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.     

Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus.

Purified virions or radiolabeled lysates of infected MA104 cells were used to characterize the structural and nonstructural polypeptides of a porcine group C rotavirus. At least six structural proteins were identified from purified group C rotavirus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these, two (37,000- and 33,000-molecular-weight polypeptides) were associated with the outer shell, as demonstrated by the ability of EDTA to remove them from the purified virion. The other four polypeptides (molecular weights, 125,000, 93,000, 74,000, and 41,000) were located in the inner shell. The structural or nonstructural nature of a 25,000-molecular-weight protein identified in our studies was unclear. Glycosylation inhibition studies with tunicamycin in infected cells demonstrated that the 37,000- and 25,000-molecular-weight proteins were glycosylated and contained mannose-rich oligosaccharides identified by radiolabeling of the infected cells with [3H]mannose. The 37,000-molecular-weight outer shell glycoprotein was shown by pulse-chase experiments to be posttranslationally processed. The kinetics of viral polypeptide synthesis in infected cells were also studied, and maximal synthesis occurred at 6 to 9 h postinfection. The 41,000-molecular-weight inner capsid polypeptide was the most abundant and was the subunit structure of a 165,000-molecular-weight protein aggregate. Two polypeptides (molecular weights, 39,000 and 35,000) appeared to be nonstructural, as determined by comparison of the protein pattern of radiolabeled infected cell lysates with that of purified virions. Radioimmunoprecipitation was used to examine the serologic cross-reactions between the viral polypeptides of a group C rotavirus with those of a group A rotavirus. No serologic cross-reactivities were detected. The polypeptides of group A and C rotaviruses are compared and discussed.     

Pol-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells prior to Nef-mediated major histocompatibility complex class I downregulation

Effective, vaccine-induced CD8+ T-cell responses should recognize infected cells early enough to prevent production of progeny virions. We have recently shown that Gag-specific CD8+ T cells recognize simian immunodeficiency virus-infected cells at 2 h postinfection, whereas Env-specific CD8+ T cells do not recognize infected cells until much later in infection. However, it remains unknown when other proteins present in the viral particle are presented to CD8+ T cells after infection. To address this issue, we explored CD8+ T-cell recognition of epitopes derived from two other relatively large virion proteins, Pol and Nef. Surprisingly, infected cells efficiently presented CD8+ T-cell epitopes from virion-derived Pol proteins within 2 h of infection. In contrast, Nef-specific CD8+ T cells did not recognize infected cells until 12 h postinfection. Additionally, we show that SIVmac239 Nef downregulated surface major histocompatibility complex class I (MHC-I) molecules beginning at 12 h postinfection, concomitant with presentation of Nef-derived CD8+ T-cell epitopes. Finally, Pol-specific CD8+ T cells eliminated infected cells as early as 6 h postinfection, well before MHC-I downregulation, suggesting a previously underappreciated antiviral role for Pol-specific CD8+ T cells.     

Biochemical and electron microscopic studies of the replication and composition of milker''s node virus

Replication of milker's node virus (MNV) DNA begins 4 to 8 h postinfection, continues to 30 to 36 h postinfection in the cytoplasm of infected, primary bovine embryonic kidney cells, and is accompanied by an inhibition of host nuclear DNA synthesis. Between 20 and 24 h postinfection, newly replicated genomes are incorporated into particles which cosediment with purified MNV. These biochemical measurements could be correlated with the development of MN virions as revealed by electron microscopic analysis of thin sections prepared from infected cells. Analysis of the DNA in purified MNV showed that the virions contained a double-stranded DNA molecule with a molecular weight of 85 x 10(6) to 87 x 10(6) and a guanine-plus-cytosine content of about 63%. After denaturation and sedimentation analysis of MNV DNA in alkaline sucrose gradients, three major DNA species were resolved. These species appeared to represent intact, terminally cross-linked genomes (approximately 75 to 80S); genomes bearing one nick (or with one cross-link removed) (60 to 65S); and complementary, denatured DNA strands released from cross-linked genomes bearing two nicks (or with both cross-links removed) (52 to 55S). Forty [35S]methionine-labeled polypeptides, ranging from approximately 200,000 daltons to 10,000 to 15,000 daltons, were detected by radioautography after polyacrylamide gel electrophoresis of the proteins present in detergent-solubilized MNV preparations. Treatment of MN virions with Nonidet P-40, beta-mercaptoethanol, and sonication released 10 polypeptides, which were apparently located on the surface of virions. Further fractionation of these released polypeptides, followed by electron microscopy and polyacrylamide gel electrophoresis, indicated that a 42,000- to 45,000-dalton polypeptide is a major component of the threadlike tubule structure present on the surface of MN virions.     

Gene protein products of SA11 simian rotavirus genome

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.     

Synthesis of plus- and minus-strand RNA in rotavirus-infected cells.

The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and minus-strand RNAs was detected initially at 3 h postinfection. The steady-state levels of plus- and minus-strand RNAs increased from this time until 9 to 12 h postinfection, at which time the levels were maximal. Pulse-labeling of infected cells with [3H]uridine showed that the ratio of plus- to minus-strand RNA synthesis changed during infection and that the maximal level of minus-strand RNA synthesis occurred several hours prior to the peak of plus-strand RNA synthesis. No direct correlation was found between the levels of plus-strand and minus-strand RNA synthesis in the infected cell. Pulse-labelling studies indicated that both newly synthesized and preexisting plus-strand RNA can act as templates for minus-strand RNA synthesis throughout infection. Studies also showed that less than 1 h was required between the synthesis of minus-strand RNA in vivo and its release from the cell within virions.     

Polypeptide Synthesis in Simian Virus 5-Infected Cells

Polypeptide synthesis in three different cell types infected with simian virus 5 has been examined using high-resolution polyacrylamide slab gel electrophoresis, and all of the known viral polypeptides have been identified above the host cell background. The polypeptides were synthesized in infected cells in unequal proportions, which are approximately the same as they are found in virions, suggesting that their relative rates of synthesis are controlled. The nucleocapsid polypeptide (NP) was the first to be detected in infected cells, and by 12 to 14 h the other virion structural polypeptides were identified, except for the polypeptides comprising the smaller glycoprotein (F). However, a glycosylated precursor (F(0)) with a molecular weight of 66,000 was found in each cell type, and pulse-chase experiments suggested that this precursor was cleaved to yield polypeptides F(1) and F(2). No other proteolytic processing was found. In addition to the structural polypeptides, the synthesis of five other polypeptides, designated I through V, has been observed in simian virus 5-infected cells. One of these (V), with a molecular weight of 24,000, was found in all cells examined and may be a nonstructural viral polypeptide. In contrast, there are polypeptides present in uninfected cells that correspond in size to polypeptides I through IV, and similar polypeptides have also been detected in increased amounts in cells infected with Sendai virus. These findings, and the fact that the synthesis of all four of these polypeptides is not increased in every cell type, suggest that they represent host polypeptides whose synthesis may be enhanced upon infection. When a high salt concentration was used to decrease host cell protein synthesis in infected cells, polypeptides IV and (to a lesser extent) I were synthesized in relatively greater amounts than other cellular polypeptides, as were the viral polypeptides. The possibility that these polypeptides may play some role in virus replication is discussed.     

Identification and characterization of a porcine parvovirus nonstructural polypeptide.

Sera from porcine parvovirus (PPV)-infected swine fetuses immunoprecipitated and 84- to 86-kilodalton polypeptide in addition to the A and B virion structural proteins. This polypeptide, designated NS-1, was present in PPV-infected cell lysates but not in purified virions. Partial proteolysis mapping revealed that NS-1 was not related to the A and B viral structural proteins. All three proteins in infected cells were phosphorylated at serine residues, and NS-1 also contained phosphothreonine. From pulse-labeling experiments with either 32Pi or [35S]methionine, NS-1 was found to first appear 5 to 7 h postinfection, whereas the viral structural polypeptides were first synthesized 9 to 11 h postinfection. Pulse-chase experiments revealed that NS-1 initially appeared as an 84-kilodalton protein and was subsequently structurally modified to forms of slower electrophoretic mobilities. The time of appearance of NS-1 after virus infection coincided with the initiation of viral DNA synthesis, suggesting that this polypeptide (and the modified forms thereof) may be involved in PPV replication.     

Proteins Specified by Herpes Simplex Virus VI. Viral Proteins in the Plasma Membrane

Previous papers in the series have shown that the surface membranes of herpesvirus-infected cells acquire new immunological specificities and that purified infected cell membrane preparations, characterized by their physical properties rather than topology in the cell, contain new glycoproteins genetically determined by the virus. In this study, we prepared purified plasma membrane identified by its 5' nucleotidase, fucose, and reduced nicotinamide adenine dinucleotide-diaphorase content. Analysis of the membrane proteins and glycoproteins by electrophoresis in acrylamide gels indicated the following. (i) Purified plasma membranes from infected cells contained two sets of proteins, i.e., host proteins were present both before and after infection and viral proteins were present only after infection. (ii) After infection, no appreciable selective or nonselective loss of host proteins from membranes was demonstrable. However, no new host proteins were made. (iii) Electropherograms of plasma membrane proteins from infected cells indicated the presence of at least 12 virus-specific proteins ranging in molecular weight from 25 x 10(3) to 126 x 10(3) daltons. Of these, at least nine were glycosylated. Proteins and glycoproteins with similar electrophoretic mobilities but in somewhat different ratios were also present in preparations of highly purified virions.     

Synthesis of Proteins and Glycoproteins in Cells Infected with Human Cytomegalovirus

In cytomegalovirus-infected cells, the rate of protein synthesis was detected as two peaks. One occurred during the early phase of infection, 0 to 36 h postinfection, and the other occurred during the late phase, after the initiation of viral DNA synthesis. Double-isotopic-label difference analysis demonstrated that host and viral proteins were synthesized simultaneously during both phases. In the early phase, approximately 70 to 90% of the total proteins synthesized were host proteins, whereas approximately 10 to 30% were viral, even at a multiplicity of infection of 20 PFU/cell. Virus-related proteins or glycoproteins were referred to as infected-cell specific (ICS). Two ICS glycoproteins (gp145 and 100) were clearly detectable and were synthesized preferentially in the early phase of infection. Their synthesis was concomitant with stimulation of the protein synthesis rate. In the late phase of infection, approximately 50 to 60% of the total protein synthesis was viral and approximately 40 to 50% was host. The ICS proteins and glycoproteins detected during the late phase of infection were viral structural proteins. Infectious virus was not detectable until 48 to 72 h postinfection. An inhibitor of viral DNA synthesis, phosphonoacetic acid, prevented the appearance of the late-phase ICS proteins and glycoproteins, but there was little or no effect on early ICS glycoprotein synthesis. Radiolabeled ICS proteins and glycoproteins were identified by their relative rates of synthesis, by their different electrophoretic mobilities compared with those of host proteins and host glycoproteins, and by their similar electrophoretic mobilities compared to those of proteins and glycoproteins associated with virions and dense bodies of cytomegalovirus. Structural viral antigens in the infected-cell extracts were removed by immunoprecipitation, using F(ab')(2) fragments of cytomegalovirus-specific antibodies, and identified as described above. The last two criteria were used to identify viral structural ICS proteins and glycoproteins. Although approximately 35 structural proteins were found to be associated with purified virions and dense bodies, the continued synthesis of host cell proteins complicated their identification in infected cells. Nevertheless, seven of the nine structural glycoproteins were identified as ICS glycoproteins.     

In vivo and in vitro synthesis of adenovirus type 2 early proteins.

The synthesis of adenovirus type 2 (Ad2)-induced early polypeptides was examined in vivo and in vitro by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis alone and specific immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of total [35S]methionine-labeled polypeptides synthesized in vivo at 3 h postinfection allowed us to detect in infected cells at lease 13 distinct polypeptides that are either absent or less conspicuous in extracts from mock-infected cells. These Ad2-induced early polypeptides have molecular weights ranging from 72 x 10(3) to 10.5 x 10(3) and have accordingly been designated as E72K to E10.5K. Nine of the in vivo synthesized early polypeptides can be precipitated specifically from infected cell extracts by antisera with specificity against early adenovirus proteins. In vitro translation of mRNA extracted from mock-infected cells and from Ad2-infected cells was carried out in preincubated Ehrlich ascites cell extracts. All the early Ad2-induced polypeptides identified in the extracts from infected cells labeled in vivo were also detected among the polypeptides immunoprecipitated specifically from the in vitro reaction mixtures programmed by RNA extracted at 4 h postinfection from Ad2-infected cells.     

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.     

Multiplication of parvovirus LuIII in a synchronized culture system. IV. Association of viral structural polypeptides with the host cell chromatin.

Newly synthesized structural polypeptides of parvovirus LuIII, VP1 (62,000 daltons) and VP2 (74,000 daltons), were detected in nuclei of synchronized, infected HeLa cells at 11 to 12 h postinfection, i.e., after cells had passed through the S phase of the cell cycle. At this time, most of intranuclear viral polypeptides were associated with the chromatin acidic proteins. However, 13 to 14 h postinfection, about one-third of intranuclear VP1 and VP2 also could be extracted in the fraction containing nuclear sap proteins. According to pulse-chase experiments, VP1 and VP2 accumulated in the chromatin with a time lag of 20 to 30 min. About 90% of these chromatin-associated viral polypeptides represented empty viral capsids. In addition, chromatin prepared at 14 h postinfection contained 90 to 95% of the total intranuclear viral 16S replicative-form DNA. Since viral replicative-form DNA and empty viral capsids seem to be associated specifically with cellular chromatin, we assume that this subnuclear structure is the site of the synthesis of progeny viral DNA and the formation of complete virions.     

Proteins Specified by bovine herpesvirus 1 (infectious bovine rhinotracheitis virus)

An electrophoretic analysis of radioactively labeled, purified, "empty" and DNA-containing infectious bovine rhinotracheitis virions revealed the presence of 25 to 33 structural (virion) polypeptides. A total of 11 of these polypeptides could be labeled with [3H]glucosamine and were identified as glycoproteins. In addition to the 25 structural polypeptides, infectious bovine rhinotracheitis virus infected cells also contained at least 15 nonstructural (nonvirion) polypeptides that were not present in purified virions. Expression of the viral polypeptides in infected cells was controlled temporally. Thus, most viral polypeptides could be categorized as "alpha" (immediate early), "beta" (early), or "gamma" (late) on the basis of their order of appearance in infected cells and whether their syntheses were dependent upon prior viral protein or DNA synthesis. None of the glycoproteins belongs to the alpha class, although at least one (GVP11) was synthesized in the absence of viral DNA synthesis. Serum from a cow in which infectious bovine rhinotracheitis virus lesions were reactivated by dexamethasone precipitated both structural and nonstructural polypeptides.     

Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: effect of tunicamycin.

We identified eight protein species in virions of mouse hepatitis virus strain A59. Based on their sizes, prosthetic groups, and locations in virions, these proteins were designated gp180/E2, gp90/E2, pp54/N, gp26.5/E1, gp25.5/E1, p24/E1, p22/X, and p14.5/Y. The positions of the last two proteins in virions are not known. Host protein synthesis in Sac(-) cells infected with mouse hepatitis virus strain A59 was inhibited, and the following novel proteins appeared: gp150, gp90, p54, gp26.5, gp25.5, p24, p22, and p14.5. Except for gp150, these polypeptides all co-electrophoresed with mouse hepatitis virus strain A59 structural proteins. In addition, all of these proteins could be immunoprecipitated with a convalescent mouse serum or a rabbit antiserum raised against purified disrupted virus. After a 15-min pulse of infected cells with radioactive amino acids at 7h postinfection, gp90 was not detected, whereas gp26.5 and gp25.5 were only labeled to a small extent. During a subsequent chase period gp150 was processed to gp90, whereas the radioactivity in gp26.5 and gp25.5 increased concomitantly with a reduction of label in p24. Tunicamycin, an antibiotic which inhibits the synthesis of glycopeptides bearing N glycosidically linked oligosaccharides, prevented the appearance of gp150 in mouse hepatitis virus strain A59-infected cells. Instead, a 110,000-dalton protein accumulated. In contrast, the syntheses of the smaller viral glycoproteins gp26.5 and gp25.5 were resistant to this drug, indicating that these glycosylations were of the O glycosidical type. Although the production of infectious virus in tunicamycin-treated cells was inhibited by more than 99%, release of noninfectious viral particles continued. An analysis of these particles revealed that they lacked the peplomeric glycoproteins gp90/E2 and gp180/E2. Obviously, although the surface projections were not essential for budding of virus particles from the cells, they were required for infectivity.     

Immune reactive measles virus polypeptides on the cell's surface: turnover and relationship of the glycoproteins to each other and to HLA determinants

To better understand the mechanism(s) whereby antibody and complement and cytotoxic lymphocytes lyse infected cells, we studied the structure, interrelationship and turnover of measles virus polypeptides expressed on the cell's surface. Of the 6 major viral structural polypeptides, L, HA, P, NC, F, and M, found in purified virions or infected cells, only 2, the HA and F, resided on the surface of infected cells. The HA was present primarily in the form of a 160k dimer, and F was identified as a 64k polypeptide migrating distinct from other viral polypeptides. With reduction, the HA migrated as a 80k monomer, and F0, after cleavage, was found to be composed of a 42k nonglycosylated polypeptide, F1, and a 24k glycosylated protein, F2. The relationship between F0 and F1 and between the HA dimer and monomer was verified by tryptic peptide mapping. The turnover of HA and F from the cell's surface was 10 and 9 hr, respectively. However, in the presence of specific antibody after a marked loss of viral antigen from the surface, the turnover for HA and F was 15 and 12 hr, respectively. Despite being independent molecules, HA and F were closely linked, as they moved together (co-capped) over the plasma membrane when incubated with monospecific or monoclonal antibody. In contrast, neither HA nor F co-capped with the major histocompatibility antigens or with other host cell proteins, which indicates a separation between these host cell proteins and measles viral glycoproteins on the cell's surface.     

Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis.

The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various times postinfection were also analyzed. At least 13 proteins labeled with [3H]glucosamine were detected in vaccinia-infected HeLa cells.