Kinetics of utilization of Sendai virus RNA and protein in the process of virion assembly.

The synthesis of the 50S genomic RNA and strucural proteins of Sendai virus was examined with respect to their utilization in virus assembly. It was found that during a single cycle of infection, 50S RNA was synthesized before the structural proteins and that both RNA and protein were synthesized 2 to 4 h before their appearance in released virions. Pulse-chase labeling indicated that the NP and P proteins synthesized early and the M and F proteins synthesized late were preferentially incorporated into virus relative to the other viral proteins. The kinetics of incorporation of pulse-labeled NP protein suggested that it was withdrawn from a relatively large pool whereas the M protein appeared to be present in a relatively small pool in the cytoplasm. Further, it was possible to chase pulse-labeled M protein, but not NP protein, from the cell during an 8-h time period.     

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.     

Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells.

We have investigated the association of the influenza virus matrix (M1) and nucleoprotein (NP) with the host cell cytoskeletal elements in influenza virus-infected MDCK and MDBK cells. At 6.5 h postinfection, the newly synthesized M1 was Triton X-100 (TX-100) extractable but became resistant to TX-100 extraction during the chase with a t1/2 of 20 min. NP, on the other hand, acquired TX-100 resistance immediately after synthesis. Significant fractions of both M1 and NP remained resistant to differential detergent (Triton X-114, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], octylglucoside) extraction, suggesting that M1 and NP were interacting with the cytoskeletal elements. However, the high-molecular-weight form of the viral transmembrane protein hemagglutinin (HA), which had undergone complex glycosylation, also became resistant to TX-100 extraction but was sensitive to octylglucoside detergent extraction, indicating that HA, unlike M1 or NP, was interacting with TX-100-insoluble lipids and not with cytoskeletal elements. Morphological analysis with cytoskeletal disrupting agents demonstrated that M1 and NP were associated with microfilaments in virus-infected cells. However, M1, expressed alone in MDCK or HeLa cells from cloned cDNA or coexpressed with NP, did not become resistant to TX-100 extraction even after a long chase. NP, on the other hand, became TX-100 insoluble as in the virus-infected cells. M1 also did not acquire TX-100 insolubility in ts 56 (a temperature-sensitive mutant with a defect in NP protein)-infected cells at the nonpermissive temperature. Furthermore, early in the infectious cycle in WSN-infected cells, M1 acquired TX-100 resistance very slowly after a long chase and did not acquire TX-100 resistance at all when chased in the presence of cycloheximide. On the other hand, late in the infectious cycle, M1 acquired TX-100 resistance when chased in either the presence or absence of cycloheximide. Taken together, these results demonstrate that M1 and NP interact with host microfilaments in virus-infected cells and that M1 requires other viral proteins or subviral components (possibly viral ribonucleoprotein) for interaction with host cytoskeletal components. The implication of these results for viral morphogenesis is discussed.     

Synthesis of Newcastle disease virus polypeptides in a wheat germ cell-free system.

We have isolated 18S RNA from cytoplasmic extracts of Newcastle disease virus-infected Chinese hamster ovary cells and tested its ability to direct protein synthesis in extracts derived from wheat germ. The products of the cell-free reaction directed by this RNA contain polypeptides that comigrate with NP, M,F, and 47K roteins from virions. In addition, the products contain a polypeptide (67K) that migrates on polyacrylamide gels slightly faster than the HN protein from virions. Tryptic peptide analysis of the cell-free products and proteins from virions confirms their identity.     

Basis for selective incorporation of glycoproteins into the influenza virus envelope.

The ability of mutant or chimeric A/Japan hemagglutinins (HAs) to compete for space in the envelope of A/WSN influenza viruses was investigated with monkey kidney fibroblasts that were infected with recombinant simian virus 40 vectors expressing the Japan proteins and superinfected with A/WSN influenza virus. Wild-type Japan HA assembled into virions as well as WSN HA did. Japan HA lacking its cytoplasmic sequences, HAtail-, was incorporated into influenza virions at half the efficiency of wild-type Japan HA. Chimeric HAs containing the 11 cytoplasmic amino acids of the herpes simplex virus type 1gC glycoprotein or the 29 cytoplasmic amino acids of the vesicular stomatitis virus G protein were incorporated into virions at less than 1% the efficiency of HAtail-. Thus, the cytoplasmic domain of HA was not required for the selection process; however, foreign cytoplasmic sequences, even short ones, were excluded. A chimeric HA having the gC transmembrane domain and the HA cytoplasmic domain (HgCH) was incorporated at 4% the efficiency of HAtail-. When expressed from simian virus 40 recombinants in this system, vesicular stomatitis virus G protein with or without (Gtail-) its cytoplasmic domain was essentially excluded from influenza virions. Taken together, these data indicate that the HA transmembrane domain is required for incorporation of HA into influenza virions. The slightly more efficient incorporation of HgCH than G or Gtail- could indicate that the region important for assembling HA into virions extends into part of the cytoplasmic domain.     

Purification of measles virus and characterization of subviral components.

Purified measles virus was obtained from [35S]methionine-labeled cells infected at 33 degrees C and maintained in the absence of fetal calf serum. The pellet that was produced by a single high-speed ultracentrifuge spin of culture medium contained virus of purity sufficient for structural analysis. Purified virions contain seven polypeptides with estimated molecular weights of: L, 200,000; G, 80,000; P2, 70,000; NP, 60,000; A, 43,000; F1, 41,000; and M, 37,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Treatment of virions with 0.25% trypsin resulted in a less dense particle which lacked polypeptides G and F1. Solubilization of the viral membrane with the detergent Triton X-100 in low-salt buffer resulted in the loss of the G polypeptide, whereas in the presence of 1 M KCl, Triton X-100 also removed most of the M polypeptide. The nucleocapsids (p = 1.3) obtained from virions treated with Triton X-100 and 1 M KCl contained the L, P2, NP, and M polypeptides. Nucleocapsids isolated from the cytoplasm of infected cells were predominantly composed of the NP polypeptide with smaller amounts of either polypeptide P2 or novel polypeptides, related to NP, with estimated molecular weights of 56,000 to 58,000 and 45,000 to 46,000. A significant amount of polypeptide L was always found in association with nucleocapsids isolated either from virions or from the cytoplasm of infected cells. A membrane component containing the viral membrane polypeptides G, F1, and M was also isolated from infected cells. The data presented here thus suggest that L is an integral part of the nucleocapsid complex. In addition, 37,000-molecular-weight polypeptide (M) appears to have the function described for the matrix proteins of other paramyxoviruses.     

Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

Molecular biology of rotaviruses. I. Characterization of basic growth parameters and pattern of macromolecular synthesis

The United Kingdom tissue-adapted bovine rotavirus growing in African green monkey kidney (BSC-1) cells was selected as a model system with which to study the detailed molecular virology of rotavirus replication. Study of the kinetics of infectious virus production revealed a fairly rapid replication cycle, with maximum yield of virus after 10 to 12 h at 37 degrees C. Progeny genome synthesis was first detected during the virus latent period at 2 to 3 h postinfection. Study of the kinetics of viral polypeptide synthesis showed that virus rapidly inhibited cellular polypeptide synthesis such that by 4 h postinfection, only virus-induced polypeptides, 15 of which were detected, were being synthesized. No qualitative changes in the pattern of viral polypeptide synthesis were observed during infection, although, based on kinetic synthesis, three quantitative classes of polypeptides were defined. Pulse-chase analysis revealed three post-translational changes in viral proteins, two of which were shown to be due to glycosylation. Tunicamycin inhibition studies were used to identify the putative non-glycosylated precursors of the two glycoproteins. Comparison of the infected-cell polypeptides with those present in purified virions revealed that mot of the virus-induced proteins were incorporated into virions, with only VP9 being a truly nonstructural protein. Some localization of the various polypeptides within the purified virion was achieved by producing viral cores.     

Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles.     

Packaging of the virion host shutoff (Vhs) protein of herpes simplex virus: two forms of the Vhs polypeptide are associated with intranuclear B and C capsids, but only one is associated with enveloped virions

The virion host shutoff (Vhs) protein (UL41) is a minor component of herpes simplex virus virions which, following penetration, accelerates turnover of host and viral mRNAs. Infected cells contain 58-kDa and 59.5-kDa forms of Vhs, which differ in the extent of phosphorylation, yet only a 58-kDa polypeptide is incorporated into virions. In pulse-chase experiments, the primary Vhs translation product comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the 58-kDa virion polypeptide, and could be chased to 59.5 kDa. While both 59.5-kDa and 58-kDa forms were found in nuclear and cytoplasmic fractions, the 59.5-kDa form was significantly enriched in the nucleus. Both forms were associated with intranuclear B and C capsids, yet only the 58-kDa polypeptide was found in enveloped cytoplasmic virions. A 58-kDa form, but not the 59.5-kDa form, was found in L particles, noninfectious particles that contain an envelope and tegument but no capsid. The data suggest that virions contain two populations of Vhs that are packaged by different pathways. In the first pathway, the primary translation product is processed to 59.5 kDa, is transported to the nucleus, binds intranuclear capsids, and is converted to 58 kDa at some stage prior to final envelopment. The second pathway does not involve the 59.5-kDa form or interactions between Vhs and capsids. Instead, the primary translation product is phosphorylated to the 58-kDa virion form and packaged through interactions with other tegument proteins in the cytoplasm or viral envelope proteins at the site of final envelopment.     

Rabies virus protein synthesis in infected BHK-21 cells.

Rabies virus specific polypeptide synthesis was examined under hypertonic conditions, which selectively inhibit cellular protein synthesis. The rabies virus proteins (L, G, N, M1, M2) were synthesized throughout the course of infection, with little change in their relative rates of synthesis. The rates of synthesis of the G and M1 polypeptides were more sensitive to increasing osmolarity than those of the L, N, and M2 polypeptides. Extrapolation to isotonicity of the results obtained under hypertonic conditions indicated that the molar ratios of the polypeptides synthesized under normal conditions were 0.4 (L), 64 (G), 100 (N), 75 (M1) and 35 (M2). A high-molecular-weight polypeptide (190,000), designated polypeptide L, was repeatedly detected both in infected cells and in extracellular virus. The estimated number of L polypeptide molecules per virion was 33. The synthesis of a viral glycoprotein precursor, designated gp78, , preceded the appearance of the mature viral glycoprotein in infected cells labeled with [3H]glucosamine under isotonic conditions. In cells labeled under hypertonic conditions, little or no mature viral glycoprotein was detected, but a virus-specific glycoprotein with an electrophoretic mobility similar to that of gp78 was observed. This glycoprotein could be chased into mature viral glycoprotein when the hypertonic conditions were made isotonic. These results suggest that a reversible block of viral glycoprotein synthesis occurs under hypertonic conditions.     

Analysis of the Inhibitory Effect of Canavanine on the Replication of Influenza RI/5+ Virus: II. Interaction of M Protein with the Plasma Membrane

When influenza A/RI/5⁺ virus-infected cells were incubated in medium to which 2 μg of canavanine (arginine analog) per ml had been added 4 hr after infection, all viral polypeptides were synthesized but the budding-like process with the appearance of extracellular virus was completely inhibited. The plasma membrane isolated from these cells contained exclusively hemagglutinin (HA), and membrane (M) protein and nucleoprotein (NP) appeared to be associated with the nucleus, in contrast to untreated cells whose plasma membrane contained abundant HA, M protein, and NP. Disruption of canavanine-treated cells by freeze-thawing generated a number of hemagglutinating membranous vesicles or fragments containing exclusively HA. By isotope labeling it was found that the M protein synthesized in the presence of canavanine, together with HA and NP, is a canavanine-substituted polypeptide. It is suggested that canavanine inhibits the formation of the mature envelope of influenza RI/5⁺, because of the inability of M protein to associate with the plasma membrane.     

Envelope Proteins of Vesicular Stomatitis Virus: Effect of Temperature-Sensitive Mutations in Complementation Groups III and V

All five major viral proteins were synthesized in chicken embryo cells infected with vesicular stomatitis virus temperature-sensitive (ts) mutants of complementation groups III and V and maintained at the nonpermissive temperature. The distribution of these proteins among cytoplasmic cellular fractions separated on discontinuous sucrose gradients was identical for wild-type and tsIII-infected cells. Strikingly different patterns were observed for the G protein in gradients from cells infected by tsV mutants; very little, if any, G protein was found in the lightest fraction. Pulse and chase experiments with wild-type, virus-infected cells showed that protein G moves from the heaviest to the lightest fraction before being incorporated into the virion. After shift down to the permissive temperature (30 C), G protein synthesized at 39.6 C in tsV-infected cells became associated with the lightest cellular fraction and later with the released virions. In contrast, M protein, synthesized at 39.6 C in tsIII-infected cells, was not incorporated into the virions after shift down. These data strongly suggest, first, that M protein is encoded by the vesicular stomatitis gene III, and second, that incorporation of G protein in the lightest cellular fraction is a necessary step of vesicular stomatitis maturation. This step is impaired by tsV mutations.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

Measles virus polypeptides in infected cells studied by immune precipitation and one-dimensional peptide mapping.

Measles virus does not turn off host cell polypeptide synthesis, making it difficult to precisely identify the polypeptides specified by the virus during the infectious cycle. By using the technique of immune precipitation with measles-specific antisera, the host cell background has been eliminated, and new observations have been made concerning measles virus polypeptides H, P, NP, F, and M. The H polypeptide is first synthesized as a monomer which is processed by further glycosylation and by the formation of disulfide-bonded dimers. Polypeptide P (70,000 daltons) has been found to occur also as a 65,000-dalton molecule, P2, and both forms of the molecule are equally phosphorylated. Polypeptide NP is processed from a cleavage-sensitive form (which undergoes cleavage during the process of isolation to form polypeptide 6 [41,000 daltons]) to a form which is resistant to this cleavage. The fusion and hemolysin polypeptide is first found in the cells as a 55,000-dalton precursor, F0, which is clearly resolved from the NP polypeptide on gel electrophoresis. The measles virus F0 protein identified in previous reports had not been resolved from the 60,000-dalton NP polypeptide. The M protein occurs in the infected cells as two distinct bands, and, as in the case of Sendai virus, one of these two M protein bands represents a phosphorylated form of the other.