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.

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.

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.

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 β signaling.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Randomization and in vivo selection reveal a GGRG motif essential for packaging human immunodeficiency virus type 2 RNA.

The packaging signal (psi) of human immunodeficiency virus type 2 (HIV-2) is present in the 5' noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5'-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3' side of pal (GCUCC-3') is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro.

Regulation of primate lentiviral RNA dimerization by structural entrapment.

Background: Genomic RNA dimerization is an important process in the formation of an infectious lentiviral particle. One of the signals involved is the stem-loop 1 (SL1) element located in the leader region of lentiviral genomic RNAs which also plays a role in encapsidation and reverse transcription. Recent studies revealed that HIV types 1 and 2 leader RNAs adopt different conformations that influence the presentation of RNA signals such as SL1.

HIV-2 RNA dimerization is regulated by intramolecular interactions in vitro.

Genomic RNA dimerization is an essential process in the retroviral replication cycle. In vitro, HIV-2 RNA dimerization is mediated at least in part by direct intermolecular interaction at stem-loop 1 (SL1) within the 5'-untranslated leader region (UTR). RNA dimerization is thought to be regulated via alternate presentation and sequestration of dimerization signals by intramolecular base-pairings.

An extended stem-loop 1 is necessary for human immunodeficiency virus type 2 replication and affects genomic RNA encapsidation.

Genomic RNA encapsidation in lentiviruses is a highly selective and regulated process. The unspliced RNA molecules are selected for encapsidation from a pool of many different viral and cellular RNA species. Moreover, two molecules are encapsidated per viral particle, where they are found associated as a dimer.

Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro.

During retroviral replication, full-length viral RNAs are encapsidated into new virus particles, while spliced RNAs are excluded. The Retroviridae are unique among viruses in that infectious viral particles contain a dimer of two identical genomic RNA strands. A variety of experimental data has suggested that dimerization and encapsidation of full-length viral RNAs are linked processes, although whether dimerization is a prerequisite for encapsidation, or conversely, dimerization follows encapsidation, has not been firmly established.