Glycoprotein synthesis in a temperature-sensitive Chinese hamster cell cycle mutant

A temperature-sensitive mutant of Chinese hamster cells is described which has two interesting properties: (1) it is a cell cycle mutant and (2) glycoprotein synthesis appears to be affected at the at the non-permissive temerature (40degreesC). Synchronized cells shifed to 40degreesC in the beginning of their G1 phase do not incorporate [3H]-thymidine into DNA during the expected S-phase, but once DNA synthesis has been initiated ( approximately 10 hours after termination of serum starvation) a shift to 40 degrees C no longer leads to an arrest of DNA synthesis. Flow microfluorimetric analysis of DNA content/cell supports this conclusion and indicates that a majority of cells become arrested in the G1 phase of the cell cycle when a non-synchronized population of cells is transferred to 40degreesC. Apparently at all times in the cell cycle there is a drastic reduction if incorporation of labeled sugars (particularly fucose) into glycoproteins. The uptake of fucose and its conversion to GDP-fucose appears to be normal at 40degreesC. Chromatographic analysis indicates that all classes of glycoproteins are affected, and we do not find any evidence for partially completed oligosaccharides at 40 degrees C. Overall protein synthesis is not reduced at he nonpermissive temperature during the time interval under consideration and the number of polysomes attached to membranes (RER) is also normal at 40degreesC. This suggests that the defect is at an early step in the synthesis or regulation of synthesis of glycoproteins. The mutation is a recessive mutation in hybrid cells and mutagen induced revertants can be obtained which grow normally at 40degreesC and in which glycoprotein synthesis at 40 degrees C is restored to normal, wild type levels.     

Regulation of polypeptide chain initiation in Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase. Changes in phosphorylation of initiation factor eIF-2 and in the activity of the guanine nucleotide exchange factor GEF

When cultures of the temperature-sensitive Chinese hamster ovary cell mutant tsH1 are shifted from 34 degrees C (permissive temperature) to 39.5 degrees C (nonpermissive temperature), protein synthesis is inhibited by more than 80%. This is due principally to a block in activity of polypeptide chain initiation factor eIF-2. In this paper we show that there is impairment of the ability of the guanine nucleotide exchange factor (GEF) to displace GDP from eIF-2 X GDP complexes in extracts from cells incubated at the nonpermissive temperature. Addition of GEF or of high concentrations of eIF-2 stimulates protein synthesis to the level observed in control cell extracts, suggesting that GEF is rate-limiting for eIF-2 activity and overall protein synthesis at the nonpermissive temperature. Analysis of eIF-2 by two-dimensional gel electrophoresis and immunoblotting reveals an increase in the proportion of the alpha subunit in the phosphorylated form from 5.5 +/- 2.4% to 17.2 +/- 3.9% on shifting tsH1 cells from 34 to 39.5 degrees C. No such effect is seen in wild-type cells, which do not exhibit temperature-sensitive protein synthetic activity. Since the primary lesion in tsH1 cells is in their leucyl-tRNA synthetase, these results suggest a role for eIF-2 phosphorylation and GEF activity in coupling the rate of polypeptide chain initiation to the activity of the chain elongation machinery.     

Role of glycosylation in transport of vesicular stomatitis virus envelope glycoprotein. A new class of mutant defective in glycosylation and transport of G protein

A temperature-sensitive mutant (ts gamma 1) of the Cocal serotype of vesicular stomatitis virus synthesizes at the permissive temperature (32 degrees C) a glycoprotein G whose size is smaller (Mr 68,000) than the wild-type (Mr 71,000) and that renders the virion thermolabile. At the nonpermissive temperature (39 degrees C), reduced amounts of noninfectious virus-like particles deficient in G protein were produced. The size of the intracellular G protein was further decreased (Mr 64,000) at the nonpermissive temperature. Biochemical studies including sugar labeling, tryptic peptide analysis, and NH2-terminal sequence analysis of the various glycoproteins suggest that at 32 degrees C a G protein containing a single glycosidic moiety is synthesized. The G protein containing only 1 oligosaccharide residue is transported to the cell surface and is incorporated in infectious virus particles. In contrast, the G protein synthesized at 39 degrees C is nonglycosylated and fails to reach the cell surface. These results suggest that glycosylation of G protein is essential for its transport to the cell surface, and the presence of a single carbohydrate chain is sufficient for this purpose.     

Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein

Maturation of the vesicular stomatitis virus (VSV) glycoprotein (G) to the cell surface is blocked at the nonpermissive temperature in cells infected with temperature-sensitive mutants in the structural gene encoding for G. We show here that these mutants fall into two discrete classes with respect to the stage of post-translational processing at which the block occurs. In all cases the mutant glycoproteins are inserted normally into the endoplasmic reticulum membrane, receive the two-high-mannose oligosaccharides, and apparently lose the NH2-terminal signal sequence of 16 amino acids. In cells infected with one class of mutants, no further processing of the glycoprotein occurs, and we conclude that the mutant protein is blocked at a pre-Golgi stage. In cells infected with ts L511(V), however, addition of the terminal sugars galactose and sialic acid occurs normally. Thus the maturation of G proceeds through several Golgi functions but is blocked before its appearance on the cell surface. The oligosaccharide chain of ts L511(V) G, accumulated at either the permissive (where surface maturation occurs) or the nonpermissive temperature, lacks one saccharide residue, probably fucose. In addition, no fatty acid residues are added to the ts L511(V) G protein at the nonpermissive temperature, although addition does occur under permissive conditions.     

Simian virus 40 mutants carrying two temperature-sensitive mutations.

Five temperature-sensitive mutants of simian virus 40 containing two temperature-sensitive mutations were isolated. The double mutant of the A and D complementation groups, like the D mutants, failed to complement by conventional complementation analysis and did not induce host DNA synthesis at 40 degrees C. However, under conditions that suppressed the D defect, the A:D double mutant expressed only the A defect. Thus, viral DNA replication dropped rapidly after this mutant was shifted from permissive to restrictive temperatures. The A:D double mutant failed to transfrom at the restrictive temperature when subconfluent Chinese hamster lung monolayers were used. Double mutants of A:B, A:C, and A:BC complementation groups, like their A parent, were defective in viral DNA replication, in the induction of host DNA synthesis and in the transformation of secondary Chinese hamster lung cells at the nonpermissive temperature.     

Temperature-sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose-regulated protein GRP78

A temperature-sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse-chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40 degrees C. However, after 3-4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose-regulated protein GRP78. The induction of this protein at 40 degrees C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.     

A temperature-sensitive mutant of the mammalian RNA helicase,DEAD-BOX X isoform,DBX, defective in the transition from G1 to S phase

ts ET24 cells are a novel temperature-sensitive (ts) mutant for cell proliferation of hamster BHK21 cells. The human genomic DNA which rescued the temperature-sensitive lethality of ts ET24 cells was isolated and screened for an open reading frame in the deposited human genomic library. X chromosomal DBX gene encoding the RNA helicase, DEAD-BOX X isoform, which is homologous to yeast Ded1p, was found to be defective in this mutant. The single point mutation (P267S) was localized between the Motifs I and Ia of the hamster DBX of ts ET24 cells. At the nonpermissive temperature of 39.5 degrees C, ts ET24 cells were arrested in the G1-phase and survived for more than 3 days. In ts ET24 cells, total protein synthesis was not reduced at 39.5 degrees C for 24 h, while mRNA accumulated in the nucleus after incubation at 39.5 degrees C for 17 h. The amount of cyclin A mRNA decreased in ts ET24 cells within 4 h after the temperature shift to 39.5 degrees C, consistent with the fact that the entry into the S-phase was delayed by the temperature shift.     

Temperature-sensitive Chinese hamster fibroblast mutant with a defect in RNA metabolism

We describe a new temperature-sensitive mutant of Chinese hamster cell fibroblasts. After a shift to the nonpermissive temperature of 40.5 degrees C, the rates of DNA, RNA, and protein synthesis declined rapidly (to < or = 50% within 12 h) and the progression of unsynchronized cells through the cell cycle was affected. We believe that DNA synthesis came to a halt after a short time, because cells no longer entered the S phase. The decrease in protein synthesis at 40.5 degrees C was shown to be a consequence of a decrease in the number of polysomes, whereas free 80S ribosomes accumulated. We concluded that the components of the protein biosynthetic machinery were intact (ribosomes and soluble factors), but synthesis was limited by a shortage of mRNA. The decline in mRNA production had a significant effect on the synthesis of proteins (e.g., heat shock proteins) translated from short-lived messages. We observed that both polyadenylated and nonpolyadenylated RNA syntheses declined at 40.5 degrees C, whereas the synthesis of small RNAs (4 to 5S) was less reduced. The argument is made that the temperature-sensitive phenotype is the result of a defect affecting mRNA synthesis.     

Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation

Homogeneous glycopeptides and glycoproteins are indispensable for detailed structural and functional studies of glycoproteins. It is also fundamentally important to correct glycosylation patterns for developing effective glycoprotein-based therapeutics. This review discusses a useful chemoenzymatic method that takes advantage of the endoglycosidase-catalyzed transglycosylation to attach an intact oligosaccharide to a polypeptide in a single step, without the need for any protecting groups. The exploration of sugar oxazolines (enzymatic reaction intermediates) as donor substrates has not only expanded substrate availability, but also has significantly enhanced the enzymatic transglycosylation efficiency. Moreover, the discovery of a novel mutant with glycosynthase-like activity has made it possible to synthesize homogeneous glycoproteins with full-size natural N-glycans. Recent advances in this highly convergent chemoenzymatic approach and its application for glycopeptide and glycoprotein synthesis are highlighted.     

Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression

In the preceding paper (Hearing, J., E. Hunter, L. Rodgers, M.-J. Gething, and J. Sambrook. 1989. J. Cell Biol. 108:339-353) we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39 degrees C in two mutant cell lines accumulate a truncated version, Man5GlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32 degrees C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39 degrees C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39 degrees C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains.     

Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine.

Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation.     

Herpes Simplex Virus Glycoproteins: Isolation of Mutants Resistant to Immune Cytolysis

Immune cytolysis mediated by antibody and complement is directed against components of the major herpes simplex virus (HSV) glycoprotein complex (molecular weight, 115,000 to 130,000), comprised of gA, gB, and gC, and against glycoprotein gD-all present on the surfaces of infected cells. Tests with a temperature-sensitive (ts) mutant of HSV-1 (tsA1) defective in glycoprotein synthesis at the nonpermissive temperature (39 degrees C) demonstrated that over 90% of mutant-infected cells maintained at 39 degrees C and treated with antibody and complement were not lysed, presumably due to the absence of viral glycoproteins on the surface of infected cells at this temperature. Furthermore, a small number of tsA1-infected cells could be detected among a large excess of wild-type virus-infected cells by virtue of their failure to be lysed at 39 degrees C by antibody and complement. Making use of the involvement of viral glycoproteins in immune cytolysis and the ability of cells infected with glycoprotein-defective mutants to escape cytolysis, we sought mutants defective in the expression of individual viral glycoproteins. For this purpose, antisera directed against the VP123 complex and against the gC and combined gA and gB glycoprotein subcomponents of this complex were first tested for their ability to lyse wild-type virus-infected cells in the presence of complement. Wild-type virus-infected cells were lysed after treatment with each of the three antisera, demonstrating that the gC glycoprotein and the combined gA and gB glycoproteins can act as targets in the immune cytolysis reaction. Next, these antisera were used to select for mutants which were resistant to immune cytolysis. Cells infected with wild-type virus which had been mutagenized with 2-aminopurine and incubated at 39 degrees C were treated with one of the three types of antisera (anti-VP123 complex, anti-gC, or anti-gAgB) and lysed by the addition of complement. Cells which survived immune cytolysis were plated, and virus in the resulting plaques was isolated. Plaque isolates were tested for temperature sensitivity of growth and altered cytopathic effects in cell culture at 34 degrees C (the permissive temperature) and 39 degrees C. A total of 73 mutants was isolated in this manner. Selection with glycoprotein-specific antisera resulted in a 2- to 16-fold enrichment for mutants compared with "mock" -selected mutants using normal rabbit serum. Phenotypically, 24 mutants were temperature sensitive for growth, 27 were partially temperature sensitive, and 22 were not temperature sensitive but exhibited markedly altered cytopathic effects at both permissive and nonpermissive temperatures. Nine mutants of each phenotype (temperature sensitive, partially temperature sensitive, and non-temperature sensitive) were selected at random for confirmatory immune cytolysis tests with the antisera used in their selection. Cells infected with eight of the nine mutants were shown to be significantly more resistant to immune cytolysis at the nonpermissive temperature than were the mock-selected mutants or the wild-type virus from which they were derived.     

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases

The regulation of polypeptide chain initiation has been investigated in extracts from a number of well-characterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyl-tRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34 degrees C to the non-permissive temperature of 39.5 degrees C. During a brief incubation with [35S]Met-tRNAMetf or [35S]methionine, formation of initiation complexes on native 40S ribosomal subunits and 80S ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5 degrees C. Wild-type cells exposed to 39.5 degrees C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40S initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 X GTP X Met-tRNAMetf ternary complex formation in postribosomal supernatants from the temperature-sensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5 degrees C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 degrees C and 39.5 degrees C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperature-sensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAMetf and form 40S initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation.     

Genetic and phenotypic analysis of herpes simplex virus type 1 mutants conditionally resistant to immune cytolysis

Nine temperature-sensitive (ts) mutants of herpes simplex virus type 1 selected for their inability to render cells susceptible to immune cytolysis after infection at the nonpermissive temperature have been characterized genetically and phenotypically. The mutations in four mutants were mapped physically by marker rescue and assigned to functional groups by complementation analysis. In an effort to determine the molecular basis for cytolysis resistance, cells infected with each of the nine mutants were monitored for the synthesis of viral glycoprotein in total cell extracts and for the presence of these glycoproteins in plasma membranes. The four mutants whose ts mutations were mapped were selected with polypeptide-specific antiserum to glycoproteins gA and gB; however, three of the four mutations mapped to DNA sequences outside the limits of the structural gene specifying these glycoproteins. Combined complementation and phenotypic analysis indicates that the fourth mutation also lies elsewhere. The ts mutations in five additional cytolysis-resistant mutants could not be rescued with single cloned DNA fragments representing the entire herpes simplex virus type 1 genome, suggesting that these mutants may possess multiple mutations. Complementation tests with the four mutants whose ts lesions had been mapped physically demonstrated that each represents a new viral gene. Examination of mutant-infected cells at the nonpermissive temperature for the presence of viral glycoproteins in total cell extracts and in membranes at the cell surface demonstrated that (i) none of the five major viral glycoproteins was detected in extracts of cells infected with one mutant, suggesting that this mutant is defective in a very early function; (ii) cells infected with six of the nine mutants exhibited greatly reduced levels of all the major viral glycoproteins at the infected cell surface, indicating that these mutants possess defects in the synthesis or processing of viral glycoproteins; and (iii) in cells infected with one mutant, all viral glycoproteins were precipitable at the surface of the infected cell, despite the resistance of these cells to cytolysis. This mutant is most likely mutated in a gene affecting a late stage in glycoprotein processing, leading to altered presentation of glycoproteins at the plasma membrane. The finding that the synthesis of both gB and gC was affected coordinately in cells infected with six of the nine mutants suggests that synthesis of these two glycoproteins, their transport to the cell surface, or their insertion into plasma membranes is coordinately regulated.     

Identification and Characterization of a Herpes Simplex Virus Gene Product Required for Encapsidation of Virus DNA

A mutant of herpes simplex virus type 1, 17tsVP1201, has a temperature-sensitive processing defect in a late virus polypeptide. Immunoprecipitation studies with monoclonal antibodies showed that the aberrant polypeptide in mutant virus-infected cells was the nucleocapsid polypeptide known as p40. Since a revertant, TS(+) for growth, processed the polypeptide normally under conditions restrictive for the mutant, the processing event must be essential for virus replication. Electron microscopic analysis of mutant virus-infected cells grown at the nonpermissive temperature revealed that the nuclei contained large aggregations of empty nucleocapsids possessing some internal structure. Therefore, although the mutant synthesized virus DNA at the nonpermissive temperature, the DNA was not packaged into nucleocapsids. When mutant virus-infected cells were shifted from 39 to 31 degrees C in the presence of cycloheximide, the polypeptide p40 was processed to lower-molecular-weight forms, and full nucleocapsids were detected in the cell nuclei. The aberrant polypeptide of the mutant, however, was not processed in cells mixedly infected with 17tsVP1201 and a revertant at the nonpermissive temperature, suggesting that the defect of the mutant was in the gene encoding p40 rather than in a gene of a processing enzyme.     

Local expression and exocytosis of viral glycoproteins in multinucleated muscle cells

We have analyzed the distribution of enveloped viral infections in multinucleated L6 muscle cells. A temperature-sensitive vesicular stomatitis virus (mutant VSV ts045) was utilized at the nonpermissive temperature (39 degrees C). As expected, the glycoprotein (G protein) of this mutant was restricted to the ER when the multinucleated cells were maintained at 39 degrees C. We demonstrate that this G protein remained localized when the infection was performed at low dose. By 4 h after infection the G protein patches spanned an average of 220 microns. The localization was independent of nuclear positions, showing that the ER was a peripheric structure. Thus, the infection did not recognize nuclear domains characteristic of nuclearly encoded proteins. After release of the 39 degrees C block, transport through a perinuclear compartment into a restricted surface domain lying above the internal G protein patch occurred. Accordingly, the transport pathway was locally restricted. After a 16-h infection the G protein spanned 420 microns, while the matrix protein occupied 700-800 microns of the myotube length. Double infection of multinucleated L6 muscle cells with Semliki Forest virus and VSV at high multiplicities showed that the glycoprotein of each virus occupied intracellular domains which were devoid of the other respective glycoprotein. Taken together, these findings indicate that the viral glycoproteins did not range far from their site of synthesis within the ER or other intracellular membrane compartments in these large cells. This result also suggests that relocation of viral RNA synthesis occurred slowly.     

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.     

Characterization of a cell cycle mutant derived from hamster fibroblast: reversion analysis

K12 is a temperature-sensitive (ts) mutant cell line derived from Chinese hamster fibroblasts. When incubated at the nonpermissive temperature, K12 cells exhibit the following properties: (a) the cells cannot initiate DNA synthesis;o (b) the synthesis of cytosol thymidine kinase is suppressed; and (c) the synthesis of three cellular proteins of molecular weights 94, 78, and 58 kdaltons is greatly enhanced. Here we characterize a spontaneous revertant clone, R12, derived from the K12 cells. We selected the revertant clone for its ability to grow at the nonpermissive temperature. Our results indicate that all the traits which constitute the K12 mutant phenotype are simultaneously reverted to the wild type in the revertant cell line, suggesting that the ts mutation of the K12 cells is of regulatory nature and exerts multiple effects on the expressed phenotypes.     

Impaired processing of precursor polypeptides of temperature-sensitive mutants of Rauscher murine leukemia virus.

The synthesis and processing of virus-specific precursor polypeptides in NIH/3T3 cells infected at the permissive temperature (31 degrees C) with temperature-sensitive (ts) mutants of Rauscher murine leukemia virus was studied in pulse-chase experiments at the permissive and nonpermissive (39 degrees C) temperatures. The newly synthesized virus-specific polypeptides were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after immunoprecipitation with polyvalent and monospecific antisera against Rauscher murine leukemia virus proteins. In cells infected with ts mutants defective in early replication steps (the early mutants ts17 and ts29), and ts mutants defective in postintegration steps (the late mutants ts25 and ts26), the processing of the primary gag gene product was impaired at the nonpermissive temperature. gag-pr75 of all four mutants was converted into gag-pr65; however, gag-pr65 accumulated at the nonpermissive temperature, and the main internal virion polypeptide p30 was not formed. Therefore, the proteolytic cleavage is blocked beyond gag-pr65. Concomitantly, the formation of the env gene-related polypeptide p12(E) of all four mutants was blocked at the restrictive temperature. In contrast, cells infected with the late mutant ts28, which produced noninfectious virions at 39 degrees C, showed a normal turnover of the gag and env precursor polypeptides.