Hepatoviruses promote very-long-chain fatty acid and sphingolipid synthesis for viral RNA replication and quasi-enveloped virus release.

Virus-induced changes in host lipid metabolism are an important but poorly understood aspect of viral pathogenesis. By combining nontargeted lipidomics analyses of infected cells and purified extracellular quasi-enveloped virions with high-throughput RNA sequencing and genetic depletion studies, we show that hepatitis A virus, an hepatotropic picornavirus, broadly manipulates the host cell lipid environment, enhancing synthesis of ceramides and other sphingolipids and transcriptionally activating acyl-coenzyme A synthetases and fatty acid elongases to import and activate long-chain fatty acids for entry into the fatty acid elongation cycle. Phospholipids with very-long-chain acyl tails (>C22) are essential for genome replication, whereas increases in sphingolipids support assembly and release of quasi-enveloped virions wrapped in membranes highly enriched for sphingomyelin and very-long-chain ceramides.

CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized.

Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT).

Nonlytic cellular release of hepatitis A virus requires dual capsid recruitment of the ESCRT-associated Bro1 domain proteins HD-PTP and ALIX.

Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown.

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C.

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells.

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection.

Basal expression of interferon regulatory factor 1 drives intrinsic hepatocyte resistance to multiple RNA viruses.

Current models of cell-intrinsic immunity to RNA viruses centre on virus-triggered inducible antiviral responses initiated by RIG-I-like receptors or Toll-like receptors that sense pathogen-associated molecular patterns, and signal downstream through interferon regulatory factors (IRFs), transcription factors that induce synthesis of type I and type III interferons. RNA viruses have evolved sophisticated strategies to disrupt these signalling pathways and evade elimination by cells, attesting to their importance. Less attention has been paid to how IRFs maintain basal levels of protection against viruses.

Redundant Late Domain Functions of Tandem VP2 YPXL Motifs in Nonlytic Cellular Egress of Quasi-enveloped Hepatitis A Virus.

The quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses.

Hepatitis A Virus Genome Organization and Replication Strategy.

Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus of the family It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses.

Protein composition of the hepatitis A virus quasi-envelope.

The are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation.

Human pDCs preferentially sense enveloped hepatitis A virions.

Unlike other picornaviruses, hepatitis A virus (HAV) is cloaked in host membranes when released from cells, providing protection from neutralizing antibodies and facilitating spread in the liver. Acute HAV infection is typified by minimal type I IFN responses; therefore, we questioned whether plasmacytoid dendritic cells (pDCs), which produce IFN when activated, are capable of sensing enveloped virions (eHAV). Although concentrated nonenveloped virus failed to activate freshly isolated human pDCs, these cells produced substantial amounts of IFN-α via TLR7 signaling when cocultured with infected cells.

Naked Viruses That Aren't Always Naked: Quasi-Enveloped Agents of Acute Hepatitis.

Historically, viruses were considered to be either enveloped or nonenveloped. However, recent work on hepatitis A virus and hepatitis E virus challenges this long-held tenet. Whereas these human pathogens are shed in feces as naked nonenveloped virions, recent studies indicate that both circulate in the blood completely masked in membranes during acute infection.

Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses.

Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV). Mother-to-child transmission of HCV occurs in 3-5% of pregnancies and accounts for most new childhood infections. HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) are vital in the clearance of acute HCV infections, but in the 60-80% of infections that persist, these cells become functionally exhausted or select for mutant viruses that escape T cell recognition.

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes.

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver.

Discovery of potent, cyclic calcitonin gene-related peptide receptor antagonists.

Calcitonin gene-related peptide (CGRP), a potent dilator of cerebral and dural vasculature, is known to be elevated in plasma and cerebral spinal fluid during migraine attacks. Selective blockade of the CGRP receptor offers the promise of controlling migraine headache more effectively and without the side-effects associated with the use of triptans. Our efforts to develop a novel, peptide-based CGRP antagonist focused on the C-terminal portion of the peptide which is known to bind the receptor but lack agonist properties.

High throughput screening of library compounds against an oligonucleotide substructure of an RNA target.

Approximately 300,000 compounds from selected libraries were screened against a subdomain of a hepatitis C viral (HCV) RNA using a high throughput flow injection mass spectrometry (FIA-MS) method with automated data storage and analysis. Samples contained 2 microM RNA target and 10 microM of each of up to ten ligands. Preliminary studies to optimize operational parameters used the binding of aminoglycosides to the A44 subdomain of bacterial RNA.

An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype.

The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable.

RNA as a target for developing antivirals.

The base of knowledge concerning RNA structure and function has been expanding rapidly in recent years. Simultaneously, an increasing awareness of the pivotal role RNA plays in viral diseases has prompted many researchers to apply new technologies in high-throughput screening and molecular modelling to the design of antiviral drugs that target RNA. While the two RNA viruses with the greatest unmet medical need, HIV and HCV, have been most actively pursued, the approaches discussed in this review are relevant to all virus infections.

Sequence requirements for viral RNA replication and VPg uridylylation directed by the internal cis-acting replication element (cre) of human rhinovirus type 14.

Until recently, the cis-acting signals required for replication of picornaviral RNAs were believed to be restricted to the 5' and 3' noncoding regions of the genome. However, an RNA stem-loop in the VP1-coding sequence of human rhinovirus type 14 (HRV-14) is essential for viral minus-strand RNA synthesis (K. L.