The landscape of antibody binding in SARS-CoV-2 infection.

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here, we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which 1 epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the 6 other known human CoVs. We also confirm reactivity against 4 of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to 9 SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

An Update on Gender Parity Trends for Invited Speakers at Four Prominent Virology Conference Series.

Historically, men rather than women have been selected for invited speaking positions at the four prominent virology conference series we have followed since the 1980s. However, the low ratio of female representation in most earlier years (20%, 1982-2010) has shown encouraging improvement (37%, 2011-2017), particularly over the last few years (48% from 2018-2020). We describe this promising rise in inclusion and discuss factors that may help perpetuate and extend this overdue transformation towards gender parity.

Solution NMR Determination of the CDHR3 Rhinovirus-C Binding Domain, EC1.

Cadherin Related Family Member 3 (CDHR3) is the identified and required cellular receptor for all virus isolates in the rhinovirus-C species (RV-C). Cryo-EM determinations recently resolved the atomic structure of RV-C15a, and subsequently, a complex of this virus bound to CDHR3 extracellular domain 1 (EC1), the N-terminal portion of this receptor responsible for virus interactions. The EC1 binds to a hypervariable sequence footprint on the virus surface, near the 3-fold axis of icosahedral symmetry. The key contacts involve discontinuous residues from 3 viral proteins, VP1, VP2 and VP3. That single cryo-EM EC1 structure, however, could not resolve whether the virus-receptor interface was structurally adaptable to accommodate multiple virus sequences. We now report the solution NMR determination of CDHR3 EC1, showing that this protein, in fact, is mostly inflexible, particularly in the virus-binding face. The new, higher resolution dataset identifies 3 cis-Pro residues in important loop regions, where they can influence both rigidity and overall protein conformation. The data also provide clarification about the residues involved in essential calcium ion binding, and a potential CDHR3 surface groove feature that may be involved in native protein interactions with cellular partners.

The landscape of antibody binding in SARS-CoV-2 infection.

The search for potential antibody-based diagnostics, vaccines, and therapeutics for pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has focused almost exclusively on the spike (S) and nucleocapsid (N) proteins. Coronavirus membrane (M), ORF3a, and ORF8 proteins are humoral immunogens in other coronaviruses (CoVs) but remain largely uninvestigated for SARS-CoV-2. Here we use ultradense peptide microarray mapping to show that SARS-CoV-2 infection induces robust antibody responses to epitopes throughout the SARS-CoV-2 proteome, particularly in M, in which one epitope achieved excellent diagnostic accuracy. We map 79 B cell epitopes throughout the SARS-CoV-2 proteome and demonstrate that antibodies that develop in response to SARS-CoV-2 infection bind homologous peptide sequences in the six other known human CoVs. We also confirm reactivity against four of our top-ranking epitopes by enzyme-linked immunosorbent assay (ELISA). Illness severity correlated with increased reactivity to nine SARS-CoV-2 epitopes in S, M, N, and ORF3a in our population. Our results demonstrate previously unknown, highly reactive B cell epitopes throughout the full proteome of SARS-CoV-2 and other CoV proteins.

Cryo-EM structure of rhinovirus C15a bound to its cadherin-related protein 3 receptor.

Infection by Rhinovirus-C (RV-C), a species of Picornaviridae Enterovirus, is strongly associated with childhood asthma exacerbations. Cellular binding and entry by all RV-C, which trigger these episodes, is mediated by the first extracellular domain (EC1) of cadherin-related protein 3 (CDHR3), a surface cadherin-like protein expressed primarily on the apical surfaces of ciliated airway epithelial cells. Although recombinant EC1 is a potent inhibitor of viral infection, there is no molecular description of this protein or its binding site on RV-C. Here we present cryo-electron microscopy (EM) data resolving the EC1 and EC1+2 domains of human CDHR3 complexed with viral isolate C15a. Structure-suggested residues contributing to required interfaces on both EC1 and C15a were probed and identified by mutagenesis studies with four different RV-C genotypes. In contrast to most other rhinoviruses, which bind intercellular adhesion molecule 1 receptors via a capsid protein VP1-specific fivefold canyon feature, the CDHR3 EC1 contacts C15a, and presumably all RV-Cs, in a unique cohesive footprint near the threefold vertex, encompassing residues primarily from viral protein VP3, but also from VP1 and VP2. The EC1+2 footprint on C15a is similar to that of EC1 alone but shows that steric hindrance imposed by EC2 would likely prevent multiprotein binding by the native receptor at any singular threefold vertex. Definition of the molecular interface between the RV-Cs and their receptors provides new avenues that can be explored for potential antiviral therapies.

Genetic susceptibility to severe childhood asthma and rhinovirus-C maintained by balancing selection in humans for 150 000 years.

Selective pressures imposed by pathogens have varied among human populations throughout their evolution, leading to marked inter-population differences at some genes mediating susceptibility to infectious and immune-related diseases. Here, we investigated the evolutionary history of a common polymorphism resulting in a Y529 versus C529 change in the cadherin related family member 3 (CDHR3) receptor which underlies variable susceptibility to rhinovirus-C infection and is associated with severe childhood asthma. The protective variant is the derived allele and is found at high frequency worldwide (69-95%). We detected genome-wide significant signatures of natural selection consistent with a rapid increase of the haplotypes carrying the allele, suggesting that non-neutral processes have acted on this locus across all human populations. However, the allele has not fixed in any population despite multiple lines of evidence suggesting that the mutation predates human migrations out of Africa. Using an approximate Bayesian computation method, we estimate the age of the mutation while explicitly accounting for past demography and positive or frequency-dependent balancing selection. Our analyses indicate a single emergence of the mutation in anatomically modern humans ~150 000 years ago and indicate that balancing selection has maintained the beneficial allele at high equilibrium frequencies worldwide. Apart from the well-known cases of the MHC and ABO genes, this study provides the first evidence that negative frequency-dependent selection plausibly acted on a human disease susceptibility locus, a form of balancing selection compatible with typical transmission dynamics of communicable respiratory viruses that might exploit CDHR3.

Simultaneous outbreaks of respiratory disease in wild chimpanzees caused by distinct viruses of human origin.

Respiratory viruses of human origin infect wild apes across Africa, sometimes lethally. Here we report simultaneous outbreaks of two distinct human respiratory viruses, human metapneumovirus (MPV; Pneumoviridae: Metapneumovirus) and human respirovirus 3 (HRV3; Paramyxoviridae; Respirovirus, formerly known as parainfluenza virus 3), in two chimpanzee (Pan troglodytes schweinfurthii) communities in the same forest in Uganda in December 2016 and January 2017. The viruses were absent before the outbreaks, but each was present in ill chimpanzees from one community during the outbreak period. Clinical signs and gross pathologic changes in affected chimpanzees closely mirrored symptoms and pathology commonly observed in humans for each virus. Epidemiologic modelling showed that MPV and HRV3 were similarly transmissible (R0 of 1.27 and 1.48, respectively), but MPV caused 12.2% mortality mainly in infants and older chimpanzees, whereas HRV3 caused no direct mortality. These results are consistent with the higher virulence of MPV than HRV3 in humans, although both MPV and HRV3 cause a significant global disease burden. Both viruses clustered phylogenetically within groups of known human variants, with MPV closely related to a lethal 2009 variant from mountain gorillas (Gorilla beringei beringei), suggesting two independent and simultaneous reverse zoonotic origins, either directly from humans or via intermediary hosts. These findings expand our knowledge of human origin viruses threatening wild chimpanzees and suggest that such viruses might be differentiated by their comparative epidemiological dynamics and pathogenicity in wild apes. Our results also caution against assuming common causation in coincident outbreaks.

CDHR3 Asthma-Risk Genotype Affects Susceptibility of Airway Epithelium to Rhinovirus C Infections.

CDHR3 (cadherin-related family member 3) is a transmembrane protein that is highly expressed in airway epithelia and the only known receptor for rhinovirus C (RV-C). A CDHR3 SNP (rs6967330) with G to A base change has been linked to severe exacerbations of asthma and increased susceptibility to RV-C infections in young children. The goals of this study were to determine the subcellular localization of CDHR3 and to test the hypothesis that CDHR3 asthma-risk genotype affects epithelial cell function and susceptibility to RV-C infections of the airway epithelia. We used immunofluorescence imaging, Western blot analysis, and transmission electron microscopy to show CDHR3 subcellular localization in apical cells, including expression in the cilia of airway epithelia. Polymorphisms in CDHR3 rs6967330 locus (G→A) that were previously associated with childhood asthma were related to differences in CDHR3 expression and epithelial cell function. The rs6967330 A allele was associated with higher overall protein expression and RV-C binding and replication compared with the rs6967330 G allele. Furthermore, the rs6967330 A allele was associated with earlier ciliogenesis and higher FOXJ1 expression. Finally, CDHR3 genotype had no significant effects on membrane integrity or ciliary beat function. These findings provide information on the subcellular localization and possible functions of CDHR3 in the airways and link CDHR3 asthma-risk genotype to increased RV-C binding and replication.

α1ACT Is Essential for Survival and Early Cerebellar Programming in a Critical Neonatal Window.

Postnatal cerebellar development is a precisely regulated process involving well-orchestrated expression of neural genes. Neurological phenotypes associated with CACNA1A gene defects have been increasingly recognized, yet the molecular principles underlying this association remain elusive. By characterizing a dose-dependent CACNA1A gene deficiency mouse model, we discovered that α1ACT, as a transcription factor and secondary protein of CACNA1A mRNA, drives dynamic gene expression networks within cerebellar Purkinje cells and is indispensable for neonatal survival. Perinatal loss of α1ACT leads to motor dysfunction through disruption of neurogenesis and synaptic regulatory networks. However, its elimination in adulthood has minimal effect on the cerebellum. These findings shed light on the critical role of α1ACT in facilitating neuronal development in both mice and humans and support a rationale for gene therapies for calcium-channel-associated cerebellar disorders. Finally, we show that bicistronic expression may be common to the voltage-gated calcium channel (VGCC) gene family and may help explain complex genetic syndromes.

Rhinoviruses and Their Receptors.

Human rhinoviruses (RVs) are picornaviruses that can cause a variety of upper and lower respiratory tract illnesses, including the common cold, bronchitis, pneumonia, and exacerbations of chronic respiratory diseases such as asthma. There are currently > 160 known types of RVs classified into three species (A, B, and C) that use three different cellular membrane glycoproteins expressed in the respiratory epithelium to enter the host cell. These viral receptors are intercellular adhesion molecule 1 (used by the majority of RV-A and all RV-B types), low-density lipoprotein receptor family members (used by 12 RV-A types), and cadherin-related family member 3 (CDHR3; used by RV-C). RV-A and RV-B interactions with intercellular adhesion molecule 1 and low-density lipoprotein receptor glycoproteins are well defined and their cellular functions have been described, whereas the mechanisms of the RV-C interaction with CDHR3 and its cellular functions are being studied. A single nucleotide polymorphism (rs6967330) in CDHR3 increases cell surface expression of this protein and, as a result, also promotes RV-C infections and illnesses. There are currently no approved vaccines or antiviral therapies available to treat or prevent RV infections, which is a major unmet medical need. Understanding interactions between RV and cellular receptors could lead to new insights into the pathogenesis of respiratory illnesses as well as lead to new approaches to control respiratory illnesses caused by RV infections.

CDHR3 extracellular domains EC1-3 mediate rhinovirus C interaction with cells and as recombinant derivatives, are inhibitory to virus infection.

Viruses in the rhinovirus C species (RV-C) are more likely to cause severe wheezing illnesses and asthma exacerbations in children than related isolates of the RV-A or RV-B. The RV-C capsid is structurally distinct from other rhinoviruses and does not bind ICAM-1 or LDL receptors. The RV-C receptor is instead, human cadherin-related family member 3 (CDHR3), a protein unique to the airway epithelium. A single nucleotide polymorphism (rs6967330, encoding C529Y) in CDHR3 regulates the display density of CDHR3 on cell surfaces and is among the strongest known genetic correlates for childhood virus-induced asthma susceptibility. CDHR3 immunoprecipitations from transfected or transduced cell lysates were used to characterize the RV-C interaction requirements. The C529 and Y529 variations in extracellular repeat domain 5 (EC5), bound equivalently to virus. Glycosylase treatment followed by mass spectrometry mapped 3 extracellular N-linked modification sites, and further detected surface-dependent, α2-6 sialyation unique to the Y529 format. None of these modifications were required for RV-C recognition, but removal or even dilution of structurally stabilizing calcium ions from the EC junctions irreversibly abrogated virus binding. CDHR3 deletions expressed in HeLa cells or as bacterial recombinant proteins, mapped the amino-terminal EC1 unit as the required virus contact. Derivatives containing the EC1 domain, could not only recapitulate virus:receptor interactions in vitro, but also directly inhibit RV-C infection of susceptible cells for several virus genotypes (C02, C15, C41, and C45). We propose that all RV-C use the same EC1 landing pad, interacting with putative EC3-mediated multimerization formats of CDHR3.

Lethal Respiratory Disease Associated with Human Rhinovirus C in Wild Chimpanzees, Uganda, 2013.

We describe a lethal respiratory outbreak among wild chimpanzees in Uganda in 2013 for which molecular and epidemiologic analyses implicate human rhinovirus C as the cause. Postmortem samples from an infant chimpanzee yielded near-complete genome sequences throughout the respiratory tract; other pathogens were absent. Epidemiologic modeling estimated the basic reproductive number (R0) for the epidemic as 1.83, consistent with the common cold in humans. Genotyping of 41 chimpanzees and examination of 24 published chimpanzee genomes from subspecies across Africa showed universal homozygosity for the cadherin-related family member 3 CDHR3-Y529 allele, which increases risk for rhinovirus C infection and asthma in human children. These results indicate that chimpanzees exhibit a species-wide genetic susceptibility to rhinovirus C and that this virus, heretofore considered a uniquely human pathogen, can cross primate species barriers and threatens wild apes. We advocate engineering interventions and prevention strategies for rhinovirus infections for both humans and wild apes.

Gender Parity Trends for Invited Speakers at Four Prominent Virology Conference Series.

Scientific conferences are most beneficial to participants when they showcase significant new experimental developments, accurately summarize the current state of the field, and provide strong opportunities for collaborative networking. A top-notch slate of invited speakers, assembled by conference organizers or committees, is key to achieving these goals. The perceived underrepresentation of female speakers at prominent scientific meetings is currently a popular topic for discussion, but one that often lacks supportive data. We compiled the full rosters of invited speakers over the last 35 years for four prominent international virology conferences, the American Society for Virology Annual Meeting (ASV), the International Herpesvirus Workshop (IHW), the Positive-Strand RNA Virus Symposium (PSR), and the Gordon Research Conference on Viruses & Cells (GRC). The rosters were cross-indexed by unique names, gender, year, and repeat invitations. When plotted as gender-dependent trends over time, all four conferences showed a clear proclivity for male-dominated invited speaker lists. Encouragingly, shifts toward parity are emerging within all units, but at different rates. Not surprisingly, both selection of a larger percentage of first-time participants and the presence of a woman on the speaker selection committee correlated with improved parity. Session chair information was also collected for the IHW and GRC. These visible positions also displayed a strong male dominance over time that is eroding slowly. We offer our personal interpretation of these data to aid future organizers achieve improved equity among the limited number of available positions for session moderators and invited speakers.IMPORTANCE Politicians and media members have a tendency to cite anecdotes as conclusions without any supporting data. This happens so frequently now, that a name for it has emerged: fake news. Good science proceeds otherwise. The underrepresentation of women as invited speakers at international scientific conferences exemplifies a present-day discussion topic usually occurring without facts to support or refute the arguments. We now provide records profiling four prominent virology conferences over the years 1982 to 2017 with the intention that the trends and accompanying analyses of the gender parity of invited speakers may allow the ongoing discussions to be informed.

Rhinovirus C targets ciliated airway epithelial cells.

BACKGROUND: The Rhinovirus C (RV-C), first identified in 2006, produce high symptom burdens in children and asthmatics, however, their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs), and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor, cadherin related family member 3 (CDHR3). METHODS: RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry. RESULTS: C15 produced a scattered pattern of infection, and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat], p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC, p = ns). CDHR3 expression was increased on ciliated epithelial cells, but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs, CDHR3 expression progressively increased and correlated with both RV-C binding and replication. CONCLUSIONS: The RV-C only replicate in ciliated AECs in vitro, leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication, and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity.

Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases.

The RNA rhinoviruses (RV) encode 2A proteases (2Apro) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2Apro sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2Apro predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/ß, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2Apro (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2Apro activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2Apro) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2Apro were measured comparatively in transformed cells programed with fluorescent reporter systems and by quantitative cell imaging, the cellular substrates, particularly in the nuclear pore complex, used by these proteases were indeed attacked at different rates and with different affinities. The importance of this finding is that it provides a mechanistic explanation for how different types (strains) of rhinoviruses may elicit different cell responses that directly or indirectly lead to distinct disease phenotypes.

Rhinovirus C, Asthma, and Cell Surface Expression of Virus Receptor CDHR3.

Human rhinoviruses (RVs) of the A, B, and C species are defined agents of the common cold. But more than that, RV-A and RV-C are the dominant causes of hospitalization category infections in young children, especially those with asthma. The use of cadherin-related family member 3 (CDHR3) by RV-C as its cellular receptor creates a direct phenotypic link between human genetics (G versus A alleles cause Cys529 versus Tyr529 protein variants) and the efficiency with which RV-C can infect cells. With a lower cell surface display density, the human-specific Cys529 variant apparently confers partial protection from the severest virus-induced asthma episodes. Selective pressure favoring the Cys529 codon may have coemerged with the evolution of RV-C and helped shape modern human genomes against the virus-susceptible, albeit ancestral Tyr529.

The Language of Life.

Science is our best current approximation of the way things work. You cannot do science unless you believe there is a discernable truth inherent to the arrangement of our tangible world. The problem is, we in our given time never know where exactly the asymptote lies or how far we are from it. My curiosity about the natural world is innate, but fate has variously gifted me with outstanding personal opportunities to indulge that curiosity through the study of viruses. To a woman of the boomer generation, professional paths were not always open-door, and to a certain extent they still aren't. Whether such points should now be viewed as obstacles or stepping stones is a matter of perspective. RNA viruses, and the multiple, seminal mentors who taught me their secrets, have defined my career. Some of their stories are told here as they dovetail with mine. If there is any unity to this, it would be a pursuit of the language of life, or sequence analysis, as taught to us by natural selection. The intent here is not a legacy but an example. Science is a beautiful fate.

Mutations in VP1 and 3A proteins improve binding and replication of rhinovirus C15 in HeLa-E8 cells.

Viruses in the rhinovirus C species (RV-C) can cause severe respiratory illnesses in children including pneumonia and asthma exacerbations. A transduced cell line (HeLa-E8) stably expressing the CDHR3-Y529 receptor variant, supports propagation of RV-C after infection. C15 clinical or recombinant isolates replicate in HeLa-E8, however progeny yields are lower than those of related strains of RV-A and RV-B. Serial passaging of C15 in HeLa-E8 resulted in stronger cytopathic effects and increased (≥10-fold) virus binding to cells and progeny yields. The adaptation was acquired by two mutations which increased binding (VP1 T125K) and replication (3A E41K), respectively. A similar 3A mutation engineered into C2 and C41 cDNAs also improved viral replication (2-8 fold) in HeLa but the heparan sulfate mediated cell-binding enhancement by the VP1 change was C15-specific. The findings now enable large-scale cost-effective C15 production by infection and the testing of RV-C infectivity by plaque assay.

Atomic structure of a rhinovirus C, a virus species linked to severe childhood asthma.

Isolates of rhinovirus C (RV-C), a recently identified Enterovirus (EV) species, are the causative agents of severe respiratory infections among children and are linked to childhood asthma exacerbations. The RV-C have been refractory to structure determination because they are difficult to propagate in vitro. Here, we report the cryo-EM atomic structures of the full virion and native empty particle (NEP) of RV-C15a. The virus has 60 "fingers" on the virus outer surface that probably function as dominant immunogens. Because the NEPs also display these fingers, they may have utility as vaccine candidates. A sequence-conserved surface depression adjacent to each finger forms a likely binding site for the sialic acid on its receptor. The RV-C, unlike other EVs, are resistant to capsid-binding antiviral compounds because the hydrophobic pocket in VP1 is filled with multiple bulky residues. These results define potential molecular determinants for designing antiviral therapeutics and vaccines.

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.