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.

Three cardiovirus Leader proteins equivalently inhibit four different nucleocytoplasmic trafficking pathways.

Cardiovirus infections inhibit nucleocytoplasmic trafficking by Leader protein-induced phosphorylation of Phe/Gly-containing nucleoporins (Nups). Recombinant Leader from encephalomyocarditis virus, Theiler׳s murine encephalomyelitis virus and Saffold virus target the same subset of Nups, including Nup62 and Nup98, but not Nup50. Reporter cell lines with fluorescence mCherry markers for M9, RS and classical SV40 import pathways, as well as the Crm1-mediated export pathway, all responded to transfection with the full panel of Leader proteins, showing consequent cessation of path-specific active import/export. For this to happen, the Nups had to be presented in the context of intact nuclear pores and exposed to cytoplasmic extracts. The Leader phosphorylation cascade was not effective against recombinant Nup proteins. The findings support a model of Leader-dependent Nup phosphorylation with the purpose of disrupting Nup-transportin interactions.

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.

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

UNLABELLED: The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 µM. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations. IMPORTANCE: Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis.

AMP-activated protein kinase phosphorylates EMCV, TMEV and SafV leader proteins at different sites.

Cardioviruses of the Encephalomyocarditis virus (EMCV) and Theilovirus species encode small, amino-terminal proteins called Leaders (L). Phosphorylation of the EMCV L (LE) at two distinct sites by CK2 and Syk kinases is important for virus-induced Nup phosphorylation and nucleocytoplasmic trafficking inhibition. Despite similar biological activities, the LE phosphorylation sites are not conserved in the Theiloviruses, Saffold virus (LS, SafV) or Theiler׳s murine encephalitis virus (LT, TMEV) sequences even though these proteins also become phosphorylated in cells and cell-free extracts. Site prediction algorithms, combined with panels of site-specific protein mutations now identify analogous, but not homologous phosphorylation sites in the Ser/Thr and Theilo protein domains of LT and LS, respectively. In both cases, recombinant AMP-activated kinase (AMPK) was reactive with the proteins at these sites, and also with LE, modifying the same residue recognized by CK2.

Modeling of the human rhinovirus C capsid suggests possible causes for antiviral drug resistance.

Human rhinoviruses of the RV-C species are recently discovered pathogens with greater clinical significance than isolates in the RV-A+B species. The RV-C cannot be propagated in typical culture systems; so much of the virology is necessarily derivative, relying on comparative genomics, relative to the better studied RV-A+B. We developed a bioinformatics-based structural model for a C15 isolate. The model showed the VP1-3 capsid proteins retain their fundamental cores relative to the RV-A+B, but conserved, internal RV-C residues affect the shape and charge of the VP1 hydrophobic pocket that confers antiviral drug susceptibility. When predictions of the model were tested in organ cultures or ALI systems with recombinant C15 virus, there was a resistance to capsid-binding drugs, including pleconaril, BTA-188, WIN56291, WIN52035 and WIN52084. Unique to all RV-C, the model predicts conserved amino acids within the pocket and capsid surface pore leading to the pocket may correlate with this activity.

Modeling of the human rhinovirus C capsid suggests a novel topography with insights on receptor preference and immunogenicity.

Features of human rhinovirus (RV)-C virions that allow them to use novel cell receptors and evade immune responses are unknown. Unlike the RV-A+B, these isolates cannot be propagated in typical culture systems or grown for structure studies. Comparative sequencing, I-TASSER, MODELLER, ROBETTA, and refined alignment techniques led to a structural approximation for C15 virions, based on the extensive, resolved RV-A+B datasets. The model predicts that all RV-C VP1 proteins are shorter by 21 residues relative to the RV-A, and 35 residues relative to the RV-B, effectively shaving the RV 5-fold plateau from the particle. There are major alterations in VP1 neutralizing epitopes and the structural determinants for ICAM-1 and LDLR receptors. The VP2 and VP3 elements are similar among all RV, but the loss of sequence "words" contributing Nim1ab has increased the apparent selective pressure among the RV-C to fix mutations elsewhere in the VP1, creating a possible compensatory epitope.

Encephalomyocarditis virus leader is phosphorylated by CK2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins.

Encephalomyocarditis virus and Theilovirus are species in the Cardiovirus genus of the Picornaviridae family. For all cardioviruses, the viral polyprotein is initiated with a short Leader (L) protein unique to this genus. The nuclear magnetic resonance (NMR) structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc finger, a central linker, and a contiguous, highly acidic motif. The theiloviruses encode the same domains, with one or two additional, COOH-proximal domains, characteristic of the human Saffold viruses (SafV) and Theiler's murine encephalomyelitis viruses (TMEV), respectively. The expression of a cardiovirus L, in recombinant form, or during infection/transfection, triggers an extensive, cell-dependent, antihost phosphorylation cascade, targeting nucleoporins (Nups) that form the hydrophobic core of nuclear pore complexes (NPC). The consequent inhibition of active nucleocytoplasmic trafficking is potent and prevents the host from mounting an effective antiviral response. For this inhibition, the L proteins themselves must be phosphorylated. In cells (extracts or recombinant form), LE was shown to be phosphorylated at Thr47 and Tyr41. The first reaction (Thr47), catalyzed by casein kinase 2 (CK2), is an obligatory precedent to the second event (Tyr41), catalyzed by spleen tyrosine kinase (Syk). Site mutations in LE, or kinase-specific inhibitors, prevented LE phosphorylation and subsequent Nup phosphorylation. Parallel experiments with LS (SafV-2) and LT (TMEV BeAn) proteins confirmed the general cardiovirus requirement for L phosphorylation, but CK2 was not the culpable kinase. It is likely that LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains. IMPORTANCE An understanding of the diverse methods used by viruses to interfere with cellular processes is important because they can teach us how to control virus infections. This report shows how viruses in the same genus use different cellular enzymes to phosphorylate their proteins. If these processes are interfered with, the viruses are severely disabled.

Evolution of teleost fish retroviruses: characterization of new retroviruses with cellular genes.

The interactions between retroviruses and their hosts can be of a beneficial or detrimental nature. Some endogenous retroviruses are involved in development, while others cause disease. The Genome Parsing Suite (GPS) is a software tool to track and trace all Retroid agents in any sequenced genome (M. A. McClure et al., Genomics 85:512-523, 2005). Using the GPS, the retroviral content was assessed in four model teleost fish. Eleven new species of fish retroviruses are identified and characterized. The reverse transcriptase protein sequences were used to reconstruct a fish retrovirus phylogeny, thereby, significantly expanding the epsilon-retrovirus family. Most of these novel retroviruses encode additional genes, some of which are homologous to cellular genes that would confer viral advantage. Although the fish divergence is much more ancient, retroviruses began infecting fish genomes approximately 4 million years ago.

Identification of novel retroid agents in Danio rerio, Oryzias latipes, Gasterosteus aculeatus and Tetraodon nigroviridis.

Retroid agents are genomes that encode a reverse transcriptase (RT) and replicate or transpose by way of an RNA intermediate. The Genome Parsing Suite (GPS) is software created to identify and characterize Retroid agents in any genome database (McClure et al. 2005). The detailed analysis of all Retroid agents found by the GPS in Danio rerio (zebrafish), Oryzias latipes (medaka), Gasterosteus aculeatus (stickleback) and Tetraodon nigroviridis (spotted green pufferfish) reveals extensive Retroid agent diversity in the compact genomes of all four fish. Novel Retroid agents were identified by the GPS software: the telomerase reverse transcriptase (TERT) in O. latipes, G. aculeatus and T. nigroviridis and a potential TERT in D. rerio, a retrotransposon in D. rerio, and multiple lineages of endogenous retroviruses (ERVs) in D. rerio, O. latipes and G. aculeatus.