Variations in cap-binding complexes from uninfected and poliovirus-infected HeLa cells

Poliovirus infection of HeLa cells results in cleavage of the p220 subunit of eukaryotic initiation factor eIF-4F and inhibits cap-dependent initiation of protein synthesis. To examine the effect of virus-induced inhibition on the structure of initiation factor complexes involved in cap binding, the polypeptide compositions of cap affinity-purified complexes from uninfected and poliovirus-infected HeLa cells were analyzed. Monoclonal antibodies directed against p220 and an eIF-3 subunit, p170, were utilized to locate eIF-3 and eIF-4F on sucrose gradients and in fractions eluting from cap analog columns. This approach resulted in the purification of several different cap-binding complexes from different cellular subfractions and revealed significant differences in their composition after infection. The results indicate that eIF-3 and eIF-4F bind to the cap structure, possibly in the form of a complex, and that a modified form of eIF-3 alone has some cap-binding activity in the complete absence of p220, eIF-4A, and eIF-4E. Ribosome-derived complexes containing cleaved p220 are no longer associated with eIF-3 or eIF-4A, and a significant amount of cleaved p220 is associated with a unique cytoplasmic cap-binding complex. The cytoplasmic complex also contains Mr = 170,000 and 80,000 polypeptides, neither of which are major components of eIF-4F. These results demonstrate significant variation in the composition of cap-binding complexes from both infected and uninfected cells. They indicate that eIF-3 might play a direct role in cap binding and suggest that poliovirus-induced cleavage of p220 results in the release of the eIF-4A subunit from eIF-4F and abolishes an association between eIF-4F and eIF-3 which may function during the multifactor steps involved in initiation of cap-mediated translation.     

Association of cap-binding protein with eucaryotic initiation factor 3 in initiation factor preparations from uninfected and poliovirus-infected HeLa cells.

Extracts from poliovirus-infected HeLa cells are unable to translate vesicular stomatitis virus or cellular mRNAs in vitro, probably reflecting the poliovirus-induced inhibition of host cell protein synthesis which occurs in vivo. Crude initiation factors from uninfected HeLa cells are able to restore translation of vesicular stomatitis virus mRNA in infected cell lysates. This restoring activity separates into the 0 to 40% ammonium sulfate fractional precipitate of ribosomal salt wash. Restoring activity is completely lacking in the analogous fractions prepared from poliovirus-infected cells. The 0 to 40% ammonium sulfate precipitates from both uninfected and infected cells contain eucaryotic initiation factor 3 (eIF-3), eIf-4B, and the cap-binding protein (CBP), which is detected by means of a cross-linking assay, as well as other proteins. The association of eIF-3 and cap binding protein was examined. The 0 to 40% ammonium sulfate precipitate of ribosomal salt wash from uninfected and infected cells was sedimented in sucrose gradients. Each fraction was examined for the presence of eIF-3 antigens by an antibody blot technique and for the presence of the CBP by cross-linking to cap-labeled mRNAs. From uninfected cells, a major proportion of the CBP cosedimented with eIF-3; however, none of the CBP from infected cells sedimented with eIF-3. The results suggest that the association of the CBP with eIF-3 into a functional complex may have been disrupted during the course of poliovirus infection.     

Presence of the cap-binding protein in initiation factor preparations from poliovirus-infected HeLa cells.

Crude preparations of initiation factors from mock-infected and poliovirus-infected HeLa cells were analyzed for the presence of proteins which could be cross-linked to the 5' cap group of mRNA. A protein having an apparent molecular weight of 26,000, similar to the cap-binding protein in rabbit reticulocytes described by Sonenberg and Shatkin (Proc. Natl. Acad. Sci. U.S.A. 75:4843-4847, 1978), was found in the ribosomal salt wash from both uninfected and infected cells. Cross-linking of this polypeptide was inhibited by the cap analog m7GMP. In addition, cross-linking of a protein having an approximate molecular weight of 60,000 was similarly inhibited by cap analog. The smaller cap-binding protein fractionated in a 0 to 40% ammonium sulfate precipitate of ribosomal salt wash; the larger protein was found in the 40 to 70% ammonium sulfate fraction. Although the cap-binding proteins were present in both mock-infected and poliovirus-infected ribosomal salt wash, only preparations from uninfected HeLa cells were able to restore translation of capped vesicular stomatitis virus mRNA by extracts prepared from poliovirus-infected cells.     

Isolation and structural characterization of cap-binding proteins from poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, poliovirus RNA is translated at times when cellular mRNA translation is strongly inhibited. It is thought that this translational control mechanism is mediated by inactivation of a cap-binding protein complex (comprising polypeptides of 24 [24-kilodalton cap-binding protein], 50, and approximately 220 kilodaltons). This complex can restore the translation of capped mRNAs in extracts from poliovirus-infected cells. We have previously shown that the virally induced defect prevents interaction between cap recognition factors and mRNA. Here, we show that the cap-binding protein complex (and not the 24-kilodalton cap-binding protein) has activity that restores the cap-specific mRNA-protein interaction when added to initiation factors from poliovirus-infected cells. Thus, the activity that restores the cap-specific mRNA-protein interaction and that which restores the translation of capped mRNAs in extracts from poliovirus-infected cells, copurify. The results also indicate, by an alternative assay, that the cap-binding protein complex is the only factor inactivated by poliovirus. We also purified cap-binding proteins from uninfected and poliovirus-infected HeLa cells. By various criteria, the 24-kilodalton cap-binding protein is not structurally modified as a result of infection. However, the 220-kilodalton polypeptide of the cap-binding protein complex is apparently cleaved by a putative viral (or induced) protease. By in vivo labeling and m7GDP affinity chromatography, we isolated a modified cap-binding protein complex from poliovirus-infected cells, containing proteolytic cleavage fragments of the 220-kilodalton polypeptide.     

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells has been studied. When these cells are synchronized for initiation by fluoride treatment and then double labeled with [³⁵S]methionine and either tritiated proline, phenylalanine, or valine for short pulses, the percentage of N-terminal methionine incorporated in the nascent peptides compared to total incorporation is significantly higher than that of the tritiated amino acids tested. The data indicate that methionine is the initiator amino acid for the synthesis of poliovirus-specific proteins.     

Localization of cap-binding protein in subcellular fractions of HeLa cells

The 26,000-M(r) cap-binding protein was analyzed by a cross-linking assay in cell fractions from uninfected and poliovirus-infected HeLa cells. Cap-binding protein was found in the postribosomal supernatant (S-200) and in the ribosomal salt wash. The cap-binding protein in the S-200 had a sedimentation coefficient of 5 to 7S and lacked the ability to restore translation in extracts of poliovirus-infected cells.     

Two forms of purified m7G-cap binding protein with different effects on capped mRNA translation in extracts of uninfected and poliovirus-infected HeLa cells

Eukaryotic mRNA cap binding proteins were purified from ribosomal salt wash in the presence of protease inhibitors by sucrose gradient sedimentation and m7GDP-Sepharose affinity chromatography. Rabbit reticulocyte and erythrocyte proteins with sedimentation constants of less than 6 S yielded a approximately 24,000-dalton cap binding protein. It stimulated capped mRNA translation in extracts of uninfected HeLa cells but did not restore capped mRNA function in extracts prepared from poliovirus-infected cells. Restoring and stimulatory activities both were associated with a larger, approximately 8-10 S complex that included the approximately 24,000-dalton polypeptide and several higher molecular mass components. The same two translational activities were also obtained in a slightly smaller approximately 5-7 S complex from uninfected HeLa cells but were absent from poliovirus-infected cell preparations.     

Monoclonal antibody-aided characterization of cellular p220 in uninfected and poliovirus-infected HeLa cells: subcellular distribution and identification of conformers.

A monoclonal antibody directed against the Mr-220,000 subunit (p220) of the mRNA cap-binding complex has been prepared and used to analyze the sucrose gradient sedimentation and subcellular location of p220 and its poliovirus-induced cleavage products. The antibody reacted with p220 on immunoblots of cell lysates from uninfected cells, but only with several smaller polypeptides, the p220 cleavage products, in cell lysates from poliovirus-infected cells. The sedimentation of p220 antigens from uninfected or infected cells was analyzed by immunoblot and by enzyme-linked immunosorbent assay (ELISA) of sucrose gradient fractions. The results indicate that antibody reactivity was partially influenced by antigen conformation. Major forms of intact p220 and cleaved p220 were identified by immunoblot, and these had similar sedimentation properties. ELISA analysis of the same gradient fractions detected only uncleaved p220; p220 cleavage products were not recognized. Furthermore, the antibody recognized two forms of native uncleaved p220, one of which appeared to bind antibody with greater affinity. This result suggested the existence of conformational variants of p220. The differential reactivity of the antibody for cleaved versus uncleaved p220 served as a useful control during indirect immunofluorescence analysis to determine the subcellular distribution of p220 antigens. The distribution of p220 in uninfected cells was mainly cytoplasmic, but some nuclear antigens were also apparent. After poliovirus infection only the nuclear pattern remained. Disappearance of the cytoplasmic pattern confirmed the inability of the antibody to react with native p220 cleavage products. The cytoplasmic pattern also disappeared after human rhinovirus 14 infection, but not after mengovirus infection, results which correlated with the ability of human rhinovirus 14 and the inability of mengovirus to induce the cleavage of p220. The results demonstrate that p220 is not likely to be associated with the cytoskeleton and hint at the possibility of a partially nuclear location.     

Translation of capped viral mRNAs in poliovirus-infected HeLa cells.

HeLa cells doubly infected with Semliki Forest virus (SFV) and poliovirus synthesize either more poliovirus proteins or more SFV late proteins depending on the time of super-infection with poliovirus. Under some conditions, the infected cells translate uncapped poliovirus mRNA and capped 26S mRNA from SFV simultaneously, even though host protein synthesis has been shut down. Vesicular stomatitis virus (VSV) protein synthesis is depressed drastically when VSV-infected cells are super-infected with poliovirus. In cells doubly infected with VSV and encephalomyocarditis (EMC) virus or with VSV and SFV, dominance of one of the viruses depends on the time of addition of the challenge virus. The influence of external conditions on the relative translation of capped or uncapped viral mRNA in doubly infected cells has also been analysed.     

Guanidine-sensitive Na+ accumulation by poliovirus-infected HeLa cells.

The Na+ content of poliovirus-infected HeLa S3 cells increased during the late phase of virus replication, after virus inhibition of host cell protein synthesis and in coincidence with late viral functions. Guanidine hydrochloride blocked the rise in Na+ content, whereas the antiguanidine agent choline fully reversed the guanidine block. Expression of one or more late viral functions was essential for Na+ accumulation to occur because accumulation was inhibited by cycloheximide or guanidine added to the infected culture during the late phase. Increased adenosine triphosphatase activity appears to be primarily responsible for Na+ accumulation by virus-infected cells.     

Monovalent cation metabolism and cytopathic effects of poliovirus-infected HeLa cells.

To better understand the significance of 22Na+ accumulation by poliovirus-infected HeLa cells (C. N. Nair, J. W. Stowers, and B. Singfield, J. Virol. 31:184, 1979), measurements of cellular Na+, K+, and Cl- contents, volume, and density were carried out at intervals after infection. In addition, the rates of 22Na+ washout from infected and control cells were determined. Starting at around 3 h postinfection, the Na+ content of infected cells increased, whereas the K+ content decreased progressively, resulting in a net loss in the monovalent cation content decreased progressively, resulting in a net loss in the monovalent cation content per cell. The loss in cellular chloride content exceeded that in monovalent cation content. The kinetics of 22Na+ washout from infected and control cells revealed the presence of an extra Na+ compartment in infected cells. A net loss in the monovalent cation activity of infected cells was indicated by the loss of cell water as reflected in a decrease in cell volume and an increase in cell density. In spite of a net loss in monovalent cation content per cell, Na+ accumulation coupled with cell shrinkage resulted in substantial increases in the concentrations of not only Na+ but also K+. The results suggested a possible role for tonicity change in the morphological lesions of poliovirus cytotoxicity.     

Differential targeting of nuclear pore complex proteins in poliovirus-infected cells

Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2A^pro to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.     

Inhibition of host cell protein synthesis in poliovirus-infected cells

Poliovirus infection of HeLa cells in culture causes rapid inhibition of host cell protein synthesis, while viral proteins are synthesized at high levels. This inhibition correlates with the inactivation of eukaryotic initiation factor 4F (eIF-4F), by proteolytic cleavage of its γ-subunit, p220. eIF-4F is required for the translation of capped mRNAs. Poliovirus RNA is uncapped and is translated by a cap independent mechanism. The poliovirus protease, 2A^pro, is required for p220 cleavage, but induces this cleavage indirectly by activating a host protease that catalyzes p220 cleavage. Eukaryotic initiation factor 3 is also required for p220 cleavage, but its role in the cleavage reaction is unknown.     

Activity of aspartate transcarbamylase in uninfected and type 5 adenovirus-infected HeLa cells

Consigli, Richard A. (University of Pennsylvania, Philadelphia), and Harold S. Ginsberg. Activity of aspartate transcarbamylase in uninfected and type 5 adenovirus-infected HeLa cells. J. Bacteriol. 87:1034-1043. 1964.-A two- to three-fold increase in aspartate transcarbamylase (ATCase) activity was observed in type 5 adenovirus-infected HeLa cells 18 hr after infection. The enhanced enzyme activity was virus-specific and dependent on biosynthesis of deoxyribonucleic acid and protein. When various characteristics as well as the kinetics of the enzymes from uninfected and infected cells were compared, ATCase from adenovirus-infected cells was shown to have an altered pH optimum, greater heat stability, increased maximal velocity, and increased K(m) value for aspartate.