Cardiovirus Leader proteins bind exportins: Implications for virus replication and nucleocytoplasmic trafficking inhibition.

Cardiovirus Leader proteins (LX) inhibit cellular nucleocytoplasmic trafficking by directing host kinases to phosphorylate Phe/Gly-containing nuclear pore proteins (Nups). Resolution of the Mengovirus LM structure bound to Ran GTPase, suggested this complex would further recruit specific exportins (karyopherins), which in turn mediate kinase selection. Pull-down experiments and recombinant complex reconstitution now confirm that Crm1 and CAS exportins form stable dimeric complexes with encephalomyocarditis virus LE, and also larger complexes with LE:Ran. shRNA knockdown studies support this idea. Similar activities could be demonstrated for recombinant LS and LT from Theiloviruses. When mutations were introduced to alter the LE zinc finger domain, acidic domain, or dual phosphorylation sites, there was reduced exportin selection. These regions are not involved in Ran interactions, so the Ran and Crm1 binding sites on LE must be non-overlapping. The involvement of exportins in this mechanism is important to viral replication and the observation of trafficking inhibition by LE.

Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells.

It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells.

Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase.

Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (L(M)) show both modifications act together to partially stabilize a short internal α-helix comprising L(M) residues 43-46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48-67 into a new interface. The full structure of L(M) as bound to Ran (unlabeled) and Ran (216 aa) as bound by L(M) (unlabeled) places L(M) into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by L(M) binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:L(M) partner as an exportin, Crm1, or CAS. A model of Ran:L(M):Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran's selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways.

Synthesis of the allergen ovomucoid by a replicating Mengo virus.

Interferons induced by viral infections can have powerful immuno- modulatory effects, and several epidemiologic studies have found an association between certain viral infections and reduced prevalence of allergy. We hypothesized that allergenic proteins could be synthesized by a replicating virus, and this construct could be useful as an immunomodulator. To test this hypothesis, we cloned an allergenic protein (ovomucoid [OVM]) into a murine picornavirus (Mengo virus) vector. This plasmid has a multicloning site surrounded by auto-catalytic sequences so that a foreign protein will be cleaved from viral proteins during replication. OVM sequences were cloned in the context of full-length viral genome cDNA, T7 RNA transcripts of this plasmid were transfected into HeLa cells, and recombinant virus plaques appeared on the second passage. Sequence analysis of recombinant viruses derived from individual plaques demonstrated that three viral isolates contained up to 2/3 of the OVM coding sequence, which was retained by the viruses after 5 additional passages in HeLa cells. The experiments verify the stable expression of immunoreactive OVM subunits by replicating viruses. These virus/allergen constructs could provide a tool to evaluate whether intracellular presentation of allergenic proteins in the context of a viral infection could prevent allergic sensitization upon re-challenge.

Dehydron: a structurally encoded signal for protein interaction.

We introduce a quantifiable structural motif, called dehydron, that is shown to be central to protein-protein interactions. A dehydron is a defectively packed backbone hydrogen bond suggesting preformed monomeric structure whose Coulomb energy is highly sensitive to binding-induced water exclusion. Such preformed hydrogen bonds are effectively adhesive, since water removal from their vicinity contributes to their stability. At the structural level, a significant correlation is established between dehydrons and sites for protein complexation, with the HIV-1 capsid protein P24 complexed with antibody light-chain FAB25.3 providing the most dramatic correlation. Furthermore, the number of dehydrons in homologous similar-fold proteins from different species is shown to be a signature of proteomic complexity. The techniques are then applied to higher levels of organization: The formation of the capsid and its organization in picornaviruses correlates strongly with the distribution of dehydrons on the rim of the virus unit. Furthermore, antibody contacts and crystal contacts may be assigned to dehydrons still prevalent after the capsid has been assembled. The implications of the dehydron as an encoded signal in proteomics, bioinformatics, and inhibitor drug design are emphasized.

Mutational analysis of the mengovirus poly(C) tract and surrounding heteropolymeric sequences.

Previously, we described three mengovirus mutants derived from cDNA plasmids, containing shortened poly(C) tracts (C8, C12, and C13UC10), that exhibited strong attenuation for virulence in mice yet grew like wild-type virus in HeLa cells. Thirteen additional mutants hav now been constructed and characterized. Five of these differ only in poly(C) length, including one with a precise deletion of the tract. The other mutants bear deletions into the regions juxtaposing poly(C). Studies with HeLa cells confirm the essential dispensability of mengovirus's poly(C) tract but reveal a subtle, measurable correlation between poly(C) length and plaque diameter. Virulence studies with mice also revealed a strong correlation between poly(C) length and virulence. For the poly(C)-flanking mutations, the 15 bases directly 5' of the tract proved dispensable for virus viability, whereas the 20 to 30 bases 3' of poly(C) were critical for growth, thus implicating this region in the basal replication of the virus.

The cellular U-particle, whose synthesis is induced by mengovirus infection, is homologous to apoferritin.

Mengo virus infection of mouse L-cells results in induction of the synthesis of a cellular protein-containing particle, 12 nm in diameter, which was designated U (Boege et al. (1987) Virology 159, 358-367). We have purified the U-particle from virus-infected cells by a series of chromatographic steps and found it to be composed of two polypeptide species (MW 23,000 and 25,000), present in a ratio of approximately 7:3. Neither of these polypeptides is measurably glycosylated or phosphorylated and the U-particle contains no detectable nucleic acid. Several amino acid sequences obtained from CNBr fragments of the U-polypeptides identified them as the H- and L-chains of mouse apoferritin. This finding was supported by immunoblotting and electron microscopy. In terms of function, the U-particle/apoferritin effectively inhibits the translation of mRNAs in reticulocyte lysates. These experiments indicate that apoferritin may perform important functions in eukaryotic cells in addition to iron storage. Finally, we propose mechanisms to explain how Mengo virus infection could specifically induce the synthesis of apoferritin and how increasing amounts of cytoplasmic apoferritin could facilitate virus replication.

Proteolytic processing of the cardioviral P2 region: primary 2A/2B cleavage in clone-derived precursors.

The primary 2A/2B cleavage within cardiovirus polyprotein was examined by construction of cDNA plasmids which linked fragments from the P2 region of encephalomyocarditis virus (EMCV) and Mengovirus genomes to the EMCV 5' nontranslated region. When RNA transcripts from these clones were tested in reticulocyte extracts, the synthesized proteins were cotranslationally processed at the 2A/2B site. No viral segments outside of the P2 region were required for this activity. Engineered deletions which removed the amino-terminal two-thirds of protein 2A or the carboxyl half of protein 2B had no effect on this scission, nor did insertions into a Ser-Ala-Phe sequence (SAF) within 2B, which is conserved in most cardio- and aphthoviruses. In contrast, mutations which disrupted a conserved Asn-Pro-Gly-Pro (NPGP) sequence abolished primary scission. Precursors thus inactivated were unable to serve as substrate when simultaneously expressed with active (wild-type) 2AB sequences. Microsequencing placed the EMCV primary cleavage site between the Gly/Pro pair within the NPGP sequence. It was also determined that endogenous viral protease 3C is the previously unidentified agent responsible for cardiovirus 1D/2A scission, a cleavage that is part of the primary processing reaction in poliovirus.

Conformational variability of a picornavirus capsid: pH-dependent structural changes of Mengo virus related to its host receptor attachment site and disassembly.

The structure of Mengo virus had been determined from crystals grown in the presence of 100 mM phosphate buffer at pH 7.4. It is shown that Mengo virus is poorly infectious at the phosphate concentration similar to that in which it was crystallized. Maximal infectivity is achieved at 10 mM phosphate or less in physiological saline. The phosphate effect is ameliorated when the pH is lowered to 4.6. Although it has not been possible to study the crystal structure of the virus at low phosphate concentrations, it is shown that increasing the Cl- concentration at pH 6.2 or decreasing the pH to 4.6 causes substantial conformational changes confined to the "pit," a deep surface depression. These structural changes involve a movement of the "FMDV loop" (GH loop) in VP1, an ordering of the "VP3 loop" (GH loop in VP3) between 3176 and 3182, the displacement of a bound phosphate near the "FMDV loop" (GH loop in VP1), and movement of the carboxy terminus of VP2. The changes in conformation are correlated with the dissociation of the virion into pentamers at pH 6.2 and 150 mM Cl-. The localization of the conformational changes and the correlated role of the phosphate in controlling infectivity support the hypothesis that the "pit" is the receptor attachment site.

Mengo virus maturation is accompanied by C-terminal modification of capsid protein VP1.

We have demonstrated that the C-terminal ends of the VP1 proteins of Mengo virus undergo a postassembly trimming of three amino acid residues. A variable proportion of the VP1 molecules isolated from purified virions terminate at Glu 277, which corresponds to the initial viral protease 3C cleavage site. The remainder of the VP1 molecules terminates at Leu 274. When chymotrypsin treatment is included in the virus purification procedure, Leu 274 is the exclusive C-terminal amino acid of the VP1 molecules. The trimming process was found to influence the pH-mediated dissociability of virions in vitro: this was demonstrated by sucrose gradient analysis and electron microscopy. Since long-term incubation of purified virions did not alter the C-terminal Glu 277/Leu 274 ratio, it appears that the trimming process is not autocatalytic.

Conservation of the putative receptor attachment site in picornaviruses.

A deep canyon or pit on the surfaces of human rhinovirus 14 and Mengo virus, respectively, has been proposed as a putative receptor binding site. Amino acids lining the surface of the canyon or pit have been identified and show greater conservation than other surface residues.

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.

Human rhinovirus protein synthesis and polyprotein cleavage in infected HeLa-RH cells. Brief report.

Precursor and mature polypeptides of four human rhinovirus types (HRV 30, 63, 81 and 88) were compared. The SDS-PAGE profiles of the HRV-specific polypeptides differed significantly from each other, as well as from those of the HRV types studied previously (HRV 1A, 2 and 14). Our results provide further evidence for considerable heterogeneity within the rhinovirus genus.

Neuraminidase-sensitive erythrocyte receptor for enterovirus type 70.

Enterovirus type 70 (EV70) agglutinated human 'O' erythrocytes at 4 degrees C as well as 22 degrees C, but visible agglutination was lost when warmed at 37 degrees C although the virus remained attached to the surface of the erythrocyte. The receptor sites for the virus were neuraminidase-sensitive. A direct involvement of sialic acid on the cell surface in virus-cell interaction was confirmed by the fact that the presence of fetuin or free N-acetylneuraminic acid inhibited the haemagglutinating activity of EV70. Similar numbers of virus particles were required for 1 haemagglutinating unit (HAU) of EV70 and 1 HAU of mengovirus, whereas 2.6-fold or more of virus particles of echovirus type 7 and type 11 gave the same activity. On the other hand, the number of receptor sites on the cell surface for EV70 was found to be sevenfold more than for mengovirus. Therefore, the erythrocyte receptor for EV70 is different from that for common enteroviruses and similar, though not identical, to the cardiovirus receptor. However, serological tests such as neutralization, complement fixation or haemagglutination inhibition did not reveal any common antigen between EV70 and cardiovirus.

Mechanism of action of antiviral hetarylhydrazones in vitro.

Three antiviral hetarylhydrazones showed a depression of 3H-uridine incorporation into RNA of FL cells as a consequence of inhibition of uridine transport into the cells. By eliminating the action of the compounds on transport processes with the prelabelling method, only one compound showed a nearly 50% reduction of cellular RNA synthesis during the whole incubation period, whereas the other two compounds had no influence during the first 6 hr. Under prelabelling conditions all three compounds completely inhibited the synthesis of Mengo virus RNA. One compound did not affect cellular DNA synthesis, but presumably depressed thymidine transport into the cells.

Replacement of mouse LM fibroblast choline by a sulfonium analog. Effects on membrane properties as determined by virus probes.

A sulfonium analog of choline ('sulfocholine', a natural phospholipid constituent of diatoms) was metabolically incorporated into mouse LM fibroblasts cultured in serum-free medium. Subconfluent cultures of LM cells were able to utilize sulfocholine as sole choline source and to increase in cell number for 3 days of incubation; thereafter a decrease in cell number was observed. In contrast, cultures of LM cells seeded to confluency showed no decrease in cell number up to at least 10 days when maintained, with daily medium changes, in medium containing either choline or the sulfonium analog. Such confluent cultures, maintained for 7 days in sulfocholine-containing medium, showed virtually complete replacement of cellular phosphatidylcholine and greater than 50% replacement of cellular sphingomyelin by their respective sulfonium analogs. The functional exchangeability of natural phosphatidylcholine and sphingomyelin with their sulfonium analogs to participate in normal cell membrane-mediated activities was demonstrated by comparatively assaying the abilities of sulfocholine- and choline-maintained cells to incorporate and replicate certain animal viruses known to possess membrane-dependent steps in various phases of their replication cycles. No difference was detected between the abilities of sulfocholine- and choline-maintained cells to take up vesicular stomatitis virus or mengo virus, or to replicate vesicular stomatitis virus, mengo virus or mouse hepatitis virus.

Studies of two temperature-sensitive mutants of Mengo virus.

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA- ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 degrees C) to the nonpermissive (39 degrees C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA- phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 degrees C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant. Subviral (53S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 degrees C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.