (protein 1) was potentiated by TSH. There is thus a correlation between the level of synthesis of these two proteins and the proliferative state of the cells, which is much greater when the stimulating agents are acting together. The induction of protein 1 synthesis by EGF was no longer observed when the cells were no longer proliferating. In the same way, TSH no longer stimulated the synthesis of protein 7 in thyroid cells at confluence. In conclusion, the present study has identified some proteins (proteins 1 and 7) which, as judged by the peculiar stimulation and the kinetics of their synthesis, could be part of the final key events triggering DNA replication in thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biosynthesis and properties of the adenovirus 2 L1-encoded 52,000- and 55,000-Mr proteins

The adenovirus type 2 L1 region, which is located at 30.7 to 39.2 map units on the viral genome, is transcribed from the major late promoter during both early and late stages of virus replication, and a 52,000-Mr (52K) protein-55K protein doublet has been translated in vitro on L1-specific RNA. To investigate the biosynthesis and properties of the L1 52K and 55K proteins, we prepared antibody against a synthetic peptide encoded near the predicted N terminus. As determined by immunoprecipitation and immunoblot analysis, the antipeptide antibody recognized major 52K and 55K proteins synthesized in adenovirus type 2-infected cells that appeared to be identical to the 52K-55K doublet translated in vitro. The immunoprecipitated 52K and 55K proteins were very closely related, as shown by a peptide map analysis. Both L1 proteins were phosphorylated, and they were phosphorylated at similar sites. No precursor-product relationship was detected between the 52K and 55K proteins by a pulse-chase analysis. Biosynthesis of the L1 52K and 55K proteins began about 6 to 7 h postinfection, after biosynthesis of the early region 1A and early region 1B 19K (175R) T antigens, and reached a maximum rate at about 15 h; the maximum rate was maintained until at least 25 h postinfection. At all times, the 55K protein appeared to be synthesized at a severalfold-higher level than the 52K protein. Both proteins were quite stable and accumulated until late times after infection. Viral DNA replication was not essential for formation of the L1 proteins. Thus, the L1 52K-55K gene appears to be regulated in a manner different from the classical early and late viral genes but similar to the protein encoded by the i-leader (Symington et al., J. Virol. 57:849-856, 1986). The L1 proteins were detected in the cell nucleus by immunofluorescence microscopy with antipeptide antibody and were found to be primarily associated with the nuclear membrane by an immunoblot analysis of subcellular fractions.     

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

rRNA accumulation and protein synthetic patterns in growing mouse oocytes

The rRNA contents of mouse primordial oocytes, three stages of growing oocytes, full-grown oocytes, and ovulated ova have been measured by hybridization of RNA samples to excess 3H-DNA complementary to rRNA. Since it was known from previous work that rRNA is stable, the results when plotted against days of oocyte growth indicated that rRNA was synthesized at a constant rate over the first 9 days of growth and about 1.5 times faster in the last 5 days. The maximum value of 0.3 ng per oocyte was attained by about 14 days of growth in oocytes 59 micrometers in diameter, well below the maximum diameter of 77 micrometers for full-grown oocytes. The stability of proteins synthesized in mid-growth phase oocytes was measured by labeling for 5 h with 35S-methionine and then following the decline of incorporated label during a 48h chase; 40% of the label decayed with a half-life of 11 h. and 60% was apparently stable. The two-dimensional electrophoretic patterns of labeled proteins synthesized by growing and full-grown oocytes were compared. The principal change was the appearance or great increase in intensity of several spots in full-grown oocytes as compared to growing oocytes. Egg proteins separated on a two-dimensional gel were visualized by silver staining. The cytoskeletal proteins actin, tubulin, and putative intermediate filament protein, as well as putative lactate dehydrogenase, were synthesized in growing and full-grown oocytes, and accumulated to form a significant portion of bulk egg protein.     

Sequential Metabolic Events During Encystment of Azobacter vinelandii

Cells of Azotobacter vinelandii are specifically induced to encyst by beta-hydroxybutyrate (BHB). The process of differentiation, which occurs over a period of 36 h, was characterized by an ordered sequence of biochemical events. Upon initiation of encystment, nitrogen fixation and glucose-6-phosphate dehydrogenase activities decreased immediately to very low levels. This was followed by an increase in the specific activities of BHB dehydrogenase, isocitrate dehydrogenase, isocitrate lyase, and malate synthase first at 3 h and then again at 21 h. The peak activity of fructose 1,6-diphosphate aldolase occurred at 6 h, and the enzyme activity then decreased gradually. Fructose 1,6-diphosphatase had peak activities at 9 and 27 h. Deoxyribonucleic acid synthesis ceased just prior to the final cell division at 4 to 6 h, but ribonucleic acid synthesis continued until the 12th h. From labeling studies and the appearance of new enzyme activities, it appeared that protein synthesis continued throughout encystment.     

Sequential Formation of Vaccinia Virus Proteins and Viral Deoxyribonucleic Acid Replication

In vaccinia virus-infected cell cultures, cellular protein synthesis was inhibited 50% at 2 hr postinfection (PI) and 80 to 90% by 4 hr PI. Input virus was responsible for this inhibition. Five early proteins, coded for by the viral genome, could be detected at 2 to 3 hr PI. Normally, their synthesis did not continue beyond 6 hr PI, at which time synthesis of a different set of proteins began. When DNA replication was blocked, synthesis of these early proteins continued until 9 to 12 hr PI. The bulk of the proteins which were incorporated into mature virus were synthesized at 8 hr PI and thereafter. The time of their formation was close to the time at which virus maturation occurred. However, 15% of the protein found in mature virus was synthesized early in the infectious cycle. The quantity of “early viral protein” which was not incorporated into mature virus was almost as large as the quantity of viral protein which did appear in mature virus. The “early” and “late” proteins could be shown to have separate and distinct immunological properties. The role of this large quantity of “early” protein is discussed.     

Polypeptide synthesis and phosphorylation in Epstein-Barr virus-infected cells

Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, results in the sequential synthesis of 29 detectable proteins, which range in molecular weight from approximately 155,000 to 21,000, and in the shutoff of the bulk of host protein synthesis within 6 to 9 h after infection. There are three classes of virus-induced proteins; these are an early class, consisting of eight proteins synthesized by 6 h postinfection, an intermediate class, containing two proteins synthesized 9 h postinfection, and a late class, consisting of five proteins synthesized 12 h postinfection. In addition, there is a fourth class of polypeptides, called persistent, that are found both before and after superinfection. The rates of synthesis of the proteins fall into three patterns; these are pattern A, in which the rate of synthesis decreases, pattern B, in which the rate of synthesis remains steady, and pattern C, in which the rate of synthesis increases after the initial appearance of the polypeptide. Both 9-(2-hydroxy-ethoxymethyl)guanine (acyclovir) and phosphonoacetic acid inhibit the appearance of one intermediate protein and at least three late proteins. Seven polypeptides are phosphorylated at different times after infection.     

Differential arrival of newly synthesized apical and basolateral plasma membrane proteins in the epithelial cell line A6

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [³⁵S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [³⁵S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed.This work was supported by grants from the American Heart Association, the National Kidney Foundation of the Delaware Valley, and from the Department of Veterans Affairs. T.R.K. is a recipient of an Established Investigatorship Award from the American Heart Association.     

Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid.

An earlier report from this laboratory documented a substantial increase in the rates of dolichol-linked oligosaccharide intermediate synthesis and protein N-glycosylation in purified murine splenic B lymphocytes (B cells) activated by treatment with bacterial lipopolysaccharide (LPS). In this study the developmental patterns for the induction of lipid-mediated protein N-glycosylation, membrane protein, and phosphatidylcholine (PC) biosynthesis were compared during the proliferative response of B cells to LPS. By electron microscopy it could be seen that a distinct endoplasmic reticulum (ER) network began to develop by 24-48 h after exposure of the purified B cells to LPS. The rate of synthesis of membrane protein increased markedly during the first 10 h after activation, reaching a maximum at 30-40 h. The induction of protein N-glycosylation was delayed slightly relative to membrane protein synthesis, with glycoprotein synthesis increasing sharply approximately 20 h after activation. When phospholipid synthesis was monitored by measuring [CH3-3H]choline incorporation into PC, the rate of labeling increased slowly during the first 35 h, but more substantially between 35 and 90 h. The incorporation of labeled choline into PC was drastically reduced by 5'-deoxy-5'-isobutylthio-3-deazaadenosine, an inhibitor of CDP-choline synthesis, indicating that the incorporation of radiolabeled choline is primarily a measurement of the rate of de novo synthesis of PC. In vitro assays revealed that while choline kinase activity was virtually unchanged, CDP-choline synthetase activity increased gradually throughout the activation period. Diacylglycerol cholinephosphotransferase activity, an ER-associated enzyme, was present at low levels between 0 and 35 h, but increased fivefold between 35 and 90 h. On the basis of the developmental patterns for the rates of protein N-glycosylation, membrane protein insertion, and PC biosynthesis determined by metabolic labeling experiments, we tentatively conclude that all of the ER-associated membrane proteins involved in these biosynthetic processes are not induced concurrently during the activation of B cells by LPS.     

Synthesis of Saint Louis Encephalitis Virus Ribonucleic Acid in BHK-21/13 Cells

Infection of baby hamster kidney cells (BHK-21/13) with Saint Louis encephalitis (SLE) virus depressed the rate of protein and ribonucleic acid (RNA) synthesis until viral RNA synthesis began 6 hr postinfection (PI). Virus-directed RNA synthesis was subsequently inhibited until 12 hr PI when virion maturation began. The rate of protein synthesis reached a peak 6 hr PI and was subsequently depressed until just before the onset of virion maturation. Density gradient analysis of phenol-extracted RNA from actinomycin-treated infected cells indicated that, at 6 to 8 hr and again at 12 to 20 hr PI, three species of viral-specific RNA were synthesized. The most rapid sedimenting form (43S) was ribonuclease-sensitive and had a base composition similar to the RNA isolated from mature virions. The 20S RNA species was ribonuclease-resistant and had a sedimentation coefficient and base composition similar to the replicative form associated with other arbovirus infections. The 26S RNA was ribonuclease-resistant (0.2 mug/ml, 0.1 m NaCl, 25 C, 30 min) and had a nucleotide base composition closer to the 20S form than to the values for 43S RNA. Five-minute pulse labeling of infected cultures during the period viral RNA synthesis was maximal resulted in labeling of only the 20S to 22S RNA fractions. With pulse-labeling periods of 10 min, both the 20S and 26S RNA species were radioactive. Periods of radioactive labeling of as long as 15 min were required before the 43S form was radioactively labeled. These results suggest that the 20S and 26S RNA may be intermediate forms in the synthesis of 43S viral RNA.     

Uterine and oviducal protein secretion during early pregnancy in the mouse

Changes in the protein composition of the embryo's environment during early development were studied by analysis of proteins synthesized and secreted by oviducal and uterine explants on Days 1-6 of pregnancy. Although secretions from ampullar and isthmic oviduct and uterus contained many proteins in common, each area also produced its own characteristic proteins. In the uterus, changes in the secretion pattern were found during the peri-implantation period, including both increases and decreases in particular proteins which appear to be dependent on the presence of embryos. Embryo-induced effects on uterine secretion began between 09:00 h of Day 4 and 09:00 h of Day 5. Oviducal secretions exhibited many of the embryo-dependent proteins found in the uterus, but the expression of these proteins did not appear to be influenced by the presence of embryos on Day 1 or Day 3. The characteristic pattern of secreted protein expression by each portion of the reproductive tract may reflect the specialization of each area for certain developmental events.     

Stage-specific protein synthesis during early embryogenesis in Drosophila melanogaster

The changes in protein species synthesized during early Drosophila embryogenesis were characterized by two-dimensional electrophoresis. Of the 261 proteins scored, 68 (26%) show dramatic changes in rates of synthesis during the first 8 h of embryogenesis. These stage-specific proteins can be classified into three categories: early, detected at 1, 2 and 3 h but not later; late, not detected at 1 h, but appearing later; and discontinuous, detected before and after, but not at 3 and 4 h. RNA was extracted from three representative stages, translated in vitro, and the translation products separated on two-dimensional gels. There was a strong correlation between the patterns of synthesis in vivo and in vitro, suggesting that the early proteins are translated from maternal mRNA, and the late proteins from zygotic mRNA. A thorough comparison was made between the proteins synthesized in wild-type and dorsal embryos, in which virtually only dorsal hypoderm differentiates. The first observed difference was a reduced synthesis of actin I at 8 h, indicating that the absence of mesodermal and endodermal tissues is not detectable at the level of moderately abundant protein until the onset of differentiation.