Synthesis and Cleavage of Influenza Virus Proteins

The NWS strain of influenza virus grows rapidly in and kills the MDCK dog kidney cell strain. Within 1 to 2 hr, the virus inhibits host cell protein synthesis and for 3 to 4 hr more it directs the synthesis of influenza virus proteins at a rate about twice that of uninfected cell synthesis. The rates of virus ribonucleic acid (RNA) and protein synthesis reach a maximum within the first few hours after infection and then drop. Plaque assays exhibit a linear dose-response, indicating that only one virion is necessary for productive infection. We have confirmed earlier reports regarding the fragmented nature of the RNA genome of purified influenza virions. However, high resolution gel electrophoresis indicated that each size class of viral RNA is heterogenous, so that there are at least 10 and probably more fragment sizes of RNA in these virions. Repeated attempts to detect infectivity in preparations of extracted viral RNA were completely negative (over a 10(8)-fold loss of infectivity after extraction). Even infection of the "infectious" RNA-treated cells with intact, related, influenza viruses failed to support infectivity of the isolated RNA or to rescue a host range genetic marker of the RNA. Purified influenza virions exhibit only three major protein peaks based on separation according to molecular weights. These three major virion proteins are the only major virion proteins synthesized in infected cells. This is true throughout the infectious cycle from several hours after infection until the cells are dying. However, the molecular weight of these virion proteins differs slightly depending upon the cell type in which the virus is grown. No host membrane proteins are incorporated into the virions as they bud through the cell membrane. Pulse-chase labeling early after infection or prolonged chase experiments indicate that influenza virus proteins are cleaved from one or more precursor polypeptides. In fact, each of the three major peaks seems to be a heterogeneous mixture of polypeptides in various stages of cleavage. Peptide analysis confirms that the three major peaks share common peptides, but the exact precursor product relationships are not clear. There may be one or several precursor proteins. Also there could be overlapping messenger RNA molecules of varying length giving rise to polypeptides of various sizes and overlapping sequences. Late in infection, amino acid labeling shows a preponderance of internal nucleocapsid protein synthesis, indicating that either this protein is much more stable to cleavage in infection or it is made from a more stable messenger. There is no obvious relationship between virion RNA fragments and viral protein sizes, so these fragments may be artifacts.     

Intracellular synthesis of measles virus-specified polypeptides.

The intracellular synthesis of measles-specified polypeptides was examined by means of polyacrylamide gel electrophoresis of cell extracts. Since measles virus does not efficiently shut off host-cell protein synthesis, high multiplicities of infection were used to enable viral polypeptides to be detected against the high background of cellular protein synthesis. The cytoplasm of infected cells contained viral structural polypeptides with estimated molecular weights of 200,000, 80,000, 70,000, 60,000, 41,000, and 37,000. All of these structural polypeptides, with the exception of P1, the only virion glycoprotein (molecular weight congruent to 80,000), were also found in the nuclei. In addition, two nonstructural polypeptides with estimated molecular weights of 74,000 and 72,000 were also present in the cytoplasm of infected cells. The initial synthesis of the smaller, nonstructural polypeptide began later in infection than the structural polypeptides. Pulse-chase experiments failed to detect any precursor-product relationships. The intracellular glycosylation and phosphorylation of the viral polypeptides were found to be similar to those found in purified virions.     

Coronavirus multiplication: locations of genes for virion proteins on the avian infectious bronchitis virus genome.

Six overlapping viral RNAs are synthesized in cells infected with the avian coronavirus infectious bronchitis virus (IBV). These RNAs contain a 3'-coterminal nested sequence set and were assumed to be viral mRNAs. The seven major IBV virion proteins are all produced by processing of three polypeptides of ca. 23, 51, and 115 kilodaltons. These are the core polypeptides of the small membrane proteins, the nucleocapsid protein, and the 155-kilodalton precursor to the large membrane proteins GP90 and GP84, respectively. To determine which mRNAs specify these polypeptides, we isolated RNA from infected cells and translated it in a messenger-dependent rabbit reticulocyte lysate. Proteins of 23, 51, and 110 kilodaltons were produced. Two-dimensional tryptic peptide mapping demonstrated that these proteins were closely related to the major virion proteins. Fractionation of the RNA before cell-free translation permitted the correlation of messenger activities for synthesis of the proteins with the presence of specific mRNAs. We found that the smallest RNA, RNA A, directs the synthesis of P51, the nucleocapsid protein. RNA C, which contains the sequences of RNA A, directs the synthesis of the small membrane protein P23. RNA E directs the synthesis of the large virion glycoproteins. These results supported a model in which only the unique 5'-terminal domain of each IBV mRNA is active in translation and enabled us to localize genes for virion proteins on the IBV genome.     

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.     

Translation of three mouse hepatitis virus strain A59 subgenomic RNAs in Xenopus laevis oocytes

We have purified the seven virus-specific RNAs which were previously shown to be induced in Sac(-) cells upon infection with mouse hepatitis virus strain A59 (W. J. M. Spaan, P. J. M. Rottier, M. C. Horzinek, and B. A. M. van der Zeijst, Virology 108:424-434, 1981). The individual RNAs, prepared by agarose gel electrophoresis of the polyadenylated RNA fraction from infected cells, were obtained pure, except for the preparations of RNAs 4, 5, and 6, which contained some contamination of RNA 7. The RNAs were microinjected into Xenopus laevis oocytes, and after incubation of these cells in the presence of [35S]methionine, the proteins synthesized were analyzed by polyacrylamide gel electrophoresis. Whereas no translation products of RNAs 1, 2, 4, and 5 were detected, the synthesis of virus-specific polypeptides coded by RNAs 3, 6, and 7 was observed. RNA 7 (0.6 X 10(6) daltons) directed the synthesis of a 54,000-molecular-weight polypeptide which comigrated with viral nucleocapsid protein and which was immunoprecipitated by antiserum from mice that had been infected with the virus. RNA 6 (0.9 X 10(6) daltons) directed the synthesis of three polypeptides with molecular weights of 24,000, 25,500, and 26,500, which migrated with the same electrophoretic mobilities as three low-molecular-weight virion polypeptides. After injection of RNA 3 (3.0 X 10(6) daltons), a polypeptide with a molecular weight of about 150,000 was immunoprecipitated. This polypeptide had no counterpart in the virion, but comigrated with a virus-specific glycoprotein present in infected cells which is immunoprecipitated by a rabbit antiserum against the mouse hepatitis virus strain A59 structural proteins. This antiserum could also immunoprecipitate the translation products of RNAs 3, 6, and 7. These results indicate that RNAs 3, 6, and 7 encode viral structural proteins. The significance of the data with respect to the strategy of coronavirus replication is discussed.     

In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells.

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.     

Interspecific variations in proteins synthesized by mammalian mitochondria.

The products of mitochondrial protein synthesis in established cell lines of various mammalian species were labelled with [35S]methionine and their number and apparent molecular weights determined by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis and fluorography. Proteins synthesized by isolated rat liver mitochondria were labelled with [3H]valine and similarly characterized. Each species had a distinctive pattern of from 10 to 13 mitochondrially synthesized proteins with apparent molecular weights between 10,000 and 50,000. No differences were detected in the number or electrophoretic mobility of the mitochondrially synthesized proteins of SV-40-transformed and nontransformed WI-38 cells.     

Action of Interferon: Kinetics and Differential Effects on Viral Functions

A highly purified rabbit interferon was tested for its capacity to inhibit various manifestations of infection of primary rabbit kidney (RK) cells with vesicular stomatitis (VS) virus. A kinetic analysis of the actinomycin-sensitive phase of interferon-induced cellular resistance revealed that RK cells could transcribe virtually all of the hypothetical antiviral messenger ribonucleic acid (mRNA) within 3 hr. Similar exposure to interferon reduced virus yield by 95 to 99% and markedly inhibited cytopathic effect on RK cells infected at a multiplicity of 10 or less. Interferon was less effective in blocking cytopathic effects when RK cells were infected at a multiplicity of 100. However, RK cells pretreated with the same amount of interferon and infected at a multiplicity of 100 failed to incorporate (3)H-amino acids into structural or nonstructural proteins of VS virus identified by polyacrylamide gel electrophoresis. Despite this inhibition of viral protein synthesis, interferon did not prevent the switch off by VS virus of cellular protein synthesis. The rapidity with which a high multiplicity of VS virus switched off cellular protein synthesis, even in cells rendered resistant to viral infection by interferon, is further evidence that this reaction is caused by an infecting virion component rather than by a newly synthesized viral product.     

The major core protein P4a (A10L gene) of vaccinia virus is essential for correct assembly of viral DNA into the nucleoprotein complex to form immature viral particles

The vaccinia virus (VV) A10L gene codes for a major core protein, P4a. This polypeptide is synthesized at late times during viral infection and is proteolytically cleaved during virion assembly. To investigate the role of P4a in the virus life cycle and morphogenesis, we have generated an inducer-dependent conditional mutant (VVindA10L) in which expression of the A10L gene is under the control of the Escherichia coli lacI operator/repressor system. Repression of the A10L gene severely impairs virus growth, as observed by both the inability of the virus to form plaques and the 2-log reduction of viral yields. This defect can be partially overcome by addition of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). Synthesis of viral proteins other than P4a occurred, although early shutoff of host protein synthesis and expression of viral late polypeptides are clearly delayed, both in the absence and in the presence of IPTG, compared with cells infected with the parental virus. Viral DNA replication and concatemer resolution appeared to proceed normally in the absence of the A10L gene product. In cells infected with VVindA10L in the absence of the inducer virion assembly is blocked, as defined by electron microscopy. Numerous spherical immature viral particles that appear devoid of dense viroplasmic material together with highly electron-dense regular structures are abundant in VVindA10L-infected cells. These regularly spaced structures can be specifically labeled with anti-DNA antibodies as well as with a DNase-gold conjugate, indicating that they contain DNA. Some images suggest that these DNA structures enter into spherical immature viral particles. In this regard, although it has not been firmly established, it has been suggested that DNA uptake occurs after formation of spherical immature particles. Overall, our results showed that P4a and/or its cleaved products are essential for the correct assembly of the nucleoprotein complex within immature viral particles.     

Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.     

Sequential Protein Synthesis Following Vaccinia Virus Infection

Inhibition of HeLa cell protein synthesis and the sequential synthesis of viral proteins were followed by pulse-labeling infected cells with (14)C-phenylalanine. Proteins were resolved by polyacrylamide gel electrophoresis. The viral origin of native proteins was confirmed by immunodiffusion. The inhibition of host protein synthesis and the synthesis of early viral proteins occur 1 to 3 hr after infection. This early sequence of events also occurs in the presence of 5-fluorodeoxyuridine, an inhibitor of deoxyribonucleic acid synthesis. Other viral proteins are synthesized at a later time. Those proteins which are not made in the absence of viral deoxyribonucleic acid synthesis can be further subdivided into intermediate and late classes. The intermediate protein is synthesized before the late proteins but does not appear to be a precursor of them. Many more viral polypeptides were resolved by polyacrylamide gel electrophoresis after solubilization of the entire cytoplasmic fraction with sodium dodecyl sulfate. Virion and nonvirion proteins were identified. Kinetic experiments suggested that certain structural proteins as well as certain nonstructural proteins are made early, whereas others of both classes are made primarily at later times.     

Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.     

Analysis of VSV mutant with attenuated cytopathogenicity: mutation in viral function, P, for inhibition of protein synthesis.

T1026, a ts mutant of VSV which is much less cytopathogenic than its parent, HR, and which can establish persistent infection under certain conditions, is a double mutant. In addition to its ts mutation in the virion RNA polymerase, T1026 has a second non-ts mutation in a viral function termed "P". This function is responsible for the inhibition of total protein synthesis in infected cells and acts chiefly at the level of translational initiation. In some cell systems, the inhibition of protein synthesis produced by P appears to be selective for cellular protein synthesis, whereas in other cell systems, both cellular and viral protein synthesis are inhibited. T1026 and its ts revertants are phenotypically P- -that is, cells infected with them show total protein synthesis rates equal to or greater than uninfected cells, while synthesizing viral proteins at the same or even greater rates than HR-infected cells. The P- mutation is correlated with failure to increase plaque size after 2-3 days of incubation. Since viral mutants obtained from persistently infected cultures in a variety of systems appear to be double mutants with a ts mutation in the virion RNA polymerase and a small plaque marker, we suggest that T1026 could represent a model for such mutants.     

Specific incorporation of host cell surface proteins into budding vesicular stomatitis virus particles

The specific incorporation of cell surface proteins into budding Vesicular Stomatitis Virus (VSV) particles was shown by two approaches. In the first, monolayer cultures of Vero or L cells were labeled by lactoperoxidase-catalyzed iodination and the cells were then infected with VSV. Approximately 2% of the cell surface ¹²⁵1 radioactivity was incorporated into particles which co-purify with normal, infectious virions by both velocity and equilibrium gradient centrifugation and which are precipitated by antiserum specific for the VSV glycoprotein. Control experiments establish that these ¹²⁵I-labeled particles are not cell debris or cellular material which aggregate with or adhere to VSV virions. VSV virions contain only a subset of the 10–15 normal ¹²⁵1-labeled cell surface polypeptides resolved by SDS gel electrophoresis; VSV grown in L cells and Vero cells incorporate different host polypeptides. In a second approach, Vero cells were labeled with ³⁵S-methione, then infected with VSV. Two predominant host polypeptides (molecular weights 110,000 and 20,000) were incorporated into VSV virions. These proteins, like VSV G protein, are exposed to the surface of the virion. They co-migrate with the major incorporated ¹²⁵1 host polypeptides. These host proteins are present in approximately 10 and 80 copies, respectively, per virion. Specific incorporation of host polypeptides into VSV virions does not require the presence of viral glycoprotein. This was shown by use of a ts VSV mutant defective in maturation of VSV G protein to the cell surface. Budding from infected cells are noninfectious particles which contain all the viral proteins except for G; these particles contain the same proportion and spectrum of ¹²⁵1-labeled host surface polypeptides as do wild-type virions. These results extend previous conclusions implicating the submembrane viral matrix protein, or the viral nucleocapsid, as being of primary importance in selecting cell surface proteins for incorporation into budding VSV virions.     

Viral proteomics: The emerging cutting-edge of virus research

Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment. They encode a wide spectrum of multifunctional proteins, which interplay with and modify proteins in host cells. Viral genomes were chronologically the first to be sequenced. However, the corresponding viral proteomes, the alterations of host proteomes upon viral infection, and the dynamic nature of proteins, such as post-translational modifications, enzymatic cleavage, and activation or destruction by proteolysis, remain largely unknown. Emerging high-throughput techniques, in particular quantitative or semi-quantitative mass spectrometry-based proteomics analysis of viral and cellular proteomes, have been applied to define viruses and their interactions with their hosts. Here, we review the major areas of viral proteomics, including virion proteomics, structural proteomics, viral protein interactomics, and changes to the host cell proteome upon viral infection.     

Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein,A6

A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.