Craniofacial chondrogenesis in organoids from human stem cell-derived neural crest cells.

Knowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete, yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress, we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens, aggrecan, perlecan, proteoglycans, and elastic fibers. We identified two populations of chondroprogenitor cells, mesenchyme cells and nascent chondrocytes, and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage, but also play a pivotal autocrine cell signaling role in determining chondrocyte fate.

Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.

Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection.

RIOK3 and Its Alternatively Spliced Isoform Have Disparate Roles in the Innate Immune Response to Rift Valley Fever Virus (MP12) Infection.

Rift Valley fever virus (RVFV) is a pathogenic human and livestock RNA virus that poses a significant threat to public health and biosecurity. During RVFV infection, the atypical kinase RIOK3 plays important roles in the innate immune response. Although its exact functions in innate immunity are not completely understood, RIOK3 has been shown to be necessary for mounting an antiviral interferon (IFN) response to RVFV in epithelial cells. Furthermore, after immune stimulation, the splicing pattern for RIOK3 mRNA changes markedly, and RIOK3's dominant alternatively spliced isoform, RIOK3 X2, exhibits an opposite effect on the IFN response by dampening it. Here, we further investigate the roles of RIOK3 and its spliced isoform in other innate immune responses to RVFV, namely the NFκB-mediated inflammatory response. We find that while RIOK3 is important for negatively regulating this inflammatory pathway, its alternatively spliced isoform, RIOK3 X2, stimulates it. Overall, these data demonstrate that both RIOK3 and its X2 isoform have unique roles in separate innate immune pathways that respond to RVFV infection.

Time-Resolved Analysis of N-RNA Interactions during RVFV Infection Shows Qualitative and Quantitative Shifts in RNA Encapsidation and Packaging.

Rift Valley fever virus (RVFV) is a negative-sense, tripartite RNA virus that is endemic to Africa and the Arabian Peninsula. It can cause severe disease and mortality in humans and domestic livestock and is a concern for its potential to spread more globally. RVFV's nucleocapsid protein (N) is an RNA-binding protein that is necessary for viral transcription, replication, and the production of nascent viral particles. We have conducted crosslinking, immunoprecipitation, and sequencing (CLIP-seq) to characterize N interactions with host and viral RNAs during infection. In parallel, to precisely measure intracellular N levels, we employed multiple reaction monitoring mass spectrometry (MRM-MS). Our results show that N binds mostly to host RNAs at early stages of infection, yielding nascent virus particles of reduced infectivity. The expression of N plateaus 10 h post-infection, whereas the intracellular viral RNA concentration continues to increase. Moreover, the virions produced later in infection have higher infectivity. Taken together, the detailed examination of these N-RNA interactions provides insight into how the regulated expression of N and viral RNA produces both infectious and incomplete, noninfectious particles.

Mutagenesis of Human Cytomegalovirus Glycoprotein L Disproportionately Disrupts gH/gL/gO over gH/gL/pUL128-131.

Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but how the gH/gL portions of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB-dependent cell-cell fusion but were still able to form gH/gL/pUL128-131 and induce receptor interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR, and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. The effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRα, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a "hyperactive" gH/gL/gO. Recently published crystallography and cryo-electron microscopy studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB. IMPORTANCE The endemic betaherpesvirus HCMV circulates in human populations as a complex mixture of genetically distinct variants, establishes lifelong persistent infections, and causes significant disease in neonates and immunocompromised adults. This study capitalizes on our recent characterizations of three genetically distinct HCMV BAC clones to discern the functions of the envelope glycoprotein complexes gH/gL/gO and gH/gL/pUL128-13, which are promising vaccine targets that share the herpesvirus core fusion apparatus component, gH/gL. Mutations in the shared gL subunit disproportionally affected gH/gL/gO, demonstrating mechanistic differences between the two complexes, and may provide a basis for more refined evaluations of neutralizing antibodies.

The Human Cytomegalovirus Protein UL116 Interacts with the Viral Endoplasmic-Reticulum-Resident Glycoprotein UL148 and Promotes the Incorporation of gH/gL Complexes into Virions.

Heterodimers of glycoproteins H (gH) and L (gL) comprise a basal element of the viral membrane fusion machinery conserved across herpesviruses. In human cytomegalovirus (HCMV), the glycoprotein UL116 assembles onto gH at a position similar to that occupied by gL, forming a heterodimer that is incorporated into virions. Here, we show that UL116 promotes the expression of gH/gL complexes and is required for the efficient production of infectious cell-free virions. UL116-null mutants show a 10-fold defect in production of infectious cell-free virions from infected fibroblasts and epithelial cells. This defect is accompanied by reduced expression of two disulfide-linked gH/gL complexes that play crucial roles in viral entry: the heterotrimer of gH/gL with glycoprotein O (gO) and the pentameric complex of gH/gL with UL128, UL130, and UL131. Kifunensine, a mannosidase inhibitor that interferes with endoplasmic reticulum (ER)-associated degradation (ERAD) of terminally misfolded glycoproteins, restored levels of gH, gL, and gO in UL116-null-infected cells, indicating that constituents of HCMV gH complexes are unstable in the absence of UL116. Further, we find that gH/UL116 complexes are abundant in virions, since a major gH species not covalently linked to other glycoproteins, which has long been observed in the literature, is detected from wild-type but not UL116-null virions. Interestingly, UL116 coimmunoprecipitates with UL148, a viral ER-resident glycoprotein that attenuates ERAD of gO, and we observe elevated levels of UL116 in UL148-null virions. Collectively, our findings argue that UL116 is a chaperone for gH that supports the assembly, maturation, and incorporation of gH/gL complexes into virions. IMPORTANCE HCMV is a betaherpesvirus that causes dangerous opportunistic infections in immunocompromised patients as well as in the immune-naive fetus and preterm infants. The potential of the virus to enter new host cells is governed in large part by two alternative viral glycoprotein H (gH)/glycoprotein L (gL) complexes that play important roles in entry: gH/gL/gO and gH/gL/UL128-131. A recently identified virion gH complex, comprised of gH bound to UL116, adds a new layer of complexity to the mechanisms that contribute to HCMV infectivity. Here, we show that UL116 promotes the expression of gH/gL complexes and that UL116 interacts with the viral ER-resident glycoprotein UL148, a factor that supports the expression of gH/gL/gO. Overall, our results suggest that UL116 is a chaperone for gH. These findings have important implications for understanding HCMV cell tropism as well as for the development of vaccines against the virus.

The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing.

In recent years, transcriptome profiling studies have identified changes in host splicing patterns caused by viral invasion, yet the functional consequences of the vast majority of these splicing events remain uncharacterized. We recently showed that the host splicing landscape changes during Rift Valley fever virus MP-12 strain (RVFV MP-12) infection of mammalian cells. Of particular interest, we observed that the host mRNA for Rio Kinase 3 (RIOK3) was alternatively spliced during infection. This kinase has been shown to be involved in pattern recognition receptor (PRR) signaling mediated by RIG-I like receptors to produce type-I interferon. Here, we characterize RIOK3 as an important component of the interferon signaling pathway during RVFV infection and demonstrate that RIOK3 mRNA expression is skewed shortly after infection to produce alternatively spliced variants that encode premature termination codons. This splicing event plays a critical role in regulation of the antiviral response. Interestingly, infection with other RNA viruses and transfection with nucleic acid-based RIG-I agonists also stimulated RIOK3 alternative splicing. Finally, we show that specifically stimulating alternative splicing of the RIOK3 transcript using a morpholino oligonucleotide reduced interferon expression. Collectively, these results indicate that RIOK3 is an important component of the mammalian interferon signaling cascade and its splicing is a potent regulatory mechanism capable of fine-tuning the host interferon response.

Tra2beta-Dependent Regulation of RIO Kinase 3 Splicing During Rift Valley Fever Virus Infection Underscores the Links Between Alternative Splicing and Innate Antiviral Immunity.

Rift Valley fever virus (RVFV) is an emerging pathogen that has potential to cause severe disease in humans and domestic livestock. Propagation of RVFV strain MP-12 is negatively impacted by the actions of RIOK3, a protein involved in the cellular immune response to viral infection. During RVFV infection, RIOK3 mRNA is alternatively spliced to produce an isoform that correlates with the inhibition of interferon ß signaling. Here, we identify splicing factor TRA2-ß (also known as TRA2beta and hTRA2-ß) as a key regulator governing the relative abundance of RIOK3 splicing isoforms. Using RT-PCR and minigenes, we determined that TRA2-ß interaction with RIOK3 pre-mRNA was necessary for constitutive splicing of RIOK3 mRNA, and conversely, lack of TRA2-ß engagement led to increased alternative splicing. Expression of TRA2-ß was found to be necessary for RIOK3's antiviral effect against RVFV. Intriguingly, TRA2-ß mRNA is also alternatively spliced during RVFV infection, leading to a decrease in cellular TRA2-ß protein levels. These results suggest that splicing modulation serves as an immune evasion strategy by RVFV and/or is a cellular mechanism to prevent excessive immune response. Furthermore, the results suggest that TRA2-ß can act as a key regulator of additional steps of the innate immune response to viral infection.

Specialization for Cell-Free or Cell-to-Cell Spread of BAC-Cloned Human Cytomegalovirus Strains Is Determined by Factors beyond the UL128-131 and RL13 Loci.

It is widely held that clinical isolates of human cytomegalovirus (HCMV) are highly cell associated, and mutations affecting the UL128-131 and RL13 loci that arise in culture lead to the appearance of a cell-free spread phenotype. The bacterial artificial chromosome (BAC) clone Merlin (ME) expresses abundant UL128-131, is RL13 impaired, and produces low infectivity virions in fibroblasts, whereas TB40/e (TB) and TR are low in UL128-131, are RL13 intact, and produce virions of much higher infectivity. Despite these differences, quantification of spread by flow cytometry revealed remarkably similar spread efficiencies in fibroblasts. In epithelial cells, ME spread more efficiently, consistent with robust UL128-131 expression. Strikingly, ME spread far better than did TB or TR in the presence of neutralizing antibodies on both cell types, indicating that ME is not simply deficient at cell-free spread but is particularly efficient at cell-to-cell spread, whereas TB and TR cell-to-cell spread is poor. Sonically disrupted ME-infected cells contained scant infectivity, suggesting that the efficient cell-to-cell spread mechanism of ME depends on features of the intact cells such as junctions or intracellular trafficking processes. Even when UL128-131 was transcriptionally repressed, cell-to-cell spread of ME was still more efficient than that of TB or TR. Moreover, RL13 expression comparably reduced both cell-free and cell-to-cell spread of all three strains, suggesting that it acts at a stage of assembly and/or egress common to both routes of spread. Thus, HCMV strains can be highly specialized for either for cell-free or cell-to-cell spread, and these phenotypes are determined by factors beyond the UL128-131 or RL13 loci.IMPORTANCE Both cell-free and cell-to-cell spread are likely important for the natural biology of HCMV. In culture, strains clearly differ in their capacity for cell-free spread as a result of differences in the quantity and infectivity of extracellular released progeny. However, it has been unclear whether "cell-associated" phenotypes are simply the result of poor cell-free spread or are indicative of particularly efficient cell-to-cell spread mechanisms. By measuring the kinetics of spread at early time points, we were able to show that HCMV strains can be highly specialized to either cell-free or cell-to-cell mechanisms, and this was not strictly linked the efficiency of cell-free spread. Our results provide a conceptual approach to evaluating intervention strategies for their ability to limit cell-free or cell-to-cell spread as independent processes.

Polymorphisms in Human Cytomegalovirus Glycoprotein O (gO) Exert Epistatic Influences on Cell-Free and Cell-to-Cell Spread and Antibody Neutralization on gH Epitopes.

Human cytomegalovirus (HCMV) glycoproteins H and L (gH/gL) can be bound by either gO or the UL128 to UL131 proteins (referred to here as UL128-131) to form complexes that facilitate entry and spread, and the complexes formed are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131, and this can impact infectivity and cell tropism. In this study, we investigated how natural interstrain variation in the amino acid sequence of gO influences the biology of HCMV. Heterologous gO recombinants were constructed in which 6 of the 8 alleles or genotypes (GT) of gO were analyzed in the backgrounds of strains TR and Merlin (ME). The levels of gH/gL complexes were not affected, but there were impacts on entry, spread, and neutralization by anti-gH antibodies. AD169 (AD) gO (GT1a) [referred to here as ADgO(GT1a)] drastically reduced cell-free infectivity of both strains on fibroblasts and epithelial cells. PHgO(GT2a) increased cell-free infectivity of TR in both cell types, but spread in fibroblasts was impaired. In contrast, spread of ME in both cell types was enhanced by Towne (TN) gO (GT4), despite similar cell-free infectivity. TR expressing TNgO(GT4) was resistant to neutralization by anti-gH antibodies AP86 and 14-4b, whereas ADgO(GT1a) conferred resistance to 14-4b but enhanced neutralization by AP86. Conversely, ME expressing ADgO(GT1a) was more resistant to 14-4b. These results suggest that (i) there are mechanistically distinct roles for gH/gL/gO in cell-free and cell-to-cell spread, (ii) gO isoforms can differentially shield the virus from neutralizing antibodies, and (iii) effects of gO polymorphisms are epistatically dependent on other variable loci.IMPORTANCE Advances in HCMV population genetics have greatly outpaced understanding of the links between genetic diversity and phenotypic variation. Moreover, recombination between genotypes may shuffle variable loci into various combinations with unknown outcomes. UL74(gO) is an important determinant of HCMV infectivity and one of the most diverse loci in the viral genome. By analyzing interstrain heterologous UL74(gO) recombinants, we showed that gO diversity can have dramatic impacts on cell-free and cell-to-cell spread as well as on antibody neutralization and that the manifestation of these impacts can be subject to epistatic influences of the global genetic background. These results highlight the potential limitations of laboratory studies of HCMV biology that use single, isolated genotypes or strains.

Transcriptome profiling in Rift Valley fever virus infected cells reveals modified transcriptional and alternative splicing programs.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus belonging to the Phenuiviridae family that infects both domestic livestock and humans. The NIAID has designated RVFV as a Category A priority emerging pathogen due to the devastating public health outcomes associated with epidemic outbreaks. However, there is no licensed treatment or vaccine approved for human use. Therefore it is of great interest to understand RVFV pathogenesis in infected hosts in order to facilitate creation of targeted therapies and treatment options. Here we provide insight into the host-pathogen interface in human HEK293 cells during RVFV MP-12 strain infection using high-throughput mRNA sequencing technology. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes showed robust innate immune and cytokine-mediated inflammatory pathway activation as well as alterations in pathways associated with fatty acid metabolism and extracellular matrix receptor signaling. We also analyzed the promoter regions of DEGs for patterns in transcription factor binding sites, and found several that are known to act synergistically to impact apoptosis, immunity, metabolism, and cell growth and differentiation. Lastly, we noted dramatic changes in host alternative splicing patterns in genes associated with mRNA decay and surveillance, RNA transport, and DNA repair. This study has improved our understanding of RVFV pathogenesis and has provided novel insight into pathways and signaling modules important for RVFV diagnostics and therapeutic development.

RNA Encapsidation and Packaging in the Phleboviruses.

The Bunyaviridae represents the largest family of segmented RNA viruses, which infect a staggering diversity of plants, animals, and insects. Within the family Bunyaviridae, the Phlebovirus genus includes several important human and animal pathogens, including Rift Valley fever virus (RVFV), severe fever with thrombocytopenia syndrome virus (SFTSV), Uukuniemi virus (UUKV), and the sandfly fever viruses. The phleboviruses have small tripartite RNA genomes that encode a repertoire of 5-7 proteins. These few proteins accomplish the daunting task of recognizing and specifically packaging a tri-segment complement of viral genomic RNA in the midst of an abundance of host components. The critical nucleation events that eventually lead to virion production begin early on in the host cytoplasm as the first strands of nascent viral RNA (vRNA) are synthesized. The interaction between the vRNA and the viral nucleocapsid (N) protein effectively protects and masks the RNA from the host, and also forms the ribonucleoprotein (RNP) architecture that mediates downstream interactions and drives virion formation. Although the mechanism by which all three genomic counterparts are selectively co-packaged is not completely understood, we are beginning to understand the hierarchy of interactions that begins with N-RNA packaging and culminates in RNP packaging into new virus particles. In this review we focus on recent progress that highlights the molecular basis of RNA genome packaging in the phleboviruses.

Scanning Mutagenesis of Human Cytomegalovirus Glycoprotein gH/gL.

UNLABELLED: The core, conserved function of the herpesvirus gH/gL is to promote gB-mediated membrane fusion during entry, although the mechanism is poorly understood. The human cytomegalovirus (HCMV) gH/gL can exist as either the gH/gL/gO trimer or the gH/gL/UL128/UL130/UL131 (gH/gL/UL128-131) pentamer. One model suggests that gH/gL/gO provides the core fusion role during entry into all cells within the broad tropism of HCMV, whereas gH/gL/UL128-131 acts at an earlier stage, by a distinct receptor-binding mechanism to enhance infection of select cell types, such as epithelial cells, endothelial cells, and monocytes/macrophages. To further study the distinct functions of these complexes, mutants with individual charged cluster-to-alanine (CCTA) mutations of gH and gL were combined to generate a library of 80 mutant gH/gL heterodimers. The majority of the mutant gH/gL complexes were unable to facilitate gB-mediated membrane fusion in transient-expression cell-cell fusion experiments. In contrast, these mutants supported the formation of gH/gL/UL128-131 complexes that could block HCMV infection in receptor interference experiments. These results suggest that receptor interactions with gH/gL/UL128-131 involve surfaces contained on the UL128-131 proteins but not on gH/gL. gH/gL/UL128-131 receptor interference could be blocked with anti-gH antibodies, suggesting that interference is a cell surface phenomenon and that anti-gH antibodies can block gH/gL/UL128-131 in a manner that is distinct from that for gH/gL/gO. IMPORTANCE: Interest in the gH/gL complexes of HCMV (especially gH/gL/UL128-131) as vaccine targets has far outpaced our understanding of the mechanism by which they facilitate entry and contribute to broad cellular tropism. For Epstein-Barr virus (EBV), gH/gL and gH/gL/gp42 are both capable of promoting gB fusion for entry into epithelial cells and B cells, respectively. In contrast, HCMV gH/gL/gO appears to be the sole fusion cofactor that promotes gB fusion activity, whereas gH/gL/UL128-131 expands cell tropism through a distinct yet unknown mechanism. This study suggests that the surfaces of HCMV gH/gL are critical for promoting gB fusion but are dispensable for gH/gL/UL128-131 receptor interaction. This underscores the importance of gH/gL/gO in HCMV entry into all cell types and reaffirms the complex as a candidate target for vaccine development. The two functionally distinct forms of gH/gL present in HCMV make for a useful model with which to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion.

Computational prediction and biochemical characterization of novel RNA aptamers to Rift Valley fever virus nucleocapsid protein.

Rift Valley fever virus (RVFV) is a potent human and livestock pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that has potential to spread to other parts of the world. Although there is no proven effective and safe treatment for RVFV infections, a potential therapeutic target is the virally encoded nucleocapsid protein (N). During the course of infection, N binds to viral RNA, and perturbation of this interaction can inhibit viral replication. To gain insight into how N recognizes viral RNA specifically, we designed an algorithm that uses a distance matrix and multidimensional scaling to compare the predicted secondary structures of known N-binding RNAs, or aptamers, that were isolated and characterized in previous in vitro evolution experiment. These aptamers did not exhibit overt sequence or predicted structure similarity, so we employed bioinformatic methods to propose novel aptamers based on analysis and clustering of secondary structures. We screened and scored the predicted secondary structures of novel randomly generated RNA sequences in silico and selected several of these putative N-binding RNAs whose secondary structures were similar to those of known N-binding RNAs. We found that overall the in silico generated RNA sequences bound well to N in vitro. Furthermore, introduction of these RNAs into cells prior to infection with RVFV inhibited viral replication in cell culture. This proof of concept study demonstrates how the predictive power of bioinformatics and the empirical power of biochemistry can be jointly harnessed to discover, synthesize, and test new RNA sequences that bind tightly to RVFV N protein. The approach would be easily generalizable to other applications.

Human Cytomegalovirus gH/gL/gO Promotes the Fusion Step of Entry into All Cell Types, whereas gH/gL/UL128-131 Broadens Virus Tropism through a Distinct Mechanism.

UNLABELLED: Interaction between gH/gL and the fusion protein gB is likely a conserved feature of the entry mechanism for all herpesviruses. Human cytomegalovirus (HCMV) gH/gL can be bound by gO or by the set of proteins UL128, UL130, and UL131, forming gH/gL/gO and gH/gL/UL128-131. The mechanisms by which these complexes facilitate entry are poorly understood. Mutants lacking UL128-131 replicate well on fibroblasts but fail to enter epithelial/endothelial cells, and this has led to the general assumption that gH/gL/UL128-131 promotes gB-mediated fusion on epithelial/endothelial cells whereas gH/gL/gO provides this function on fibroblasts. This was challenged by observations that gO-null mutants were defective on all of these cell types, suggesting that entry into epithelial/endothelial cells requires both of the gH/gL complexes, but the severe replication defect of the gO mutants precluded detailed analysis. We previously reported that the ratio of gH/gL/gO and gH/gL/UL128-131 in the virion envelope varied dramatically among HCMV strains. Here, we show that strains not only differ in the ratio, but also vary in the total amount of gH/gL in the virion. Cell-type-specific particle-to-PFU ratios of HCMV strains that contained different amounts of gH/gL/gO and gH/gL/UL128-131 were determined. Infection of both fibroblasts and epithelial cells was generally correlated with the abundance of gH/gL/gO, but not with that of gH/gL/UL128-131. The low infectivity of virions rich in gH/gL/UL128-131 but low in gH/gL/gO could be overcome by treatment with the chemical fusogen polyethylene glycol (PEG), strongly arguing that gH/gL/gO provides the conserved herpesvirus gH/gL entry function of promoting gB-mediated fusion for entry into all cell types, whereas gH/gL/UL128-131 acts through a distinct mechanism to allow infection of select cell types. IMPORTANCE: The functions of HCMV gH/gL complexes in entry are unclear. Unlike the well-studied Epstein-Barr virus (EBV), where gH/gL and gH/gL/gp42 complexes both seem capable of promoting gB fusion during entry into different cell types, our studies here suggest that for HCMV, gH/gL/gO promotes gB fusion on all cell types, whereas gH/gL/UL128-131 broadens virus tropism through a distinct, as yet unknown mechanism. To our knowledge, this is the first suggestion of a herpesvirus gH/gL that does not act by promoting gB fusion, which might make HCMV a useful model to study the fundamental mechanisms by which herpesvirus gH/gL regulates gB fusion. Moreover, gH/gL/UL128-131 is a candidate vaccine target. Our findings help to explain the cell-type-dependent virus neutralization exhibited by anti-gH/gL/UL128-131 antibodies and underscore the importance of gH/gL/gO as another important part of vaccine or therapeutic strategies.

Inhibition of Rift Valley fever virus replication and perturbation of nucleocapsid-RNA interactions by suramin.

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. There are currently no proven safe and effective treatment options for RVFV infection. Inhibition of RNA binding to RVFV nucleocapsid protein (N) represents an attractive antiviral therapeutic strategy because several essential steps in the RVFV replication cycle involve N binding to viral RNA. In this study, we demonstrate the therapeutic potential of the drug suramin by showing that it functions well as an inhibitor of RVFV replication at multiple stages in human cell culture. Suramin has been used previously to treat trypanosomiasis in Africa. We characterize the dynamic and cooperative nature of N-RNA binding interactions and the dissociation of high-molecular-mass ribonucleoprotein complexes using suramin, which we previously identified as an N-RNA binding inhibitor in a high-throughput screen. Finally, we elucidate the molecular mechanism used by suramin in vitro to disrupt both specific and nonspecific binding events important for ribonucleoprotein formation.

Identification of Rift Valley fever virus nucleocapsid protein-RNA binding inhibitors using a high-throughput screening assay.

Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.

Characterization of RNA aptamers directed against the nucleocapsid protein of Rift Valley fever virus.

Nucleocapsid protein (N) is an essential RNA binding protein in many RNA viruses. During replication, N protein encapsidates viral genomic and antigenomic RNA, but not viral mRNA or other cellular RNAs. To discriminate between different species of RNA in a host cell, it is likely that N interacts with specific sequences and/or secondary structures on its target RNA. In this study, we explore the RNA binding properties of N using both natural and artificially selected RNAs as ligands. We found that N binds to RNAs that resemble the terminal panhandle structures of RVFV genomic and antigenomic RNA. Furthermore, we used SELEX to isolate RNA aptamers that bound N with high affinity and determined that N specifically recognizes and binds to GAUU and pyrimidine/guanine motifs. Interestingly, BLAST analysis revealed the presence of these motifs within the coding region of the viral genome, suggesting that N may interact with non-terminal viral RNA sequences during replication. Finally, the aptamer RNAs were used to construct a sensitive fluorescence based sensor of N binding with potential applications for drug screening and imaging methodologies.

Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication.

The 5' UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 5' untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The "winner" viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication.

A 5'UTR-spliced mRNA isoform is specialized for enhanced HIV-2 gag translation.

Full-length unspliced genomic RNA plays critical roles in HIV replication, serving both as mRNA for the synthesis of the key viral polyproteins Gag and Gag-Pol and as genomic RNA for encapsidation into assembling viral particles. We show that a second gag mRNA species that differs from the genomic RNA molecule by the absence of an intron in the 5' untranslated region (5'UTR) is produced during HIV-2 replication in cell culture and in infected patients. We developed a cotransfection system in which epitopically tagged Gag proteins can be traced back to their mRNA origins in the translation pool. We show that a disproportionate amount of Gag is translated from 5'UTR intron-spliced mRNAs, demonstrating a role for the 5'UTR intron in the regulation of gag translation. To further characterize the effects of the HIV-2 5'UTR on translation, we fused wild-type, spliced, or mutant leader RNA constructs to a luciferase reporter gene and assayed their translation in reticulocyte lysates. These assays confirmed that leaders lacking the 5'UTR intron increased translational efficiency compared to that of the unspliced leader. In addition, we found that removal or mutagenesis of the C-box, a pyrimidine-rich sequence located in the 5'UTR intron and previously shown to affect RNA dimerization, also strongly influenced translational efficiency. These results suggest that the splicing of both the 5'UTR intron and the C-box element have key roles in regulation of HIV-2 gag translation in vitro and in vivo.