Maturation of viral proteins in cells infected with temperature-sensitive mutants of vesicular stomatitis virus.

Maturation of viral proteins in cells infected with mutants of vesicular stomatitis virus was studied by surface iodination and cell fractionation. The movement of G, M, and N proteins to the virion bud appeared to be interdependent. Mutations thought to be in G protein prevented its migration to the cell surface, allowed neither M nor N protein to become membrane bound, and blocked formation of viral particles. Mutant G protein appeared not to leave the endoplasmic reticulum at the nonpermissive temperature, but this defect was partially reversible. In cells infected with mutants that caused N protein to be degraded rapidly or prevented its assembly into nucleocapsids, M protein did not bind to membranes and G protein matured to the cell surface, but never entered structures with the density of virions. Mutations causing M protein to be degraded prevented virion formation, and G protein behaved as in cells infected by mutants in N protein. These results are consistent with a model of virion formation involving coalescence of soluble nucleocapsid and soluble M protein with G protein already in the plasma membrane.     

Newcastle Disease Virus Infection of L Cells

Newcastle disease virus (NDV) California strain reportedly grows poorly in L cells but replicates very well in chicken embryo cells. NDV-infected L cell cultures show a characteristic virus growth curve with respect to uridine incorporation, but plaque assays of the virus produced 24 h postinfection (PI) show no infectious particles when assayed on L cell monolayers and only a very low titer on chick cell monolayers. Plasma membranes isolated and purified from infected L cells 8 h PI contain all of the major virion proteins. In addition, NDV-infected L cells show a 50% loss of H-2 antigenic activity, a phenomenon previously observed in cells productively infected with vesicular stomatitis virus. These results suggest that at least part of the normal process of NDV maturation occurs in NDV-infected L cells. Sodium dodecyl sulfate-polyacrylamide gel patterns of supernatant virus purified from cells radiolabeled with amino acids from 3 to 24 h PI in the presence of actinomycin D show that all the major NDV structural proteins are present. Electron micrographs of NDV-infected L cells show extensive virus maturation at cell membranes. It can be concluded that infection of L cells with NDV results in a normal production of virus-specific RNA, synthesis of all the major structural proteins, association of the viral envelope proteins with the L cell plasma membrane, and the loss of cell surface H-2 antigenic activity. However, most of the virus particles produced are noninfectious.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Localization and putative function of the UL20 membrane protein in cells infected with herpes simplex virus 1.

The UL20 protein of herpes simplex virus 1, an intrinsic membrane protein, is required in infected Vero cells in which the Golgi apparatus is fragmented for the transport of virions from the space between the inner and outer nuclear membranes and for the transport of fully processed cell membrane-associated glycoproteins from the trans-Golgi to the plasma membrane. It is not required in the human 143TK- cell line, in which the Golgi apparatus remains intact. We report the following. (i) The UL20 protein was detected in infected cells beginning at 6 h postinfection and was regulated as a gamma 1 gene. (ii) Pulse-chase experiments revealed no detectable alteration in the mobility of the UL20 protein in polyacrylamide gels. (iii) In both infected Vero and infected 143TK- cells, the UL20 protein was detected by immunofluorescence in association with nuclear membranes and in the cytoplasm. Some of the cytoplasmic fluorescence colocalized with beta-COP, a protein associated with Golgi-derived transport vesicles. UL20 protein was present in virions purified from the extracellular space but could not be detected in the plasma membrane. These results are consistent with the hypothesis that UL20 is a component of virion envelopes and membranes of virion transport vesicles and is selectively retained from the latter in a Golgi compartment.     

Proteins Specified by Herpes Simplex Virus: II. Viral Glycoproteins Associated with Cellular Membranes

Membranes prepared from HEp-2 cells infected with herpes simplex virus and free from soluble proteins, virus, ribosomes, and other cellular constituents were solubilized and subjected to electrophoresis on acrylamide gels. The electropherograms showed the following. (i) The synthesis of host proteins and glycoproteins ceases after infection. However, the spectrum of host proteins in membranes remains unaltered. (ii) Between 4 and 22 hr postinfection, at least four glycoproteins are synthesized and bound to the smooth cytoplasmic membranes. On electrophoresis, these glycoproteins form two major and two minor bands in the gel and migrate with proteins ranging from 50,000 to 100,000 daltons in molecular weight. (iii) The same glycoproteins are present in all membranes fractionated by density and in partially purified virus. The implications of the data are discussed.     

Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly

Poxvirus acquires its primary envelope through a process that is distinct from those of other enveloped viruses. The molecular mechanism of this process is poorly understood, but several poxvirus proteins essential for the process have been identified in studies of vaccinia virus (VACV), the prototypical poxvirus. Previously, we identified VACV A6 as an essential factor for virion morphogenesis by studying a temperature-sensitive mutant with a lesion in A6. Here, we further studied A6 by constructing and characterizing an inducible virus (iA6) that could more stringently repress A6 expression. When A6 expression was induced by the inducer isopropyl-β-D-thiogalactoside (IPTG), iA6 replicated normally, and membrane proteins of mature virions (MVs) predominantly localized in viral factories where virions were assembled. However, when A6 expression was repressed, electron microscopy of infected cells showed the accumulation of large viroplasm inclusions containing virion core proteins but no viral membranes. Immunofluorescence and cell fractionation studies showed that the major MV membrane proteins A13, A14, D8, and H3 did not localize to viral factories but instead accumulated in the secretory compartments, including the endoplasmic reticulum. Overall, our results show that A6 is an additional VACV protein that participates in an early step of virion membrane biogenesis. Furthermore, A6 is required for MV membrane protein localization to sites of virion assembly, suggesting that MV membrane proteins or precursors of MV membranes are trafficked to sites of virion assembly through an active, virus-mediated process that requires A6.     

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis.

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.     

Synthesis of Capsid and Noncapsid Viral Proteins in Response to Encephalomyocarditis Virus Ribonucleic Acid in Animal Cell-Free Systems

The polypeptide products formed in two cell-free protein-synthetic systems programmed with encephalomyocarditis (EMC) virus ribonucleic acid (RNA) have been compared with the virus-specific proteins found in EMC-infected cells and with the capsid proteins of the purified virion. Tryptic peptides of (35)S-methioninelabeled proteins from these three sources were compared by co-chromatography and electrophoresis and by isoelectric focussing. Fifty-two methionine-containing peptides were resolved in digests of material from infected cells, of which about one-third were also clearly present in digests of the virion capsid proteins. The product formed in response to EMC RNA in cell-free systems from Krebs mouse ascites tumor cells yielded 26 to 29 such peptides. Most of these peptides were shown to behave identically with virus-specific peptides from infected cells, whereas just under half of them appeared to be identical with peptides from the virion capsid proteins. The product formed in response to EMC RNA in the L-cell cell-free system was similar, whereas six additional EMC-specific peptides were detected in mixed Krebs L-cell systems. The results indicate that the EMC RNA genome is partially translated in the mouse cell-free systems used to yield products containing both virion capsid and virus-specific noncapsid polypeptides.     

Isolation and characterization of a membrane-bound population of group B coxsackieviruses.

HeLa cells infected with several group B coxsackieviruses contain a previously undetected, virus-specific ribonucleoprotein particle which we designated membrane-bound virion (MBV). MBVs of B5 virus have a pronounced polygonal appearance and are slightly smaller than virions. The particles sediment more slowly (at about 107S) and have a lower buoyant density (about 1.30). They contain 35S virion RNA; only three, and not four, capsid proteins; and at least seven additional proteins with apparent molecular weights of 21,000 to 92,000. Three of the latter proteins appear to be of host origin; the rest may be precursors of virion capsid proteins. The RNA is resistant to digestion by RNase, and EDTA treatment disrupts the particle. MBVs are infectious, although significantly less so than virions. Cells infected with MBVs produce both types of progeny, virions and MBVs. In coinfected cultures, the yield of progeny is lower than in cells infected with virions alone, suggesting interference by MBVs. Synthesis of both types can be detected within 3.5 h after infection, and synthesis continues for 24 h.     

Adenovirus gene function required for induction of nuclear acidic protein synthesis: binding of these proteins to adenovirus DNA.

Two different viral DNA-defective temperature-sensitive mutants of adenovirus 12 (H 12) were defective in their ability to induce the synthesis of various molecular weight classes of nuclear acidic proteins, both virion and nonvirion components, after lytic infection of human embryo kidney (HEK) cells at the restrictive temperature. This finding indicates that the induction of nuclear acidic protein synthesis is an adenovirus gene function(s). Treatment of infected cells with actinomycin D at an early stage of virus maturation suppressed the synthesis of an acidic virion protein (hexon), but allowed the synthesis of other classes of nuclear nonvirion acidic proteins during the subsequent late maturation period, suggesting that different mechanisms control virion and nonvirion polypeptide synthesis. The interaction of the nuclear acidic proteins isolated from H 12-infected cells with native-labeled H 12 DNA was studied using the membrane filter technique. Measurements of the ability of different DNA preparations to inhibit the H 12 DNA-acidic protein complex formation suggest that the nuclear acidic proteins bound to native H 12 or HEK cell DNA with much higher affinity than to native calf thymus DNA. Moreover, native H 12 DNA was able to bind the acidic proteins more efficiently than did denatured H 12 DNA. The acidic proteins isolated from the cytoplasm of H 12-infected cells bound approximately 100-fold less to native H 12 DNA than did the nuclear proteins. Furthermore, the H 12 DNA binding affinity of the nuclear acidic proteins from uninfected cells, or from H 12-infected and 1-beta-D-arabinofuranosylcytosine-treated cells, was somewhat lower than that of the nuclear proteins from infected (untreated) cells.     

Separate pathways of maturation of the major structural proteins of vesicular stomatitis virus.

Cell fractionation and protein electrophoresis were used to study the intracellular sites of synthesis and intermediate structures in the assembly of the virion proteins of vesicular stomatitis virus. Each of the three major virion proteins assembled into virions through a separable pathway. The nucleocapsid (N) protein was first a soluble protein and later incorporated into free, cytoplasmic nucleocapsids. A small amount of N protein was bound to membranes at later times, presumably representing either nucleocapsids in the process of budding or completed virions attached to the cell surface. The matrix (M) protein also appeared to be synthesized as a soluble protein, but was then directly incorporated into membranous structures with the same density as whole virus. Very little M protein was ever found in membranes banding at the density of plasma membranes. The M protein entered extracellular virus very quickly, as though it moved directly from a soluble state into budding virus. In contrast, the glycoprotein (G) was always membrane bound; it appeared to be directly inserted into membranes during its synthesis. Glycosylation of the G protein was completed only in smooth membrane fractions, possibly in the Golgi apparatus. After a minimum time of 15 min following its synthesis, G protein was incorporated into the surface plasma membrane, from which it was slowly shed into virions. These multiple processing steps probably account for its delayed appearance in virus. From this work it appears that the three major structural proteins come into the surface budding structure through independent pathways and together they coalesce at the plasma membrane to form the mature virion.     

Isolation and Properties of an RNA Polymerase from Influenza Virus-Infected Cells

Structures with RNA polymerase activity were isolated from influenza virus-infected cells, and consisted of ribonucleoprotein (RNP) complexes, similar in morphology to the viral internal component or nucleocapsid. The isolation procedure involved fractionation of infected cells in a discontinuous sucrose gradient, in which enzyme activity was concentrated in a fraction of intermediate density which contains both smooth and rough cytoplasmic membranes. The RNPs with polymerase activity were further purified in a velocity gradient, after which the peak fractions showed a 35-fold purification of the polymerase activity when compared with cytoplasmic extracts. The NP polypeptide, which is the subunit of the virion RNP, was the only virus-specific polypeptide detected in these RNP structures.     

Translation of Sindbis virus 26 S RNA and 49 S RNA in lysates of rabbit reticulocytes

Sindbis virus-specific polypeptides were synthesized in lysates of rabbit reticulocytes in response to added 26 S or 49 S RNA. Sindbis 26 S RNA was translated into as many as three polypeptides which co-migrate in acrylamide gels with proteins found in infected cells.Wild type 26 S RNA was translated primarily into two polypeptides, which appear to be the Sindbis nucleocapsid protein (mol. wt 30,000) and the precursor of the two glycoproteins of the virion (mol. wt 100,000). A larger polypeptide (mol. wt 130,000) was synthesized in response to ts2 26 S RNA, a species of RNA which was isolated from cells infected with the ts2 mutant of Sindbis virus. This large polypeptide is apparently the protein which accumulates in cells infected with the mutant virus and which is thought to be a precursor of all three viral structural proteins.These results support the hypothesis that 26 S RNA is the messenger for the three structural proteins of the virion and that the RNA codes for one large polypeptide precursor. The precursor may then be cleaved at a specific site to yield the nucleocapsid protein and a second polypeptide which, in infected cells, is cleaved in a series of steps to yield the two glycoproteins of the virion.Sindbis 49 S RNA was translated into eight or nine polypeptides ranging from 60,000 to 180,000 molecular weights. The viral structural proteins, as such, were not synthesized in response to the added 49 S RNA.     

The herpes simplex virus UL20 protein compensates for the differential disruption of exocytosis of virions and viral membrane glycoproteins associated with fragmentation of the Golgi apparatus.

The Golgi apparatus is fragmented and dispersed in Vero cells but not in human 143TK- cells infected with wild-type herpes simplex virus 1. Moreover, a recombinant virus lacking the gene encoding the membrane protein UL20 (UL20- virus) accumulates in the space between the inner and outer nuclear membranes of Vero cells but is exported and spreads from cell to cell in 143TK- cell cultures. Here we report that in Vero cells infected with UL20- virus, the virion envelope glycoproteins were of the immature type, whereas the viral glycoproteins associated with cell membranes were fully processed up to the addition of sialic acid, a trans-Golgi function. Moreover, the amounts of viral glycoproteins accumulating in the plasma membranes were considerably smaller than those detected on the surface of Vero cells infected with wild-type virus. In contrast, the amounts of viral glycoproteins present on the plasma membranes of 143TK- cells infected with wild-type or UL20- virus were nearly identical. We conclude that (i) in Vero cells infected with UL20- virus the block in the export of virions is at the entry into the exocytic pathway, and a second block in the exocytosis of viral glycoproteins associated with cytoplasmic membranes is due to an impairment of transport beyond Golgi fragments containing trans-Golgi enzymes and not to a failure of the Golgi oligosaccharide-processing functions; (ii) these defects are manifested in cells in which the Golgi apparatus is fragmented; and (iii) the UL20 protein compensates for these defects by enabling transport to and from the fragmented Golgi apparatus.     

The membrane fusion step of vaccinia virus entry is cooperatively mediated by multiple viral proteins and host cell components

For many viruses, one or two proteins allow cell attachment and entry, which occurs through the plasma membrane or following endocytosis at low pH. In contrast, vaccinia virus (VACV) enters cells by both neutral and low pH routes; four proteins mediate cell attachment and twelve that are associated in a membrane complex and conserved in all poxviruses are dedicated to entry. The aim of the present study was to determine the roles of cellular and viral proteins in initial stages of entry, specifically fusion of the membranes of the mature virion and cell. For analysis of the role of cellular components, we used well characterized inhibitors and measured binding of a recombinant VACV virion containing Gaussia luciferase fused to a core protein; viral and cellular membrane lipid mixing with a self-quenching fluorescent probe in the virion membrane; and core entry with a recombinant VACV expressing firefly luciferase and electron microscopy. We determined that inhibitors of tyrosine protein kinases, dynamin GTPase and actin dynamics had little effect on binding of virions to cells but impaired membrane fusion, whereas partial cholesterol depletion and inhibitors of endosomal acidification and membrane blebbing had a severe effect at the later stage of core entry. To determine the role of viral proteins, virions lacking individual membrane components were purified from cells infected with members of a panel of ten conditional-lethal inducible mutants. Each of the entry protein-deficient virions had severely reduced infectivity and except for A28, L1 and L5 greatly impaired membrane fusion. In addition, a potent neutralizing L1 monoclonal antibody blocked entry at a post-membrane lipid-mixing step. Taken together, these results suggested a 2-step entry model and implicated an unprecedented number of viral proteins and cellular components involved in signaling and actin rearrangement for initiation of virus-cell membrane fusion during poxvirus entry.     

Role of cytoplasmic vacuoles in varicella-zoster virus glycoprotein trafficking and virion envelopment.

Varicella-zoster virus (VZV) encodes several glycoproteins which are present on both mature viral envelopes and the surfaces of infected cell membranes. Mechanisms of VZV glycoprotein transport and virion envelopment were investigated by both continuous radiolabeling and pulse-chase analyses with tritiated fucose in VZV-infected cells. We studied in detail the large cytoplasmic vacuoles which were present in infected cells but absent from uninfected cells. The specific activity in each subcellular compartment was defined by quantitative electron microscope autoradiography, using a cross-fire probability matrix analysis to more accurately assess the individual compartment demarcated by the silver grains. By these techniques, we documented a progression of activity originating in the Golgi apparatus and traveling through the post-Golgi region into virus-induced cytoplasmic vacuoles and finally to areas of the cellular membrane associated with the egress of viral particles. Significant amounts of radiolabel were not observed in the nucleus, and only low levels of radiolabel were associated with the cellular membrane not involved with the egress of viral particles. In addition, immunolabeling of Lowicryl-embedded VZV-infected cells demonstrated the presence of VZV glycoproteins within cytoplasmic vacuole membranes as well as on virion envelopes. These observations suggested that cytoplasmic vacuoles harbored VZV-specified glycoproteins and were also the predominant site of VZV virion envelopment within the infected cell. Neither enveloped nor unenveloped viral particles were observed within the Golgi apparatus itself.     

The herpes simplex virus 1 UL11 proteins are associated with cytoplasmic and nuclear membranes and with nuclear bodies of infected cells.

Earlier studies have shown that the UL11 gene of herpes simplex virus encodes a myristylated virion protein and that the UL11 gene enables efficient virion envelopment and export from infected cells. A rabbit polyclonal antibody directed against an affinity-purified UL11-glutathione-S-transferase fusion protein was made and used to study the properties of the UL11 protein and its distribution in infected cells. We report the following: (i) UL11 protein formed up to five bands (apparent M(r)s, 17,000 to 22,000) in denaturing polyacrylamide gels; (ii) fluorescent-antibody studies revealed the presence of UL11 protein in the perinuclear space and in sites within the nucleus; (iii) immune electron microscopic studies indicated that the UL11 gene products were associated with the inner nuclear membrane, with cytoplasmic membranes and ribbon-like cytoplasmic structures resembling membranous organelles, with nuclear bodies shown by fluorescence microscopy to be different from nucleoli in which US11 protein accumulates, and with enveloped virions but not with nuclear capsids; and (iv) the nuclear bodies containing UL11 protein were reminiscent both of type IV morphotypes consisting of an electron-dense core containing the UL11 proteins surrounded by a more electron-transluscent core and of type V morphotypes consisting of material homogenous in electron opacity. We conclude that (i) the UL11 protein is processed after synthesis; (ii) the localization of UL11 protein with virions and membranes is consistent with the hypothesis that UL11 plays a role in the transport of virions to the extracellular space; and (iii) although the significance of the association of UL11 proteins with nuclear bodies is unknown, the results indicate that nuclear bodies differ with respect to their morphologies and contents of viral protein and suggest that UL11 protein may have more than one function in the infected cell.     

L-particle production during primary replication of pseudorabies virus in the nasal mucosa of swine

Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.     

Proteins specified by herpes simplex virus. XIII. Glycosylation of viral polypeptides.

In the course of herpes simplex virus 1 (HSV-1) replication in human epidermoid carcinoma no. 2 cells, the synthesis and glycosylation of host cell proteins ceases and is replaced by the synthesis and glycosylation of virus-specified polypeptides. Analyses of the synthesis of viral glycoproteins show that the glycosylation of viral polypeptides occurs late in the virus growth cycle and that certain of the precursors to major vital glycoproteins are members of the gamma group of polypeptides, i.e., polypeptides synthesized at increasing rates until 12 to 15 h postinfection. Viral glycoproteins are formed by stepwise additions of heterosaccharide chains to completed precursor polypeptides. The precursor and the highly glycosylated product are separable by gel electrophoresis and are localized in different fractions of infected cells. Within 15 min of their synthesis, precursor polypeptides acquire heterosaccharide chains of about 2,000 molecular weight, which contain glucosamine but little or nor fucose or sialic acid. Both precursor and product of this first stage of glycosylation are absent or present in low concentrations in the surface membranes of the infected cell and in the virion. The partially glycosylated product is then conjugated further in a slow, discontinuous process to form the mature glycoprotein of the virion and plasma membrane. These mature products bear large heterosaccharide units with molecular weights greater than 4,000 to 5,000; these contain fucose and sialic acid as well as glucosamine. Heterosaccharide chains from infected and uninfected cells are distributed among discrete size classes and the smallest chains consist of multiple saccharide residues.