Oncornavirus budding: kinetics of formation and utilization of viral membrane glycoprotein.

Previous studies have revealed two distinct pathways leading to the assembly and extrusion of Moloney sarcoma-leukemia virus (MSV(MLV)) from a constitutively infected cell line, 78A1 (Witte, O. N., and Weissman, I. L. (1974). Virology61, 575–587). Core synthesis and appearance of recently synthesized products in extracellular virions is rapid, whereas appearance of labeled major membrane glycoproteins (gp69,71) in extracellular virions is delayed. In this paper, we have used experiments involving pulse-chase incorporation of labeled polypeptide and carbohydrate precursors into intracellular virion components isolated by immunoprecipitation to investigate the mechanism of this delay. The major intracellular component precipitated by anti-gp69,71 antisera is a 69,000–71,000-dalton form(s) which contains both amino acid (leucine) and carbohydrate (glucosamine) precursors. Following a short (10 min) pulse label with [³H]leucine, these 69,000–71,000-molecular weight forms rapidly appear and rise to their maximum intracellular specific activity within 10–20 min of chase. This maximum specific activity remains constant for 1 hr of chase. During this period little immunoprecipitable gp69,71 is released into the supernatant fluid. During later chase intervals (to 3 hr), the intracellular gp69,71-specific activity falls, while the extracellular supernatant gp69,71 rises proportionally. We conclude from these experiments that the delay in appearance of label in extracellular gp69,71 is due to some postsynthetic process involving passage of these recently synthesized glycoproteins through one or more large intermediary pools prior to availability for the budding process. Lactoperoxidase-catalyzed pulse radioiodination of log-phase 78A1 cells followed by analysis of subsequently budded virions reveals such a large pool of these glycoproteins on the cell membrane and, as well, provides evidence for a striking selection of available cell membrane proteins for inclusion in the budding site.     

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.     

The polypeptides of adenovirus. VI. Early and late glycopolypeptides

Two glucosamine-labeled macromolecules, EG at early stages and LG at late stages, of infection appeared after infection of HeLa cells by type 2 adenovirus.EG was detected at maximal level by pulse-labeling with ³H-glucosamine from 5 to 9 hr post infection. It was preferentially recovered from the cytoplasmic fraction of detergent-lysed cells and had the same electrophoretic mobility as reference virion polypeptide VII. These characteristics of EG were so similar to those of E₂, one of the three early polypeptides (E₁, E₂, E₃) reported previously, that we tentatively concluded that EG was E₂, and was a glycoprotein. Although E₂ (or EG) and virion polypeptide VII showed very similar electrophoretic mobility, the presence or absence of glucosamine label distinguished them, A pulse-chase experiment with ¹⁴C-amino acids showed that the amount of radioactivity in the 3 early polypeptides did not change after 10 or 20 hr of chase. E₂ and E₃ were produced in infected human cells cultures in the presence of cytosine arabinoside, and in infected rat embryo cell culture. EG was not labeled with fucose.The subunit of fiber (polypeptide IV) was demonstrated to be LG. Limited acid hydrolysis was used to release glucosamine from fiber protein which was qualitatively identified by paper chromatography. and quantitated by two different methods. In the amino acid composition of fiber protein histidine was the amino acid present at the lowest level, but at about 2 times the level of glucosamine residues. The ¹⁴C-glycopeptides obtained by extensive Pronase digestion of fiber protein or virion labeled with ¹⁴C-glucosamine eluted just after a tetrasaccharide marker, stachyose, from a Sephadex G-25 column. Treatment of ¹⁴C-glucosamine-labeled fiber protein or virion with 0.1 N NaOH in the presence of 1 M NaBH₄ released ¹⁴C-material which coeluted with N-acetylglucosaminitol from a Sephadex G-15 column. Radioactive fucose, galactose, and mannose did not label any virion polypeptide significantly. From these results we concluded that the fiber polypeptide, whether integrated into virions or not, has a sugar residue related to glucosamine, probably N-acetylglucosamine, linked to the polypeptide chain with a weak alkali-sensitive bond (e.g., an O-glycosidic bond to the β-hydroxy group of serine or threonine). Two N-acetylglucosamine and 4 histidine residues per fiber polypeptide yielded the simplest approximation to the molecular weight estimated by other methods.     

Isolation and characterization of vaccinia virus "nucleoids".

After treating vaccinia virions labeled with [³H]thymidine in their nucleic acid and with ¹⁴C-labeled amino acids in their polypeptides with 1% sodium dodecyl sulfate (SDS) at room temperature, the products of the reaction were subjected to sedimentation analysis in 15–30% sucrose-SDS gradients. Particles were isolated containing all of the virion DNA but only 25–30% of the virion polypeptides. Negatively stained preparations showed that most of the particles were spherical and about 300 nm in diameter. These particles lacked the palisade layer seen on viral cores as well as the core membrane and are therefore subcore particles or nucleoids. When the DNA from nucleoids was analyzed, it was found to co-sediment with DNA from mature virions at 68 S in neutral sucrose gradients. When digested with DNase, only 15–20% of the [³H]thymidine-labeled DNA in nucleoids was rendered acid soluble. Analysis of the DNA from DNase-treated preparations in neutral sucrose gradients showed the DNA had been cleaved into segments which sedimented at about 10–15 S relative to adenovirus 2 DNA. Acrylamide-gel electrophoretic analysis of the polypeptides in nucleoids showed that in addition to the surface polypeptides of mature virions, two major core polypeptides, VP-4A and VP-8, were removed by treatment with SDS, as well as components from the VP-1 and VP-2 polypeptide groups.Morphologically, the nucleoids resembled SubV-4, an intermediate in the uncoating of virions detected in the cytoplasm of HeLa cells early after infection. SubV-4 particles, labeled with ¹⁴C-labeled amino acids, were isolated from the cytoplasm of HeLa cells and purified by sedimentation in 20–60% sucrose-D₂O gradients. Polyacrylamide-gel electrophoretic analysis of the polypeptides in SubV-4 particles showed that their polypeptide composition was like that of nucleoids prepared in vitro.     

A structural model for picornaviruses as suggested from an analysis of urea-degraded virions and procapsids of coxsackievirus B3

Urea treatment (3 M, 15 min, 37 °C, pH 9) of coxsackievirus B3 inactivated virus infectivity and degraded the virus capsid into substructures recoverable on sucrose gradients. One substructure which sedimented around 20 S contained VP1 and VP3, the other substructures which sedimented at 5 S contained VP2 and VP4, respectively, as analyzed by SDS polyacrylamide electrophoresis. The VP2 and VP4 polypeptides in the 5 S peak were probably separate since their molar ratios differed over the peak, and VP4 could be removed by dialysis. Treatment of the virions with only 1 M urea for 5 min yielded four peaks of radioactivity on sucrose gradients which sedimented at about 150 S (undegraded virions), 75–80 S (capsids minus VP4), and the 20 S and 5 S structures referred to above, suggesting a stepwise degradation of B3 virions by urea. The procapsids also were degraded into substructures which were separated on sucrose gradients; one sedimenting at around 40 S containing mostly VPO, and the other sedimenting around 20 S containing only VP1 and VP3. When coxsackievirus B3-³⁵S cysteine-labeled virions were disrupted and analyzed on SDS gels, all polypeptides except VP4 were labeled, suggesting that VP2 and VP4 are distinct polypeptides. Analysis of urea-disrupted coxsackievirus B3 substructures provides the basis for a T = 3 structural model of the picornaviruses, with 12 pentamers (VP2 and VP4) and 20 hexamers (VP1 and VP3) per virion.     

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.     

Structural proteins of Tacaribe and Tamiami virions

Tacaribe and Tamiami viruses were grown in BHK-21 monolayers and purified by centrifugation in combination glycerol-tartrate gradients followed by sucrose gradient centrifugation. The major polypeptides of Tacaribe and Tamiami virions were shown to differ from those of Pichinde virions when suitably labeled preparations of the three viruses were coelectrophoresed in SDS-polyacrylamide gels. Tacaribe and Tamiami virions each contained two major polypeptides, a nonglycosylated protein of molecular weight 68,000 and 66,000, respectively, and a single glycoprotein size-class of molecular weight 42,000 and 44,000, respectively. The nonglycosylated protein is associated with the viral RNA in a nucleoprotein structure which can be isolated by equilibrium centrifugation in CsCl. The glycoproteins were selectively removed from virions by chymotrypsin treatment, which produces essentially spikeless particles. Tacaribe and Tamiami virions also contain a minor polypeptide species of molecular weight 79,000 and 77,000, respectively.     

Structure of the Mengo virion. V. Distribution of the capsid polypeptides with respect to the surface of the virus particle.

The disposition of the Mengo capsid polypeptides (α, β, γ, and δ) with respect to the external surface of the virion has been investigated by measuring their relative susceptibilities to lactoperoxidase-catalyzed iodination and their reactivities in immunological tests with specific antisera. When intact virions were subjected to iodination for a brief period of time (1 min), radioactive iodine was incorporated predominantly into the a polypeptides and to a lesser extent into β polypeptides. Only with longer incubation times (15 min or more) did label appear in the γ and δ polypeptides; and this coincided with a progressive loosening and ultimate collapse of the viral capsid. Antisera specific for each of the capsid polypeptide species were produced in rabbits using isolated proteins as antigens. Reaction of virions with these antisera in plaque-neutralization and hemagglutination-inhibition tests showed that only the anti-α serum was capable of blocking virus-cell interactions. Complement-fixation and immunodiffusion tests confirmed the observations that the α polypeptides occupy most of the external surface of the virus particle and that the β polypeptides are partially exposed. The γ and δ polypeptides apparently occupy internal locations in the capsid of the Mengo virion.     

Proteins of equine herpesvirus type 3. I. Polypeptides of the purified virion.

Enveloped virions of equine herpesvirus type 3 (EHV-3) were purified from the extracellular fluids of infected horse embryo fibroblast cell cultures (KyED) by sequential banding in dextran-10 and potassium tartrate density gradients. The preparations of purified virus consisted of enveloped herpesvirus particles with little extraneous cellular material demonstrable by electron microscopy or by overt addition of labeled cellular proteins prior to purification. Structural polypeptides of the purified EHV-3 virions were analyzed by electrophoresis in SDS-polyacrylamide slab gels cross-linked with N,N′-diallytartardiamide. Thirty-four polypeptides, ranging in molecular weight from 14 × 10³ to 220 × 10³ were resolved in Coomassie brilliant blue-stained electropherograms of the purified virions. Ten of these proteins were larger than 100 × 10³ and two were larger than 200 × 10³. A 148 × 10³ capsid protein was a major structural polypeptide of the EHV-3 virion. Coelectrophoresis of the proteins of EHV-3 virions with those of the genetically unrelated equine herpes-virus type 1 (EHV-1) revealed a similarity in size range and number of the virion structural proteins; however, the molecular weights and proportional composition of the majority of EHV-3 polypeptides differed significantly from those of the virion proteins of EHV-1.     

Structural proteins of HADEN virus

Purified HADEN virus was disrupted and separated into three polypeptide types in 7.5% neutral sodium dodecyl sulfate-polyacrylamide gels. The major polypeptide (HVP1) had a molecular weight of about 67,000 and accounted for 75–83% of the virion protein. The two minor components (HVP2 and HVP3) had molecular weights of about 77,000 and 85,500, respectively. When HADEN virus polypeptides were compared to the polypeptides of adenovirus-associated virus similar profiles were noted, but the polypeptides of the two viruses were distinguishable when coelectrophoresed on a single gel. Purified low density noninfectious HADEN particles were found to contain three polypeptides with the same molecular weights and concentrations as those found in the infectious virions.     

In vitro insertions and deletions in the G segment of phage mu DNA do not abolish the inversion process

The growth of rabies virus at a low pH of the culture medium resulted in the production of a large number of spikeless virions which lacked glycoprotein protruding from the viral surface. These virions were first evident at a pH below 7.0 and reached a maximum at pH 6.7 to 6.8. The spikeless virion exhibits a lower sedimentation velocity than the infectious virion, but is similar to the infectious virion in shape and size. Our present working hypothesis is that these spikeless virions are formed by proteolytic digestion of a large portion of glycoprotein after the assembly of standard virions at the cellular membrane. This is based on the following findings: (1) Electron micrographs of infected cells in which two-thirds of the progeny virions belonged to the spikeless type revealed that spikes were observed on most cell-associated virions; (2) SDS-PAGE of the spikeless virions showed a new low molecular weight polypeptide, and a polypeptide showing a similar mobility was also detected in the infectious virions in which spikes had been cleaved off by Pronase treatment; and (3) peptide mappings of both low molecular weight polypeptides revealed similar findings. The significance of the low pH of the culture medium in producing spikeless virus is under investigation.     

Determination by peptide mapping of the unique polypeptides in Sendai virions and infected cells.

Peptide mapping of all of the Sendai virion polypeptides and the viral polypeptides synthesized in infected cells has shown that C (MW ～22,000), which has been found only in infected cells, is unique and is thus a candidate for a nonstructural polypeptide. It has also been found that the L polypeptide of Sendai virus is unique, that polypeptide 5 is derived from NP, that polypeptide A is host-cell actin, and that the polypeptides L, P, HN, F₀, NP, and M have the same peptide composition in virions as in infected cells.     

Comparative analysis of the enveloped virions of two granulosis viruses of the armyworm, Pseudaletia unipuncta

The enveloped virions of two strains of a granulosis virus, a synergistic Hawaiian (GVH) and a nonsynergistic Oregonian (GVO) strain which infect the armyworm, Pseudaletia unipuncta, were liberated from their capsules with 0.02 N NaOH and purified by filtration through membrane filters. The envelopes were solubilized with 0.1% Triton X-100. The enveloped virions, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), had 9 to 12 polypeptides, 3 of which were associated with the naked virion. The two strains differed in their electrophoretic patterns mainly in the presence of two heavy polypeptides (89,000 and 97,000 daltons) and a 52,000-dalton polypeptide in GVH and in the absence of the heavy polypeptides and the presence of a 57,000-dalton polypeptide in GVO. The two heavy polypeptides were sensitive to proteinase. Two two-dimensional electrophoreses (first dimension with agarose gel electrophoresis and second dimension with SDS-PAGE and immunoelectrophoresis) of the envelope of GVH resolved two polypeptides, 37,000 and 48,000 daltons, which were serologically related to the synergistic factor present in the capsule of GVH; however their molecular weights differed from that of the GVH synergistic factor. In GVO, no polypeptide serologically related to the synergistic factor was detected in the envelope. The enveloped virions purified by differential filtration were infectious when fed to armyworm larvae, but the naked virions, free of envelopes, were not infectious.     

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.     

A comparison of the polypeptides of four measles virus strains

The polypeptides of measles virions from four different sources have been analyzed and compared by polyacrylamide gel electrophoresis in both phosphate buffer and discontinuous Tris buffer systems. A similar pattern of six polypeptides was found with the four different virus strains. Only one polypeptide (G) was found to contain detectable amounts of carbohydrate label; this was the largest virion polypeptide with an estimated molecular weight of 80,000. Slight differences in electrophoretic mobilities of the nucleocapsid protein subunits (NP) were found among the different strains; with estimated molecular weights in the 60,000 to 62,000 range. Another polypeptide with a molecular weight of ～ 70,000 has been found to be associated with the nucleocapsids isolated from infected cells. The three remaining polypeptides identified have estimated molecular weights of 55,000, 42,000, and 37,000, and the smallest of these has been designated M, by analogy to other paramyxoviruses. The differences between the present results and previous findings with measles virus, and the possible relationship of measles virus polypeptides to those of other paramyxoviruses, have been discussed.     

Kinetics of the Incorporation of Tropoelastin into Elastic Fibers in Embryonic Chick Aortad

Matrix-free cells isolated by enzymic digestion of chick embryo aortas were labeled with [¹⁴C]proline for 20 to 60 min and the kinetics of the secretion of tropoelastin were followed by chasing the label and assaying [¹⁴C]tropoelastin in the cells and in the medium. The results indicated that secretion of tropoelastin followed the kinetics of a single first order process with a half time of 60 min. In parallel experiments tissue explants of chick embryo aortas were labeled with [¹⁴Clproine and the kinetics for the incorporation of tropoelastin into elastic fibers were followed by chasing the label and assaying the soluble [¹⁴C]tropoelastin and insoluble [¹⁴C]elastin in tissues. It was found that the incorporation of tropoelastin into elastic fibers also followed a single first order process with a half time of 85 min, similar to the secretion of tropoelastin from cells. In further studies, antibodies directed against tropoelastin were utilized to isolate soluble[^14C]elastin components in the tissues after 0 to 4 hr chase of the ^14CC label. The results demonstrated that all soluble elastin components were recovered as monomeric tropoelastin and no soluble oligomeric elastin could be detected. These results are consistent with the proposition that elastic fiber growth occurs by addition of individual tropoelastin molecules to existing fibers and that oligomers of elastin were not intermediates in the process.     

Machupo virus polypeptides: Identification by immunoprecipitation

Summary The most abundant protein in purified Machupo virions (Corvallo strain) labelled with¹⁴C-Protein hydrolysate is a 64K polypeptide which is associated with virion RNAs. Another structural polypeptide, 37K, solubilized by nonionic detergent seems to be a major surface glycoprotein. In addition to this, a 78K polypeptide and a minor 50K polypeptide have been detected.In Machupo virus infected cells three virus-specific polypeptides similar in size to those described for structural polypeptides were immunoprecipitated with anti-Machupo virus serum. The most abundant virus-specific polypeptide was nonglycosylated (64K, NP), and the others were glycosylated polypeptides (78K and 37K). The synthesis of NP and 78K polypeptides was recognized at the beginning of a log phase of virus replication. Pulse-chase experiments as well as experiments with an arginine analogue, canavanine (to block proteolytic processing) suggest that 78K is a precursor for structural glycoproteins of Machupo virions.With 4 Figures     

Temperature-sensitive defect of influenza A/Ann Arbor/6/60 cold-adapted variant leads to a blockage of matrix polypeptide incorporation into the plasma membrane of the infected cells

A temperature-sensitive (ts) defect in growth of the A/Ann Arbor/6/60 (A/AA/60) cold-adapted (ca) and ts variant strain has been studied. At the restrictive temperature of 38.5 degrees C, the variant synthesized all the viral polypeptides in normal amounts within the infected cells, but the virions released into the culture fluid contained greatly reduced amounts of the matrix (M1) polypeptide and showed significantly low infectivity per unit hemagglutinin activity. Cell fractionation experiments revealed that incorporation of the M1 polypeptide into plasma membranes of the variant-infected cells was selectively reduced at 38.5 degrees C, whilst it occurred normally at 34 degrees C. The ts reassortants between the A/AA/60 variant and the A/AA/1/80 wild type (wt) strain (non-ts), which had the M gene derived from the wt parent, also showed similar patterns. These results suggest that the ts defect of the variant and its ts reassortants involves the process of incorporation of the M1 polypeptide into the plasma membranes of the infected cells and that this defect is not attributable to the M gene of the variant.     

The cellular site of sulfation of influenza viral glycoproteins.

The incorporation of ³⁵SO₄^2? into viral polypeptides in MDBK cells infected with influenza virus was analyzed by SDS-polyacrylamide gel electrophoresis. When infected cells were labeled in the absence of calf serum, three polypeptides, HA, NP, and M, were resolved in isolated plasma membranes, and HA was the only polypeptide in which ³⁵SO₄^2? incorporation was detected. Sulfation of HA was also demonstrated in both smooth and rough cytoplasmic membranes, whereas there was no detectable ³⁵SO₄^2? incorporation into unglycosylated proteins. These results indicate that at least partial sulfation of HA is already completed at rough membranes. However, when infected cells were doubly labeled with [³H]leucine and ³⁵SO₄^2?, the ${}^{\mathrm{<mtext>35</mtext><mtext>S</mtext>}}{}^3\text{H}$ ratio in the HA polypeptide was not uniform in virions and subcellular components. The ratio was highest in virions, and decreased in the order of virions, plasma membranes, smooth membranes, and rough membranes, suggesting that further sulfate incorporation may occur in smooth membranes and plasma membranes as well as in rough membranes. The 355/3H ratio of HA associated with plasma membranes varied with the length of labeling; higher ratios were observed with shorter labeling periods. This observation may be explained by sulfate incorporation into performed HA. Significant amounts of ³⁵SO₄^2? incorporation into HA were found in the presence of cycloheximide, at concentrations wich completely inhibited the synthesis of viral polypeptides. Further, pulse-labeling of infected cells with ³⁵SO₄^2? at various times after inhibition of protein sy thesis by cycloheximide showed that sulfation of HA polypeptides continues to occur as long as 30 min or more after synthesis, which also suggests that ³⁵SO₄^2? continues to be incorporated into HA polypeptides even after they migrate from rough membranes. The acceptors for sulfation appear to be oligosaccharide units of viral glycoproteins since almost all ³⁵S label was recovered in association with glycopeptides after exhaustive digestion of virions with Pronase followed by gel filtration. As was observed for HA, the incorporation of ³⁵SO₄^2? into cellular mucopolysaccharides was also observed in every subcellular fraction tested. Further, when either smooth or rough cytoplasmic membranes isolated from infected cells were incubated with 3′-phosphoadenosine-5′-phosphosulfate ([³⁵S]PAP) in vitro, sulfate incorporation into mucopolysaccharide was detected, which suggests that sulfation of mucopolysaccharide occurs in both smooth and rough membranes in vivo. Additionally, it was found that the rate of incorporation of ³⁵SO₄^2? into cellular mucopolysaccharide was markedly inhibited by influenza virus infection.