Studies on the assembly of the envelope of Newcastle disease virus.

The association of the envelope proteins of Newcastle disease virus with membranes of infected BHK 21-F cells and their incorporation into mature envelopes has been investigated in a study employing cell fractionation. The principal fractions obtained by sucrose density gradient centrifugation of cytoplasmic extracts were rough endoplasmic reticulum and smooth membranes derived predominantly from smooth endoplasmic reticulum and Golgi apparatus. Furthermore, by adsorption to red blood cells it was possible to isolate virions and a hemadsorptive fraction of smooth membranes believed to be immediate precursors of mature envelopes. In addition to the cytoplasmic fractions, plasma membranes obtained as cell ghosts have been analyzed. Each fraction showed a distinct pattern of virus-specific proteins. Pulse-chase experiments indicated that glycoprotein HN and F_o were synthesized on the rough endoplasmic reticulum and transferred from there via smooth intracellular membranes to the plasma membrane and into virions. In the course of migration, F_o is converted to F. In contrast to the glycoproteins, protein M was found to be incorporated into the plasma membrane immediately after synthesis. Pulse-chase experiments also demonstrated that this protein appears in the hemagglutinating fraction of smooth membranes and in mature virions more rapidly than the glycoproteins. These results suggest that M is incorporated into membranes that contain already viral glycoproteins and that this process is one of the last steps in envelope assembly.     

Synthesis of the oligosaccharides of influenza viral glycoproteins.

Glycosylation of influenza viral glycoproteins was investigated by pulse-labeling of infected BHK21-F cells with radioactive sugar precursors and by cell fractionation and analysis of Pronase-digested viral glycopeptides by gel filtration. The results with short pulses of [³H]mannose suggested that the initial event in glycosylation is the en bloc transfer of oligomamiosyl cores to viral glycoproteins associated with rough membranes. The molecular weight of the glycopeptides which represent the cores was estimated to be approximately 1600–2200. Some mannose residues appear to be subsequently removed from oligosaccharide cores. [³H]mannose-labeled glycopeptides obtained either from cells pulsed for brief periods or from rough membranes, which contain predominantly oligosaccharide cores, were sensitive to digestion by endo-β-N-acetylglucosaminidase H (endo-H). On the other hand, glycopeptides larger than oligosaccharide cores, which appeared during chases or after migration of viral glycoproteins from rough to smooth membranes, were resistant to endo-H treatment. The branched sugars (glucosamine, galactose, and fucose), which are contained only in the complex (type I) oligosaccharide chains of virions, appear to be added in a stepwise manner to the trimmed oligosaccharide cores primarily on smooth membranes. Mannoserich glycopeptides of virions (type II) are similar in size to oligosaccharide cores detected in infected cells and are totally sensitive to endo-H, suggesting that type II glycopeptides may represent oligomannosyl cores which escape trimming as well as addition of branched sugars. Comparison of glycopeptides of infected and uninfected BHK21-F cells suggests that influenza viral glycoproteins contain oligosaccharide chains similar in size to those of host cells except for the absence of sialic acid in viral glycoproteins. Further, we observed that intracytoplasmic membranes from infected cells contain much less sialic acid than those from uninfected cells, indicating that viral neuraminidase present in the interior of infected cells possesses enzymatic activity.     

The synthesis of Sendai virus polypeptides in infected cells. II. Intracellular distribution of polypeptides.

The intracellular distribution and the kinetics of association of Sendai virus polypeptides with cytoplasmic fractions and plasma membranes have been studied. The viral surface glycoproteins HN and F₀ have been found in pulse-chase experiments to migrate from rough to smooth membranes and to the plasma membrane. The M protein was found in varying amounts in most cell fractions but was predominantly associated with smooth membranes; there was no evidence of its migration from rough to smooth membranes. The L, P, and NP polypeptides were found with the rough membrane and free ribosome fractions. Polypeptides B and C, which were previously found in extracts of whole infected cells, were found to be unstable during cell fractionation. In the presence of protease inhibitors, polypeptide C, which is thought to be a virus-specific nonstructural protein, was found with the rough membrane fraction. Thus its instability in cell fractions appears to be due to proteolytic digestion. Polypeptide B was not found in cell fractions even in the presence of protease inhibitors. Evidence reported in the following paper has indicated that B is a phosphorylated form of polypeptide M and that its instability is presumably due to loss of phosphate. Polypeptides I–IV, which the available evidence suggests are cellular polypeptides whose synthesis may be enhanced in infected cells, were found in significant amount in soluble form after cell fractionation, although IV was also associated with most membrane fractions.     

Structural maturation of rubella virus in the Golgi complex

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.     

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.     

The polypeptides of influenza virus. VII. Synthesis of the hemagglutinin

Post-translational cleavage of the influenza viral glycoprotein HA occurs to different extents in different systems, varying not only for a particular virus strain grown in different host cells, but also for two strains of virus grown in the same host cell. Preparations of virus in which the HA is not substantially cleaved contain hemagglutinating and infectious virions. Cleavage occurs at different sites in the HA molecule of different virus strains.The hemagglutinin glycoprotein HA is always found in association with cytoplasmic membranes and becomes rapidly incorporated into plasma membranes. Following a 10-min pulse-label, there is already about half as much HA in preparations of plasma membranes as eventually accumulates there during a 90-min chase. Membrane preparations which appear to be mainly composed of smooth endoplasmic reticulum are greatly enriched for HA while plasma membranes contain HA and the other major viral proteins. At 24 hr after infection, the amount of HA or its cleavage products in BHK21 cells infected with Bel or WSN represents a much smaller proportion of the total viral protein than the proportion of HA in purified virions. The same is true for the membrane protein, M, whereas NP is present in excess in the infected cell.Inhibition of protein synthesis by puromycin stops the incorporation of glucosamine into Bel-infected HeLa cells almost immediately, suggesting that glycosylation of HA occurs quickly. However, fucose continues to be incorporated for apporoximately 10–15 min after protein synthesis has been blocked by puromycin or after glycosiliation has been inhibited by glucosamine hydrochloride.     

Influenza virus proteins. II. Association with components of the cytoplasm

Cytoplasmic extracts of influenza virus-infected BHK21-F and MDBK cells were separated by equilibrium sedimentation into fractions containing smooth membranes, rough membranes, and free ribosomes and polysomes. Analysis by polyacrylamide gel electrophoresis revealed that viral polypeptides were associated with all cytoplasmic fractions. Smooth membranes contained large amounts of viral glycoproteins, as well as the nonglycosylated polypeptide (M) thought to be associated with the viral membrane. Rough membranes also contained the uncleaved hemagglutinin glycoprotein (HA), and results of pulse-chase experiments suggest that this polypeptide is synthesized in association with the rough membranes and accumulates in smooth membranes. The nucleoprotein subunit NP was located mainly in the soluble fraction as well as a cell fraction of intermediate density. The nonstructural viral polypeptide NS (～25,000 MW) appeared to be associated specifically with ribo-some-containing fractions.     

In vitro assembly of polioviruses. 3. Assembly of 14 S particles into empty capsids by poliovirus-infected HeLa cell membranes

Rough membranes obtained from poliovirus-infected HeLa cells have the capacity to assemble 14 S particles into 73 S empty capsids in vitro. A corresponding fraction from uninfected cells did not possess this activity. When labeled 14 S particles were incubated with smooth membranes obtained from infected cells, a significant amount of radioactivity sedimented as heterogeneous material in the 30–70 S region of the gradient.Sucrose gradient analysis of ¹⁴C-labeled rough and smooth membrane fractions lysed with deoxycholate (DOC) demonstrated the presence of 14 S particles, 73 S empty capsids, and a 110 S structure in the rough membrane fraction. These structures were found only in trace amounts in the smooth membrane fraction. In the absence of DOC treatment, no 14 S particles were found in any of the membrane fractions. The 73 S empty capsids, on the other hand, were detected in the presence or absence of DOC treatment in the rough-membrane fraction. Therefore, it appears that 14 S particles are associated with the rough membranes where they are assembled into 73 S empty capsids and/or complete virions.     

Differences in membrane association and sub-cellular distribution between NS2-3 and NS3 of bovine viral diarrhoea virus

The sub-cellular location and mechanism of membrane association of NS3 and NS2-3 polypeptides of bovine viral diarrhoea virus (BVDV) have been examined. Both NS3 and NS2-3 proteins were detected in post-nuclear membrane fractions but not in cytosolic fractions of BVDV infected cells; a proportion of NS3, but not NS2-3, could be dissociated from the membranes with 800 mM KCl or at pH 11. Following extraction with 1% Triton X-114, NS3 was predominantly present in the aqueous phase, but NS2-3 was only recovered in the detergent phase. Confocal microscopy showed that in BVDV infected cells, NS3 and/or NS2-3 co-localise with the endoplasmic reticulum (ER) protein, ERP60, but not Golgi or lysosomal proteins. Sub-cellular fractionation analysis demonstrated that NS2-3 was almost exclusively associated with the rough ER membrane but a significant proportion of NS3 was present in the smooth ER membrane fractions in addition to the rough ER membrane. These differences in the distribution of NS2-3 and NS3 on ER membranes in cells infected with cytopathogenic (CP) strains of BVDV were also observed using confocal microscopy and antibodies that are specific to either NS2 or NS3. This distinct distribution of NS3 and NS2-3 on the ER membrane has revealed a further difference between CP and non-cytopathogenic (NCP) strains of BVDV.     

Association of influenza virus proteins with cytoplasmic fractions

Cytoplasmic extracts of chick embryo fibroblasts infected with fowl plague virus were separated into fractions containing smooth membranes, rough membranes, free ribosomes and polysomes, and a soluble fraction. Viral proteins were analyzed in these fractions by polyacrylamide gel electrophoresis. The hemagglutinin glycoproteins were found to be associated with rough and smooth membranes, and pulse-chase experiments revealed that the large glycoprotein HA migrates from rough to smooth membranes where it is cleaved into glycoproteins HA₁ and HA. The nonglycosylated envelope polypeptide M was located predominantly in smooth membranes, the nucleocapsid protein NP in a fraction of intermediate density, and the nonstructural polypeptide NS in the fractions containing ribosomes. A large amount of protein P was found in the soluble fraction.If glycoprotein synthesis was inhibited by glucosamine or deoxyglucose the predominant virus-specific component located on smooth membranes was protein HA₀ thought to be the unglycosylated or incompletely glycosylated polypeptide of glycoprotein HA. Like HA, HA₀ migrated from rough to smooth membranes where it was also cleaved into two fragments. These data show that polypeptide HA₀ has a high affinity for membranes and they further underline the close relationship between proteins HA and HA₀.     

Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves.

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.     

Cytoplasmic localization of the HTLV-III 3' orf protein in cultured T cells

HTLV-III, the etiological agent of the acquired immunodeficiency syndrome, contains in its genome coding regions for several novel proteins. One of these, the 3' open reading frame (3'orf) encodes proteins of 26-27 kDa which are expressed in infected cells both in vivo and in vitro. A specific antiserum has been raised against the recombinant 3'orf protein synthesized in bacteria and used to localize the viral proteins by subcellular fractionation and immunofluorescence on HTLV-III infected cells. The antiserum specifically immunoprecipitated the 26- to 27-kDa proteins from both the cytoplasmic (S100) and the membrane fractions, with an enrichment in the latter. The proteins were not detected in the nucleus or organelle (S100 pellet) fractions. These proteins were also recognized in the same subcellular fractions by human sera from patients with AIDS. Indirect immunofluorescence on fixed infected cells confirmed the presence of the proteins in the cytoplasm. Immunoprecipitation and Western blot analysis of total proteins from disrupted HTLV-III virions with the specific antiserum failed to detect the 3'orf protein products, suggesting that they are not a major component of mature virions and may be involved in the intracellular regulation of viral replication.     

Cellular pools of viral proteins in 3T3 cells chronically infected with Moloney-murine leukemia virus

The kinetics of incorporation of [³H]leucine into proteins of mature Moloney-MuLV was followed to estimate the cellular pools of the viral proteins, and their precursors in 3T3 cells chronically infected with M-MuLV. Viruses were isolated by isopycnic density gradient centrifugation, and their proteins were separated by SDS-polyacrylamide gel electrophoresis. Entrance of labelled proteins into complete virions reached the maximal rate at different times after addition of labelled amino acids. A 33,000 dalton protein (33k) was the first and p30 was the second to enter complete virions 1.5 to 2 hr after introduction of label. A 19,000-dalton protein and gp72 were the last, reaching maximal values for entrance into virions 6 hr after labelling. These lag periods indicate that the budding process of virions takes less than 1.5 hr, whereas modification plus assembly of some proteins into mature virions takes 2–4 hr in exponentially growing cells. The more rapid appearance of label in 33k raises the question as to whether this protein has a regulatory role in virus formation.     

Ultrastructural localization by immunoperoxidase techniques of influenza virus antigens in abortive infection of L cells

An abortive infection was induced in L cells by influenza virus A/Hong Kong/68 (H3N2). With the use of antibody and peroxidase-labelled protein A, the localization of virus protein synthesis but not the maturation of virus particles was demonstrated at the ultrastructural level. Five days after inoculation (p.i.), the synthesis of viral haemagglutinin was localized in the region of the rough endoplasmic reticulum; at late intervals p.i., haemagglutinin accumulated in the plasma membranes, where membrane vesicles, containing haemagglutinin in their membranes, were released from the cell surface. The cytoplasmic viral ribonucleoprotein was localized in the region of free cytoplasmic ribosomes and that of the outer sheet of the nuclear membrane. Viral proteins were detected in the cytoplasm and plasma membranes also after 70 and 390 days of passaging of the cells or of their long-term cultivation with regular change of medium.     

Assembly of Sendai virus: M protein interacts with F and HN proteins and with the cytoplasmic tail and transmembrane domain of F protein

Sendai virus matrix protein (M protein) is critically important for virus assembly and budding and is presumed to interact with viral glycoproteins on the outer side and viral nucleocapsid on the inner side. However, since M protein alone binds to lipid membranes, it has been difficult to demonstrate the specific interaction of M protein with HN or F protein, the Sendai viral glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and flotation in sucrose gradients, we report that the membrane-bound M protein expressed alone or coexpressed with heterologous glycoprotein (influenza virus HA) was totally TX-100 soluble but the membrane-bound M protein coexpressed with HN or F protein either individually or together was predominantly detergent-resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of F protein facilitated binding of M protein to detergent-resistant membranes. Analysis of the membrane association of M protein in the early and late phases of the Sendai virus infectious cycle revealed that the interaction of M protein with mature glycoproteins that associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of the membrane-bound M protein. Immunofluorescence analysis by confocal microscopy also demonstrated that in Sendai virus-infected cells, a fraction of M protein colocalized with F and HN proteins and that some M protein also became associated with the F and HN proteins while they were in transit to the plasma membrane via the exocytic pathway. These studies indicate that F and HN interact with M protein in the absence of any other viral proteins and that F associates with M protein via its cytoplasmic tail and transmembrane domain.     

Studies with inhibitors of oligosaccharide processing indicate a functional role for complex sugars in the transport and proteolysis of Friend mink cell focus-inducing murine leukemia virus envelope proteins

The functions of asparagine-linked oligosaccharides on the PrENV protein of Friend mink cell focus-inducing (FrMCF-1) murine leukemia virus were investigated by examining the effect of two inhibitors of different stages of the biosynthetic pathway of these sugar substituents on the synthesis and processing of the viral proteins. Treatment of virus-producing cells with tunicamycin totally inhibited the glycosylation of PrEnv, and resulted in the formation of a nonglycosylated form of the protein of molecular weight 62 kDa. This component was not proteolytically processed inside the cells, and neither it nor any derivative proteins were incorporated into extracellular virions. Treatment of cells with 1-deoxynojirimycin (DNM), which inhibits the cellular glucosidases normally involved in removal of the three glucose residues present on the initially transferred oligosaccharide chains, resulted in the intracellular accumulation of a slightly larger than normal form of PrENV, and decreased levels of cell-associated gp70. Only gp70 was detected on the cell surface. The bulk of the gp70 produced in the presence of the drug was aberrantly glycosylated, and contained decreased levels of complex and increased numbers of high mannose oligosaccharides; almost all of the gp70 molecules however, contained at least one complex sugar chain. Decreased incorporation of both env and gag proteins into extracellular virions was observed, despite the fact that the gag proteins were processed normally intracellularly; in contrast, DNM treatment of Gazdar murine sarcoma virus-infected HTG2 cells, which produce only gag but not env proteins, did not inhibit the release of extracellular virus. Ultrastructural examination of FrMCF-infected cells treated with DNM indicated the presence of large numbers of intracytoplasmic vacuoles, many of which contained viral particles. These studies indicate that the normal maturation process involved in the formation of complex oligosaccharides is necessary to obtain efficient transport to the plasma membrane and proteolysis of PrEnv, and also provide evidence suggesting a role for the env proteins in regulating assembly of gag proteins into virions.     

Studies on the role of M protein in virus assembly using a ts mutant of HVJ (Sendai virus).

A temperature-sensitive mutant of HVJ, HVJ cl.151, was isolated from BHK cells persistently infected with HVJ and characterized. HVJ c1.151 virion had an M polypeptide different in apparent molecular weight from that of HVJ wild-type, that is 36,000 and 34,000 daltons, respectively. HVJ c1.151 was blocked in a late function required for virus maturation. M protein antigen of HVJ c1.151 was detected in infected cells by immunofluorescent microscopy only at permissive temperature but not at nonpermissive temperature, although GP and NP antigens were detected at both temperatures. Further, analysis of the infected cells by SDS-polyacrylamide gel electrophoresis showed that viral structural polypeptides P, F₀, NP, and F and nonstructural polypeptide C were synthesized in infected cells at nonpermissive temperature and these structural polypeptides were incorporated into virions upon temperature shiftdown, whereas polypeptides HN and M, which may be synthesized at nonpermissive temperature, were not able to be incorporated into virions upon temperature shift down. Thus, the temperature-sensitive lesion of HVJ c1.151 is considered to be in HN and M proteins. Membrane fluorescense, immunoferritin electron microscopy, and SDS-polyacrylamide gel electrophoresis of plasma membranes showed that migration of F₀, and F to the cell surface occurred normally even at nonpermissive temperature. Immunoferritin electron microscopy also demonstrated that the viral glycoproteins which arrived at the plasma membrane were dispersed on the entire surface of the membrane at the nonpermissive temperature and that viral components synthesized at this temperature could not assemble at the plasma membrane. In addition, it was found that antibody-induced redistribution of viral glycoproteins on the surface of cells infected with HVJ c1.151 and incubated at nonpermissive temperature occurred very rapidly; in contrast, such redistribution of viral glycoproteins occurred more slowly and less completely in cells incubated at permissive temperature, suggesting that viral glycoproteins on the plasma membrane of cells at nonpermissive temperature have a high degree of mobility in the plane of the membrane as compared with those on the plasma membrane of cells at permissive temperature. These results suggest strongly that a function which fixes the viral glycoproteins at restricted areas of plasma membrane to form a viral envelope is blocked in HVJ c1.151-infected cells at nonpermissive temperature. Analysis of plasma membrane by SDS-polyacrylamide gel electrophoresis showed that viral glycopolypeptides but no NP were present on membranes isolated from HVJ cl.151-infected cells at nonpermissive temperature in spite of the presence of a large amount of NP in the whole cells, whereas plasma membranes isolated from cells at permissive temperature contained all viral structural polypeptides. The possible roles of M protein of HVJ in formation of the viral envelope and association of nucleocapsid with the envelope during assembly are discussed based on these results.     

Morphogenesis of sandfly viruses (Bunyaviridae family)

The events occurring in the morphogenesis of sandfly fever viruses have been examined by thin-section electron microscopy and by an analysis of the association of virus-specific polypeptides with membranes of infected cells. Two representative sandfly fever viruses have been studied, Karimabad virus (KV) and Punta Toro virus (PTV), which appeared indistinguishable both in terms of virion structure, as monitored by negative staining, and morphogenesis, as observed in thin sections of infected Vero cells. Ammonium molybdate negative staining of purified, glutaraldehyde-fixed virions revealed essentially spherical particles, 87 nm in diameter, in which the surface proteins were constructed into closely packed, hollow, cylindrical subunits measuring 10–11 nm in diameter and 9–10 nm in length. These surface units are located peripherally to a 7-nm membrane bilayer which surrounds a nucleoid of variable electron density. As seen in thin sections of infected cells, the assembly of these particles was first detected at 12 hr after infection, occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Morphologically mature particles were formed by continuous involution (budding) of modified membrane segments into the lumen of these vesicles. Viral ribonucleoprotein (RNP), which was not observed free in the cytoplasm, condensed at the cytoplasmic face of these vesicles at areas at which viral spike structures could be observed at the contralateral (luminal) face. Neither RNP nor spike structures could be observed on adjacent sections of the vesicular membrane, or other membranes, which were not directly involved in the assembly of a budding virion. Analysis of the viral polypeptides in unfractionated membrane vesicles prepared from infected cells demonstrated that KV-specific envelope and nucleocapsid proteins rapidly became membrane bound, whereas a nonstructural polypeptide was found only in cytoplasmic fractions. Chymotrypsin treatment of these vesicles has indicated that at least one viral glycoprotein is inserted into cellular membranes such that approximately 12% of its sequence remains at the cytoplasmic membrane face. Proteolytic removal of this sequence generated an immunoprecipitable, glycosylated fragment which was protected from further proteolysis by its orientation within the vesicle. These morphologic and biochemical data have been used to construct a model for the assembly of these viruses. Virus particles are released from infected cells by exocytosis, a process which does not appear to result in significant modification of cell surface membranes with viral antigens.