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

Coronaviruses are large, enveloped RNA viruses of both medical and veterinary importance. Interest in this viral family has intensified in the past few years as a result of the identification of a newly emerged coronavirus as the causative agent of severe acute respiratory syndrome (SARS). At the molecular level, coronaviruses employ a variety of unusual strategies to accomplish a complex program of gene expression. Coronavirus replication entails ribosome frameshifting during genome translation, the synthesis of both genomic and multiple subgenomic RNA species, and the assembly of progeny virions by a pathway that is unique among enveloped RNA viruses. Progress in the investigation of these processes has been enhanced by the development of reverse genetic systems, an advance that was heretofore obstructed by the enormous size of the coronavirus genome. This review summarizes both classical and contemporary discoveries in the study of the molecular biology of these infectious agents, with particular emphasis on the nature and recognition of viral receptors, viral RNA synthesis, and the molecular interactions governing virion assembly.

The molecular biology of coronaviruses.

The SARS-CoV-2 pandemic and its unprecedented global societal and economic disruptive impact has marked the third zoonotic introduction of a highly pathogenic coronavirus into the human population. Although the previous coronavirus SARS-CoV and MERS-CoV epidemics raised awareness of the need for clinically available therapeutic or preventive interventions, to date, no treatments with proven efficacy are available. The development of effective intervention strategies relies on the knowledge of molecular and cellular mechanisms of coronavirus infections, which highlights the significance of studying virus-host interactions at the molecular level to identify targets for antiviral intervention and to elucidate critical viral and host determinants that are decisive for the development of severe disease. In this Review, we summarize the first discoveries that shape our current understanding of SARS-CoV-2 infection throughout the intracellular viral life cycle and relate that to our knowledge of coronavirus biology. The elucidation of similarities and differences between SARS-CoV-2 and other coronaviruses will support future preparedness and strategies to combat coronavirus infections.

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Coronavirus biology and replication: implications for SARS-CoV-2.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. The disease was initially described in the 16th century and was the first animal pathogen identified as a virus. Recent FMD outbreaks in developed countries and their significant economic impact have increased the concern of governments worldwide. This review describes the reemergence of FMD in developed countries that had been disease free for many years and the effect that this has had on disease control strategies. The etiologic agent, FMD virus (FMDV), a member of the Picornaviridae family, is examined in detail at the genetic, structural, and biochemical levels and in terms of its antigenic diversity. The virus replication cycle, including virus-receptor interactions as well as unique aspects of virus translation and shutoff of host macromolecular synthesis, is discussed. This information has been the basis for the development of improved protocols to rapidly identify disease outbreaks, to differentiate vaccinated from infected animals, and to begin to identify and test novel vaccine candidates. Furthermore, this knowledge, coupled with the ability to manipulate FMDV genomes at the molecular level, has provided the framework for examination of disease pathogenesis and the development of a more complete understanding of the virus and host factors involved.

Foot-and-Mouth Disease

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.

Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B., 33:233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.

Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, and altered cellular membranes, is a characteristic feature of plus-strand RNA viruses. Here, we demonstrate the presence of a specific membrane alteration, designated the membranous web, that contains hepatitis C virus (HCV) nonstructural proteins, as well as viral plus-strand RNA, in Huh-7 cells harboring autonomously replicating subgenomic HCV RNAs. Metabolic labeling with 5-bromouridine 5'-triphosphate in the presence of actinomycin D revealed that the membranous web is the site of viral RNA synthesis and therefore represents the replication complex of HCV.

https://www.researchgate.net/profile/Volker_Lohmann/publication/10807977_Identification_of_the_hepatitis_C_virus_RNA_replication_complex_in_Huh-7_cells_harboring_subgenomic_replicons/links/00b7d531d90a40d548000000.pdf

Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harboring Subgenomic Replicons

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5′-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

Membrane Rearrangement and Vesicle Induction by Recombinant Poliovirus 2C and 2BC in Human Cells

Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography.

Denise Egger

Papers

Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex

Identification of the Hepatitis C Virus RNA Replication Complex in Huh-7 Cells Harboring Subgenomic Replicons

RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

Membrane Rearrangement and Vesicle Induction by Recombinant Poliovirus 2C and 2BC in Human Cells

Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography.