FLAVIVIRUSES 1103 Background and Classification 1103 Structure and Physical Properties of the Virion 1104 Binding and Entry 1105 Genome Structure 1106 Translation and Proteolytic Processing 1107 Features of the Structural Proteins 1108 Features of the Nonstructural Proteins 1109 RNA Replication 1112 Membrane Reorganization and the Compartmentalization of Flavivirus Replication 1112 Assembly and Release of Particles from Flavivirus-infected Cells 1112 Host Resistance to Flavivirus Infection 1113

Flaviviridae :T he Viruses and Their Replication

The interferon (IFN) system is an extremely powerful antiviral response that is capable of controlling most, if not all, virus infections in the absence of adaptive immunity. However, viruses can still replicate and cause disease in vivo, because they have some strategy for at least partially circumventing the IFN response. We reviewed this topic in 2000 [Goodbourn, S., Didcock, L. & Randall, R. E. (2000). J Gen Virol 81, 2341-2364] but, since then, a great deal has been discovered about the molecular mechanisms of the IFN response and how different viruses circumvent it. This information is of fundamental interest, but may also have practical application in the design and manufacture of attenuated virus vaccines and the development of novel antiviral drugs. In the first part of this review, we describe how viruses activate the IFN system, how IFNs induce transcription of their target genes and the mechanism of action of IFN-induced proteins with antiviral action. In the second part, we describe how viruses circumvent the IFN response. Here, we reflect upon possible consequences for both the virus and host of the different strategies that viruses have evolved and discuss whether certain viruses have exploited the IFN response to modulate their life cycle (e.g. to establish and maintain persistent/latent infections), whether perturbation of the IFN response by persistent infections can lead to chronic disease, and the importance of the IFN system as a species barrier to virus infections. Lastly, we briefly describe applied aspects that arise from an increase in our knowledge in this area, including vaccine design and manufacture, the development of novel antiviral drugs and the use of IFN-sensitive oncolytic viruses in the treatment of cancer.

Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures.

https://www.cell.com/immunity/pdf/S1074-7613(06)00394-3.pdf

Type I Inteferon Gene Induction by the Interferon Regulatory Factor Family of Transcription Factors

The interferon regulatory factor (IRF) family, consisting of nine members in mammals, was identified in the late 1980s in the context of research into the type I interferon system. Subsequent studies over the past two decades have revealed the versatile and critical functions performed by this transcription factor family. Indeed, many IRF members play central roles in the cellular differentiation of hematopoietic cells and in the regulation of gene expression in response to pathogen-derived danger signals. In particular, the advances made in understanding the immunobiology of Toll-like and other pattern-recognition receptors have recently generated new momentum for the study of IRFs. Moreover, the role of several IRF family members in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers.

The IRF Family Transcription Factors in Immunity and Oncogenesis

Tick-borne encephalitis

The tick-borne encephalitis (TBE) complex of viruses, genus Flavivirus, can cause severe encephalitis, meningitis, and/or hemorrhagic fevers. Effective interferon (IFN) responses are critical to recovery from infection with flaviviruses, and the mosquito-borne flaviviruses can inhibit this response. However, little is known about interactions between IFN signaling and TBE viruses. Langat virus (LGTV), a member of the TBE complex of viruses, was found to be highly sensitive to the antiviral effects of IFN. However, LGTV infection inhibited IFN-induced expression of a reporter gene driven by either IFN-α/β- or IFN-γ-responsive promoters. This indicated that LGTV can inhibit the IFN-mediated JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway of signal transduction. The mechanism of inhibition was due to blocks in the phosphorylation of both Janus kinases, Jak1 and Tyk2, during IFN-α signaling and at least a failure of Jak1 phosphorylation following IFN-γ stimulation. To determine the viral protein(s) responsible, we individually expressed all nonstructural (NS) proteins and examined their ability to inhibit signal transduction. Expression of NS5 alone inhibited STAT1 phosphorylation in response to IFN, thus identifying NS5 as a potential IFN antagonist. Examination of interactions between NS5 and cellular proteins revealed that NS5 associated with IFN-α/β and -γ receptor complexes. Importantly, inhibition of JAK-STAT signaling and NS5-IFN receptor interactions were demonstrated in LGTV-infected human monocyte-derived dendritic cells, important target cells for early virus replication. Because NS5 may interfere with both innate and acquired immune responses to virus infection, this protein may have a significant role in viral pathogenesis.

Inhibition of Interferon-Stimulated JAK-STAT Signaling by a Tick-Borne Flavivirus and Identification of NS5 as an Interferon Antagonist

Rotaviruses are the most important cause of severe, often life-threatening gastroenteritis in infants and children under 2 years of age (33). These viruses are ubiquitous in nature and are also responsible for a significant proportion of neonatal diarrheal illness in domestic animals, particularly in bovine and porcine species (18, 40). Substantial research efforts have thus focused on understanding the correlates of a protective immune response to rotavirus infection and the molecular mechanisms of virus replication so that efficacious vaccines can be developed.

The rotavirus segmented double-stranded RNA genome encodes six structural proteins (VP) and six nonstructural proteins (NSP) (reviewed in reference 17). The structural proteins VP1, VP2, VP3, VP4, VP6, and VP7 are well characterized in terms of their antigenic, structural, and biochemical properties. The functions of the rotavirus nonstructural proteins NSP1 to NSP6 are less well defined with regard to the roles that these proteins play in the rotavirus replication cycle. Intriguing functions have recently been described for some. NSP3 binds the 3′ consensus sequence of viral mRNAs (37) and acts as a functional analog of poly(A) binding protein through its interaction with eIF4GI (36). NSP4 is both an intracellular glycoprotein receptor for maturating rotavirus particles that bud through the endoplasmic reticulum (3, 32) and a viral enterotoxin that induces diarrhea in mice in the absence of any other viral protein (5). Functions of the remaining nonstructural proteins, NSP1, NSP2, NSP5, and NSP6, have been proposed based predominately on biochemical properties and activities of recombinant proteins (reviewed in reference 17).

NSP1 displays several interesting properties that warrant investigation. NSP1 has a calculated molecular weight of approximately 54,000 and is the least conserved protein encoded by the rotavirus genome when NSP1s of different strains are compared (23, 34). The N terminus contains a conserved zinc finger motif that binds zinc and viral mRNA in vitro (10, 22). Immunofluorescent staining showed NSP1 to be localized throughout the cytoplasm, in contrast to most other rotavirus proteins, which concentrate in viroplasms (24). NSP1 is also found associated with the cytoskeleton when analyzed by subcellular fractionation (24). NSP1 apparently is not required for rotavirus replication because strains with rearrangements in gene 5 that result in the synthesis of truncated NSP1 have been isolated from animals and from both immune-deficient and immune-competent children (1, 7, 15, 25, 35, 44, 45). Each of the described strains replicates in cell culture to titers close to those of their wild-type counterparts, but they yield small- to minute-plaque phenotypes. These observations suggest that NSP1 plays a role in regulating the efficiency of viral gene expression or in modulating host cell responses. We addressed the second possibility by constructing an MA104 cell cDNA library into the activation domain vector of the Matchmaker 3 yeast two-hybrid interaction trap (Clontech). We screened the library with NSP1 as bait to identify candidate partners of NSP1 that would provide clues to its function in rotavirus-infected cells.

Polyadenylated mRNA was isolated from MA104 cells in exponential growth phase with TriZol reagent (Life Technologies) and purified by column chromatography with the mRNA purification system from Amersham Pharmacia. cDNA was synthesized according to the protocol supplied with reagents from Clontech, with modifications. Briefly, 5 μg of mRNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase and an oligo(dT)-XhoI linker primer. Double-stranded cDNA was generated with DNA polymerase I, and the ends were blunted with Pfu DNA polymerase (Stratagene). Double-stranded cDNA fragments ligated to EcoRI adapters were size fractionated with ChromaSpin 400 columns (Clontech) and ligated into the Matchmaker pGADT7 vector prepared by EcoRI-XhoI digestion. Transformations that were sufficient to obtain 2.5 × 106 CFU were performed. The colonies were collected into Luria broth, and DNA was purified by two sequential centrifugations through CsCl density gradients. The library was quantified by measuring the absorbance at 260 nm, aliquoted, and stored at −80°C.

Gene 5 encoding NSP1 of bovine rotavirus strain B641 was cloned by reverse transcriptase PCR (unpublished data). The nucleotide sequence of B641 gene 5 is 98% identical to that of gene 5 of bovine strain RF (9). A plasmid containing gene 5 of murine strain EW was a generous gift of Harry Greenberg, Stanford University School of Medicine. Gene 5 cDNAs from B641 and EW were cloned into pGBKT7 by standard cloning techniques to generate pGBK-bNSP1 (bovine) and pGBK-mNSP1 (murine), respectively. All yeast transformations followed the lithium acetate-polyethylene glycol (PEG) method described by Gietz and Woods (19). The DNA binding domain vector pGBKT7 carries the TRP1 nutritional selection marker, and colonies containing bait plasmids were isolated by culture on synthetic complete medium lacking tryptophan (SC−W medium). The Matchmaker activation domain vector pGADT7 carries the LEU2 nutritional marker for selection of yeast.

Positive two-hybrid interactions were scored by determining colony growth on SC medium without leucine and tryptophan (SC−L−W medium) and activation of the reporter genes HIS3, ADE2, and MEL1. Reporter gene activation was indicated by growth in the absence of histidine and adenine and by the ability to metabolize the chromogenic substrate X-α-Gal(α-5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ICN).pGBK-bNSP1 yeast cells (6 × 109) cultured in SC−W liquid medium were transformed with 120 μg of the cDNA library by the lithium acetate-PEG procedure. Transformants were plated on SC medium without leucine, tryptophan, histidine, and adenine (SC−L−W−H−A medium) and cultured for 2 to 4 days at 30°C. Colonies measuring 2 to 4 mm in diameter were restreaked on SC−L−W−H−A plus X-α-Gal medium and again incubated for 2 to 4 days at 30°C. Plasmid DNA was isolated by electroporation into DH10B cells (Life Technologies) and cultured on antibiotic selection media.

Interferon Regulatory Factor 3 Is a Cellular Partner of Rotavirus NSP1

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern, to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection and that the alpha variant outcompeted the lineage A strain.

https://www.tandfonline.com/doi/pdf/10.1080/22221751.2022.2037397?needAccess=true

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant

Rotavirus mRNAs are transcribed from 11 genomic dsRNA segments within a subviral particle. The mRNAs are extruded into the cytoplasm where they serve as mRNA for protein synthesis and as templates for packaging and replication into dsRNA. The molecular steps in the replication pathway that regulate the levels of viral gene expression are not well defined. We have investigated potential mechanisms of regulation of rotavirus gene expression by functional evaluation of two differentially expressed viral mRNAs. NSP1 (gene 5) and VP6 (gene 6) are expressed early in infection, and VP6 is expressed in excess over NSP1. We formulated the hypothesis that the amounts of NSP1 and VP6 were regulated by the translational efficiencies of the respective mRNAs. We measured the levels of gene 5 and gene 6 mRNA and showed that they were not significantly different, and protein analysis indicated no difference in stability of NSP1 compared with VP6. Polyribosome analysis showed that the majority of gene 6 mRNA was present on large polysomes. In contrast, sedimentation of more than half of the gene 5 mRNA was subpolysomal. The change in distribution of gene 5 mRNA in polyribosome gradients in response to treatment with low concentrations of cycloheximide suggested that gene 5 is a poor translation initiation template compared with gene 6 mRNA. These data define a regulatory mechanism for the difference in amounts of VP6 and NSP1 and provide evidence for post-transcriptional control of rotavirus gene expression mediated by the translational efficiency of individual viral mRNAs.

Translational regulation of rotavirus gene expression.

Abstract A cell line was established from Culex tarsalis Coquillett embryonated eggs and designated as CxTr. The cell line is heterogeneous, composed predominantly of small, round cells, and spindle-shaped cells with a doubling time of approximately 52–60 h. The identity of the cell line was verified as Cx. tarsalis by sequencing of cytochrome oxidase I and the cells were found to be free of contaminating cells, bacteria, fungi, and mycoplasma. The permissiveness of CxTr cells to arbovirus infection was investigated with vaccine and wildtype arboviruses from four viral families: Flaviviridae (Japanese encephalitis virus), Phenuiviridae (Rift Valley fever phlebovirus), Rhabdoviridae (vesicular stomatitis virus), and Togaviridae (Mayaro virus). All viruses were able to infect and replicate within CxTr cells.

Dana N. Mitzel

Papers

Inhibition of Interferon-Stimulated JAK-STAT Signaling by a Tick-Borne Flavivirus and Identification of NS5 as an Interferon Antagonist

Interferon Regulatory Factor 3 Is a Cellular Partner of Rotavirus NSP1

Susceptibility of sheep to experimental co-infection with the ancestral lineage of SARS-CoV-2 and its alpha variant

Translational regulation of rotavirus gene expression.

Establishment of a Culex tarsalis (Diptera: Culicidae) Cell Line and its Permissiveness to Arbovirus Infection