Equine herpesvirus in vivo: cyclic production of a DNA density variant with repetitive sequences.

The DNA of a strain of equine herpesvirus type 1 passed more than 500 times in Syrian hamsters (EHV-1ha) has been analyzed by CsCl equilibrium density gradient ultracentrifugation, analytical sedimentation, and DNA-DNA reassociation kinetics. The viral DNA consisted of light and heavy species having densities in CsCl of 1.716 and 1.724 g/cm³, which correspond to guanine plus cytosine contents of 56 and 64%, respectively. These values were confirmed by T_m measurements. Similar molecular weight values were obtained by analytical sedimentation for the light (87.9 × 10⁶) and heavy (81.8 × 10⁶) DNA species. The heavier species was produced in a cyclic manner. Hamsters infected with virus containing a high proportion of the heavy species gave reduced virus yields and survived longer. The genetic relatedness of the two viral DNA species of EHV-1ha was compared by examining the ability of each to reanneal with ³²P-labeled viral DNA of the tissue culture strain (L-M cell) of EHV-1 (EHV-1tc). The lighter (1.716 g/cm³) species of EHV-1ha was composed of unique sequences completely homologous to the entire EHV-1tc genome, while the heavier species (1.724 g/cm³) consisted of sequences homologous to approximately 50% of the EHV-1tc genome. Of these homologous sequences, 40–60% (20–30% of the entire EHV-1tc genome) were reiterated. Further, analyses of the EHV-1tc genome (fragmented and unfragmented) by thermal chromatography on hydroxylapatite and in neutral preparative CsCl equilibrium density gradients revealed considerable intramolecular heterogeneity in nucleotide distribution. Finally, analysis of the structural polypeptides of virions of EHV-1ha which contained the heavy and light DNA species revealed that the following two major viral proteins were missing from virions containing the heavier DNA species: VP8, an envelope protein with a molecular weight of 173,000, and VP23, a nucleocapsid protein with a molecular weight of 38,000.     

Equine cytomegalovirus: Cultural characteristics and properties of viral DNA

Equine cytomegalovirus (equine herpesvirus type 2; ECMV) exhibited cultural characteristics typical of the cytomegalovirus group. Ninety-six to one hundred twenty hours were required to reach a maximum titer of 1 × 10⁷ PFU/ml in infected cells, from which no more than 50% of infectious virus produced was released into the supernatant fluid. Only cells of equine or rabbit origin were permissive for virus replication. Ultrastructural investigation of ECMV-infected cells revealed the presence of three types of intranuclear nucleocapsids (empty capsidc, capsidc with a cross-shaped, electron-lucent core, and mature capsidc with an electron-dense core). The mature capsids appeared to acquire their envelope at the nuclear membrane. Infected cells were characterized by nuclei containing marginated chromatin in a large, electron-dense inclusion substance. Viral DNA extracted and purified from virions, nucleocapsids, or infected cells (Hirt fractionation) demonstrated an average density of 1.716 g/cm³ which corresponds to a G+C content of 57.7%. Sedimentation analyses of ECMV DNA in neutral sucrose gradients using phage T4 and equine herpesvirus type 1 (EHV-1) DNA as markers indicated a sedimentation coefficient of approximately 61 S. Sedimentation in alkaline sucrose suggested that the ECMV genome is a non-cross-linked, double-stranded DNA, possibly possessing nonligated areas either within the sugar phosphate backbone of the molecule or within specific alkali-labile regions. Sedimentation analyses of ECMV DNA yielded a molecular weight of approximately 121 × 10⁶ which was confirmed by restriction endonuclease analyses which indicated a value of 126 × 10⁶ Determination of the number, size, and molarity of ECMVDNA fragments generated by digestion with restriction enzymes revealed that ECMV DNA differs markedly in molecular structure from the genome of EHV-1.     

Biological and biochemical properties of defective interfering particles of equine herpesvirus type 1.

Defective interfering (DI) particles of equine herpesvirus type 1(EHV-1) were purposely generated in an in vitro system of L-M cells by repeated high-multiplicity, serial, undiluted passage. Quantitation of infectious virus revealed a definite cyclic pattern which increased in magnitude with continued passage; additional experiments indicated that these fluctuations in virus titer were due to the presence of a population of DI particles as judged by interference assays. Attempts to separate standard and defective EHV-1 were unsuccessful. Analysis of DNA isolated from mixed populations of these virions revealed the presence of a high-density (H) variant DNA (? = 1.724 g/cm³) in addition to standard EHV-1 DNA (? = 1.716 g/cm³). Furthermore, it was found that the relative amount of this H-DNA in each passage corresponded very closely to the fluctuations in infectious virus titer. Sedimentation velocity studies of DNA isolated from populations of virions rich in H-DNA (>99%) indicated that the variant genomes were the same size as the standard EHV-1 genome (50–55 S). Comparisons of purified virion populations from 17 high-multiplicity passages with regard to particle counts, relative amount of H-DNA, and infectious virus titer indicated that the relative interference capacity of EHV-1 DI particles increased significantly with continued passage. Although the factor(s) responsible for the increased interference activity is unknown, DNA-DNA hybridization analyses of selected passages rich in H-DNA indicated that the genomes of EHV-1 DI particles became genetically less complex with passage and contained significant amounts of reiterated sequences. The possible mechanisms of the evolution of EHV-1 DI particles and their role in the interference process are discussed.     

Identification of the envelope surface glycoproteins of equine herpesvirus type 1

The structural polypeptides of purified enveloped virions of the Army 183 strain of equine herpesvirus type 1 (EHV-1) were examined by different analytical techniques to identify the envelope glycoproteins. Glycoproteins were identified by electrophoretic analysis in polyacrylamide slab gels of virus labelled in vivo with [3H]glucosamine or labelled enzymically in vitro with either UDP-[14C]galactose or sodium [3H]borohydride. Fluorograms revealed eleven glycoproteins (mol. wt. 260000, 150000, 138000, 90000, 87000, 65000, 62000, 60000, 50000, 46000, and 24000). These glycoproteins probably correspond to virion protein (VP) 1-2, 9b, 10, 13, 14, 16, 17, 18, 21, 22a and 25 respectively, as designated in two other EHV-1 strains. In addition, a poorly resolved glucosamine-rich region (mol. wt. 250000 to 200000) corresponded to VP 3 to 8. The two isotopic surface labelling methods revealed that all the virus glycoproteins were exposed on the envelope surface.     

Purification and characterization of equine herpesvirus-induced DNA.

Infection of cells with equine herpesvirus type 1 (EHV-1) or type 3 (EHV-3) resulted in the induction of a DNA polymerase activity distinguishable from host cell DNA polymerases by its high salt requirement for maximal activity. By column chromatography on DEAE-cellulose, DNA-cellulose, phosphocellulose, and hydroxyapatite, the EHV-1-induced polymerase was purified 500-fold with 1–2% recovery of total activity from the nuclei of infected hamster livers. The final enzyme preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Calculations based on Stokes radius, sedimentation coefficient, and electrophoretic mobility indicated that the native enzyme is composed of a single subunit having a molecular weight of 160,000. The purified enzyme exhibited anomalous gel filtration behavior indicating molecular asymmetry. It required Mg²⁺, dithiothreitol, alkaline pH (8–9), all four deoxyribonucleoside triphosphates, and 150 mM salt [K₂SO₄, (NH₄)₂SO₄, or K₂HPO₄] for maximal activity, and utilized templates in the following order of preference: activated DNA (100%), poly(dA)·oligo(dT) (40%), poly(dC)·oligo(dG) (21%), native DNA (7%), denatured DNA (4%), and poly(rA)·oligo(dT) (3%). N-Ethylmaleimide, Zn²⁺, and phosphonoacetate acted as inhibitors of the EHV-1-induced DNA polymerase. Antiserum elicited against the EHV-1 DNA polymerase induced in hamsters inactivated the viral enzyme from infected mouse and equine cell cultures. However, the DNA polymerase induced by EHV-3 and DNA polymerases present in uninfected cells were not inhibited by the antiserum. These results support the hypothesis that a new, virus-coded DNA polymerase is induced after equine herpesvirus infection.     

Equine cytomegalovirus: structural proteins of virions and nucleocapsids

Enveloped virions and nucleocapsids of equine cytomegalovirus (ECMV; equine herpesvirus type 2) have been purified from the supernatants and the nuclear extracts of infected rabbit kidney (RK) cells, respectively, and their structural protein compositions have been analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that ECMV nucleocapsids were composed of nine proteins (average molecular weights = 148K, 52K, 49.5K, 46K, 43.5K, 38.5K, 27K, 20K, and 18K), which together constituted 89% of the total nucleocapsid protein on the basis of incorporated 3H-labeled amino acids. The 148K protein comprised 47.3% of the total protein and thus appeared to be similar in molecular weight and proportional composition to the major capsid proteins of other herpesviruses. Purified virions were composed of 37 proteins whose average molecular weights ranged from 14K to greater than 200K. Three intense glycoprotein bands (83K, 78K, and 73.5K) as well as four less intensely labeled glycoproteins were detected in [3H]glucosamine-labeled virion preparations. At least 14 structural proteins were readily detected in extracts of infected cells which had been [35S]methionine labeled late in infection, and 11 of these were immunoprecipitated by rabbit antiserum against purified virions. The protein composition of ECMV differs substantially from those of equine herpesvirus type 1 and type 3 as well as from those of other herpesviruses.     

Translation of eggplant mosaic virus RNA in wheat germ extracts and reticulocyte lysates

Eggplant mosaic virus (EMV) RNA extracted from virions (virion EMV RNA) is a good messenger in wheat germ extracts and reticulocyte lysates but directs surprisingly little synthesis of coat protein. Large amounts of coat protein, however, are synthesized when a low molecular weight RNA purified from virion RNA is used as messenger. Heavy virion RNA directs the synthesis of two proteins of high molecular weight in reticulocyte lysates. The larger of these proteins accounts for almost the entire coding potential of 2 × 10⁶ dalton viral RNA.     

Purification and some physicochemical properties of sonchus yellow net virus

Sonchus yellow net virus (SYNV) was purified from a Nicotiana hybrid by Celite filtration and sucrose density gradient centrifugation. Infectious preparations sedimented at 1044 S in linear-log gradients and banded at 1.183 g/ml in sucrose equilibrium gradients. Electron microscopy of purified preparations revealed bacilliform particles (94 × 248 nm). The virions had internal cross striations with a periodicity of about 4.1 nm and surface projections about 6 nm long. The molecular weight of the virion, estimated from size and density, was about 9 × 10⁸. Nucleic acid from sodium dodecyl sulfate-disrupted virions was susceptible to RNase, sedimented in sucrose gradients at 44 S, and had a molecular weight of 4.42 × 10⁶ as estimated by polyacrylamide-gel electrophoresis. Four major polypeptides with average molecular weights of 76,800, 63,800, 45,500, and 39,500 were detected by gel electrophoresis. SYNV preparations reacted in gel diffusion tests with a homologous antiserum but not with antisera to broccoli necrotic yellows virus, lettuce nectrotic yellows virus, or sow thistle yellow vein virus.     

Studies of the molecular anatomy of the L-M cell strain of equine herpes virus type 1: proteins of the nucleocapsid and intact virion

The structural components of purified enveloped virions and of purified nucleocapsids of the tissue culture strain (L-M cell) of equine herpes virus type 1 (EHV-1L) were analyzed by discontinuous sodium dodecyl sulfate polyacryalmide gel electrophoresis. Enveloped virions were comprised of 28 structural proteins of average molecular weights ranging from 270,000 to 16,000. Twelve of the proteins exhibited molecular weights of greater than 100,000, and six of these were above 200,000. Utilizing radioactively labeled compounds (³H-glucosamine and ³H-choline), four glycoproteins, four lipoproteins, and nine glycolipoproteins were shown to be present in the virions. Purified nucleocapsids, isolated from nuclear extracts of infected L-M cells, contained five major structural proteins with average molecular weights of 148,000, 59,000, 46,000, 36,000, and 18,000. These five proteins comprised greater than 96% of total nucleocapsid protein, on the basis of radioactivity. The 148,000 MW protein (VP 9) accounted for approximately 65% of the total nucleocapsid protein and was the major structural protein of both nucleocapsids and intact virions. None of these proteins corresponded to glycoproteins or lipoproteins present in enveloped virions, indicating that glycoproteins and lipoproteins are components of the envelope. The remaining 4% consisted of eight structural proteins ranging from 140,000 to 30,000 MW and were judged to be minor structural components, reproducibly present in all preparations of nucleocapsids.     

Cell-free synthesis of equine herpesvirus type 3 nucleocapsid polypeptides.

Rate-velocity centrifugation in sucrose gradients of nuclear lysates from cells infected with equine herpesvirus type 3 (EHV-3) demonstrated two major species of viral nucleocapsids that differed in buoyant density, sedimentation velocity, and DNA content. Nucleocapsids present in the bottom sucrose band (B capsids) comprised 62% of the total nucleocapsid protein and possessed a higher density and seven times more DNA than nucleocapsids isolated from the top sucrose band (A capsids). Analysis of B-capsid proteins in SDS-polyacrylamide gels revealed the presence of five major polypeptides with molecular weights of 151,000, 59,000, 48,000, 40,000, and 30,000. In vitro synthesis of EHV-3 nucleocapsid polypeptides was demonstrated in a cell-free translation system consisting of (1) 150,000 g supernatant from rabbit reticulocytes; (2) preincubated ribosomes from the cells of a continuous equine cell line (ETCC); (3) factors dissociated from infected ETCC cell ribosomes with high salt; (4) RNA extracted from the cytoplasm of cells infected with EHV-3. EHV-3 nucleocapsid polypeptides were selected from the total in vitro synthesized products by indirect immunoprecipitation with specific antiviral rabbit serum and antiglobulin goat serum. Polyacrylamide-gel electrophoretic analysis of the cell-free products synthesized in response to poly(A)-rich RNA from EHV-3 infected cells and precipitated with immune serum prepared against purified viral nucleocapsids indicated that four of the five major nucleocapsid polypeptides were made in vitro.     

Tick-borne viruses structurally similar to orthomyxoviruses

Members of the Orthomyxoviridae are characterized at the structural level as enveloped, negative sense, RNA viruses that bud from the outer plasma membranes of infected cells and have seven or eight species of single-stranded RNA. None of the three types of orthomyxoviruses (influenza A, B, C) has been shown to be transmitted by arthropods; rather, the viruses are transmitted directly or indirectly from one infected vertebrate to another. Analyses of the virion RNA species and polypeptides of the tick-borne Thogoto and Dhori viruses indicate that they have structural characteristics similar to accepted members of the Orthomyxoviridae. For example, the viruses have seven size classes of single-stranded RNA with 3′ end consensus sequences of _HOUCGUUG (or U or A) UUGUUC…. The viruses contain 54–56 × 10³ Da nucleocapsid protein, an internal 28 × 10³ Da putative matrix protein plus minor 85–90 × 10³ Da proteins, and a major outer 65 × 10³ Da glycoprotein. In addition to their sensitivity to actinomycin D and α-amanitin the viruses morphologically and morphogenetically resemble orthomyxoviruses.     

The virion and soluble antigen proteins of variola,monkeypox, and vaccinia viruses

The structural proteins in purified preparations of variola, monkeypox, and vaccinia viruses were separated and compared by using a high resolution SDS-polyacrylamide gel electrophoresis system. About 30 proteins were resolved for each virus by autoradiography of longitudinally-sliced gel rods. Although the autoradioelectropherograms of each virus were similar, it was possible to differentiate them by their unique protein pattern in the 30,000 to 40,000 molecular weight region of the gels. A single virion glycoprotein (mol. wt. = 38 × 10³) and a virion phosphoprotein (mol. wt. = 12 × 10³) were associated with each of the virus preparations. Cross-absorbed monospecific immune sera against variola, monkeypox, and vaccinia virus-infected cells were used in immunodiffusion tests to precipitate radiolabeled, homologous, soluble antigen proteins. The predominant antigen protein associated with each immunospecific precipitate had a molecular weight of approximately 73,000.     

Characterization of three species of nucleocapsids of equine herpesvirus type-1 (EHV-1).

Three distinct species of nucleocapsids of equine herpesvirus type-1 (EHV-1) were isolated from infected L-M cell nuclei. The particles were classified on the basis of their densities in Renografin gradients as Light (L; ? = 1.237 g/cc), Intermediate (I; ? = 1.244 g/cc), and Heavy (H; ? = 1.258 g/cc). Analysis of the three nucleocapsid species, radioactively labeled with an ³H-labeled or ¹⁴C-labeled amino acid mixture, by discontinuous SDS-polyacrylamide-gel electrophoresis revealed significant and reproducible differences in their structural protein compositions. H nucleocapsids were comprised of six major proteins (I, II, III, IV, IVa, and V) with respective molecular weights of 148,000, 59,000, 46,000, 37,000, 30,000, and 18,000; these proteins comprised 63.7, 9.3, 6.0, 10.8, 5.3, and 4.1%, respectively, of total protein. These six proteins were also present in I nucleocapsids, however nucleocapsid protein IVa (MW, 30,000) was present only as a minor component and comprised less than 1% of total protein. L nucleocapsids were comprised of only four of these major structural proteins, as proteins III (MW, 46,000) and IVa (MW, 30,000) were absent. In addition, several minor proteins, each comprising less than 1% of total nucleocapsid protein, were present in each nucleocapsid species.Electrophoretic analysis of nucleocapsids labeled with [³H]arginine indicated that proteins extremely rich in arginine are not present in these three species. Phosphoproteins both of enveloped virions and of nucleocapsids were identified by electrophoretic analysis of particles radioactively labeled with inorganic phosphate (H₃³²PO₄). Twelve virion structural proteins were phosphorylated in vivo; five of these, including the major virion phosphoprotein VP 10 (MW, 127,000), were envelope specific proteins. Four of these 12 viral proteins were also phosphorylated in nucleocapsids, however the pattern of phosphorylation of nucleocapsid proteins varied among the three species. The two major phosphoproteins of nucleocapsids were proteins III and IVa which are absent in L nucleocapsids; protein V is phosphorylated only in H nucleocapsids.Analysis of the DNA content of the three nucleocapsid species indicated that preparations of H nucleocapsids contain more DNA than do those of the I and L species. Electron microscopic analysis of the nucleocapsids supported these results, as L and I nucleocapsids lack a dense inner nucleoid structure characteristic of the H species.     

Structural components of mouse mammary tumor virus. I. Polypeptides of the virion.

Mouse mammary tumor virus (mMTV) obtained from MJY-alpha cell cultures and analyzed by polyacrylamide gel electrophoresis possesses four major polypeptides and six to eight minor components. Three of the major proteins, with estimated molecular weights of 60,000, 52,000 and 37,000, are glycoproteins, as demonstrated by labeling with [³H]glucosamine. Labeling with [³⁵S]sulfate revealed significant amounts of sulfate associated with the 60,000 (gp60) and 52,000 (gp52) glycoproteins, whereas sulfate was minimally incorporated into the 37,000 (gp37) dalton glycoprotein. At least three glycopeptide species containing both [³H]glucosamine and [³⁵S]sulfate labels were obtained after extensive Pronase digestion of mMTV, indicating that [³⁵S]sulfate is covalently linked to the carbohydrate component of mMTV glycoproteins. Variations in the polypeptide pattern of mMTV were observed when virions were grown and labeled under different culture conditions. The amount of gp60 decreased substantially, with an apparent increase in a minor polypeptide at 33,000 daltons, if virions were harvested from stationary cultures or if culture medium was not changed daily during viral harvests. When virions containing gp60 were incubated with medium from stationary cultures or with stationary cell layers, the level of gp60 decreased with a corresponding increase in the amount of radioactivity at 33,000 daltons. Cleavage of gp60 and gp52 was demonstrated by treatment of purified mMTV virions with trypsin or alpha-chymotrypsin (1–150 μg/ml) for 1 min to 3 hr. Virions were morphologically unaltered after incubation with either protease; however, both gp60 and gp52 were absent from the polypeptide patterns. The data suggest that gp52 is cleaved to form 33,000, 22,000, and possibly 37,000 dalton glycoproteins which remain associated with virions. Other mMTV virion polypeptides were resistant to treatment with protease.     

Construction and characterization of an equine herpesvirus 1 glycoprotein C negative mutant

An equine herpesvirus 1 (EHV-1) strain RacL 11 mutant was constructed that carries the Escherichia coli LacZ gene instead of the open reading frame encoding glycoprotein C (gC). The engineered virus mutant (L11(delta)gC) lacked codons 46-440 of the 1404 bp gene. On rabbit kidney cell line Rk13 and equine dermal cell line Edmin337, the L11(delta)gC virus grew to titers which were reduced by approximately 5- to 10-fold compared with wild-type RacL11 virus or a repaired virus (R-L11(delta)gC). However, when L11(delta)gC growth properties were analyzed on primary equine cells a decrease of viral titers was observed such that extracellular L11(delta)gC titers were reduced by 48- to 210-fold compared with those of wild-type or repaired virus. Heparin sensitive and heparin resistant attachment was assessed by binding studies using radiolabeled virion preparations. These studies revealed that EHV-1 gC is important for heparin sensitive attachment to the target cell. Similar results were obtained when cellular glycosaminoglycan (GAG) synthesis was inhibited by chlorate treatment or when cells defective in GAG synthesis were used. L11(delta)gC also exhibited significantly delayed penetration kinetics on Rk13 and primary equine cells. Infection of mice with L11(delta)gC did not cause EHV-1-related disease, whereas mice infected with either RacL11 or R-L11(delta)gC exhibited massive bodyweight losses, high virus titers in the lungs, and viremia. Taken together, EHV-1 gC was shown to play important roles in the early steps of infection and in release of virions, especially in primary equine cells, and contributes to EHV-1 virulence.     

Incorporation of sulfate into influenza virus glycoproteins.

The glycoproteins of the influenza virion are labeled with [³⁵S]O₄, whereas the nonglycosylated virion polypeptides are unlabeled. Virions grown in the absence of serum contain sulfate label incorporated into both the hemagglutinin (HA) and neuraminidase (NA) polypeptides; in the presence of serum, the HA₁ and HA₂ cleavage products also both contain sulfate label, with HA, containing three to five times more label than HA₂. In addition to the glycoproteins of the virion, sulfate is incorporated into a component with an electrophoretic mobility lower than that of any of the virion proteins in sodium dodecyl sulfate-polyacrylamide gels. Unlike the virion glycoproteins, this component is also labeled when virions are grown in cells preincubated with [³⁵S]O₄ prior to infection, and it appears to be a host cell-derived sulfated mucopolysaccharide. Labeling of the glycoproteins and the mucopolysaccharide with [³⁵S]O₄ was observed in virions grown in both MDBK and BHK21-F cells. It was estimated that at least ～0.5 mole of sulfate is incorporated per mole of HA polypeptide in virions grown in MDBK cells.Treatment of purified virions with Triton X-100, which solubilizes the glycoproteins, does not solubilize the sulfated polysaccharide or the nonglycosylated polypeptides. After acid hydrolysis of the isolated glycoproteins, the sulfate label is precipitable with BaCl₂, indicating that it is incorporated as sulfate per se rather than a metabolic product.Treatment with the protease bromelain, which produces spikeless particles lacking the glycoproteins, also removes the polysaccharide, indicating that it is located on the external surface of the viral envelope. The polysaccharide is also removed by mild trypsin treatment, under conditions where HA₁ and HA₂ remain intact.     

Adenovirus-associated virus polypeptides synthesized in cells coinfected with either adenovirus or herpesvirus.

One major and two minor structural polypeptides of adenovirus-associated virus (AAV) were synthesized in AAV-infected cells coinfected with either adenovirus or herpesvirus as helper. The molar proportions of these polypeptides appeared to be the same as those found in the virion. Transport of these polypeptides from the cytoplasm to the nucleus occurred rapidly. No evidence was found for the synthesis of a large precursor protein in either system. Since complete AAV virions are not found in AAV-infected cells coinfected with herpesvirus, these findings indicate a failure in virus assembly in this system. This failure may be expressed at the level of DNA strand segregation or encapsidation of DNA, or in faulty capsid assembly.     

Synthesis of tacaribe viral proteins.

The synthesis of Tacaribe virus-specific proteins in infected BHK-21 cells has been analyzed by polyacrylamide slab gel electrophoresis and fluorography. The two major structural polypeptides of the virion were observed above the host cell background by pulse labeling with amino acid or sugar precursors. In addition to the virion glycoprotein (G), another major virus-specific glycoprotein (MW = 70,000) was detected in infected cells. The nucleoprotein (N) was first detected in infected cells around 24–34 hr postinfection (pi). It appeared to be synthesized as a primary gene product, since no larger precursor was detected by short pulse labeling. The rate of synthesis of N increased until 48 hr pi followed by a slight decrease by 72 hr pi. The structural and nonstructural glycoproteins were detected around 48 hr pi and their rate of synthesis increased up to 60 hr pi. The nucleocapsid protein, the minor nonglycosylated virion protein (P), as well as the structural and nonstructural glycoprotein could be specifically precipitated from infected cells using serum from rabbits hyperimmunized with Tacaribe virions. These results as well as pulse-chase experiments experiments suggest that the nonstructural glycoprotein may be a precursor of the virion glycoprotein.     

Biosynthesis of Rauscher leukemia viral proteins: presence of p30 and envelope p15 sequences in precursor polypeptides.

Viral structural polypeptides p30 and a 17,000-dalton polypeptide, termed envelope p15, are formed in Rauscher leukemia virus (RLV)-infected N.I.H. Swiss mouse embryo fibroblasts by cleavage of high molecular weight precursor polypeptides. The evidence for this conclusion is based on the analysis of polypeptides precipitated from RLV-infected cells by antiserum directed against RLV structural proteins. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE) of such immune precipitates from infected cells pulse-labeled with [³⁵S]methionine or pulse-labeled and then chased in unlabeled medium provides evidence that three size classes of unstable polypeptides are precursors to virion p30. They are: two polypeptides with an approximate molecular weight of 200,000 (termed Pr1a and b), an 80,000-dalton polypeptide (Pr3) and a 65,000-dalton polypeptide (Pr4). Ion-exchange chromatography of tryptic digests showed that methionine-containing tryptic peptides of p30 are present in these precursor polypeptides. Methionine-labeled tryptic peptide sequences of envelope p15 were present in a 90,000-dalton peptide fraction containing two components (Pr2a and b). The latter polypeptides comigrated with viral specific fucose-free glycoproteins not present in virions or uninfected cells.