Reoviridae Molecular Virology

Reoviridae Respiratory enteric orphan (呼腸孤病毒) Depends on the presence of a segmented, dsRNA genome the structure characters replication strategy Reoviridae consists of 12 genera Mammalian orthoreovirus prototypes Type 1 (Lang, T1L):from a healty child Type 2 (Jones, T2L):from a child with diarrhea Type 3 (Dearing, T3D): from a child with diarrhea or (Abney, T3A) an upper respiratory illness Avian reoviruses Viral arthritis (VA) Pale bird syndrome (PBS)

dsRNA Viruses Family Genera Genome Segments Hosts Reoviridae 12 10, 11, or 12 (See individual genera)       Turreteda   Orthoreovirus 10 Mammals, Birds, Reptiles Aquareovirus 11 Fish, Molluscs Cypovirus Insects Idnoreovirus Fijivirus Plants, Insectsb Oryzavirus Mycoreovirus 11 or 12 Fungi       Nonturreteda Rotavirus Mammals, Birds Orbivirus Mammals, Birds, Arthropodsb Coltivirus Mammals, Arthropodsb Phytoreovirus Seadornavirus Mammals, Insectsb

dsRNA Viruses Family Genera Genome Segments Hosts Birnaviridae 3 2 Birds, Fish, Insects Totiviridae 1 Fungi, Protozoa Partitiviridae Fungi, Plants Chrysoviridae 4 Hypoviridae Fungi Cystoviridae Bacteria aProposed division based on presence or absence of turret-like protein projecting around fivefold axes from innermost capsid layer. bServe as vectors for transmission to other hosts.

Mammalian v.s. Avian reovirus Cell fusion － ＋ HA Fusogenic reovirus Nonfusogenic reovirus Avian reovirus can grow in mammalian cell line

Properties of nonfusogenic mammalian reovirus Genome Perfect double-stranded RNA 10 gene segments in three size classes (L, M, S) Total size ~23,500 base pairs Gene segments encode either one or two proteins each Gene segments are transcribed into full-length mRNAs Plus strands of gene segments have 5’caps Nontranslated regions at segment termini are short Gene segments can undergo reassortment between virus strains Short subgenomic RNA

Properties of nonfusogenic mammalian reovirus Particles Spherical, with icosahedral (5:3:2) symmetry Nonenveloped Total diameter ~85 nm (excluding σ 1 fibers) Two concentric protein capsids: outer capsid subunits in T = 13 lattice, arrangement of inner capsid subunits in T = 1 8 structural proteins: 4 proteins in outer capsid [λ 2, μ1 (mostly as cleavage fragments μ 1N and μ 1C), σ 1, and σ 3] and 4 proteins in inner capsid (λ 1, λ 3, μ 2, and σ 2) Subviral particles (ISVPs and cores) can be generated from fully intact particles (virions) by controlled proteolysis Cell-attachment protein σ 1 can extend from the virion and ISVP surface as a long fiber Protein λ 2 forms pentamers that protrude from the core surface

Properties of nonfusogenic mammalian reovirus Replication Fully cytoplasmic Sialic acid can serve as a cell surface receptor for recognition by cell-attachment protein σ 1. Proteolytic processing of outer capsid proteins σ 3 and μ 1/μ 1C is essential to infection and can occur either extracellularly or in endo/lysosomes. Uncoating of parent particles is incomplete: genomic dsRNA does not exit particles to enter the cytoplasm. Transcription and capping of viral mRNAs occur within particles and are mediated by particle-associated enzymes. Segment assortment and packaging involves mRNAs. Minus-strand synthesis occurs within assembling particles. Mature virions are inefficiently released from infected cells by lysis.

Ten Segmented dsRNA

Gene organization

Viral protein Mono- Di- cistronic S1 Tri- RNA FIGURE 52.2 Coding strategies of the ten genome segments of reovirus type 3 Dearing. Segments are drawn approximately to scale and are oriented so that left-to-right corresponds to 5??to 3??for the protein-coding plus strands. The segment names and lengths in nucleotides (nuc #) are listed at left. The portion of each segment encompassed by protein-coding sequences is hatched. Short nontranslated regions at the ends of each segment remain unshaded, and the lengths of these regions in nucleotides (nuc #) are designated above. Names of encoded proteins and their lengths in amino acids (aa #) are listed at right. The S1 segment encodes two proteins as shown: The ?1s protein (offset, lighter hatching) initiates at a second initiator codon in a different reading frame from ?1 (darker hatching). A second protein, 繕NSC (lighter hatching) also arises from the M3 segment but in its case is thought to initiate at a second initiator codon in the same reading frame as 繕NS (darker hatching).

Structure of virus particles

Structural composition of orthoreovirus

Structural proteins of reovirus Core proteins consisting of λ1, λ3, μ2 and σ2 proteins form internal transcriptase complexes Outer capsid proteins consisting of λ2, μ1C, σ1 and σ3 proteins form external turrets and nodules

Core structural proteins Encoding Segment Protein Mass (kd) Presence in Particle Formsa Function or Property L1 λ3 142 V, I, C RNA-dependent RNA polymerase L3 λ1 143 Binds RNA, Zn metalloprotein, NTPase, RNA helicase, RNA triphosphatase M1 μ2 83 Binds RNA, NTPase, a MAP S2 σ2 47 Binds dsRNA aV.I.C.: virus particle ISVP, core.

Core structural proteins Unclear in the complete core organization σ2/λ1 is 2/1 and forms a complex Iodination is less easily carried out on σ2 than λ1 Contains transcriptase and replication activity

Outer capsid structural proteins Encoding Segment Protein Mass (kd) Presence in Particle Formsa Function or Property L2 λ2 145 V, I, C Guanylyltransferase, methyltransferases, capping M2 μ1/μ1C 76 V, I N-myristoylated, cleaved into fragments, role in penetration, role in transcriptase activation S1 σ 1 49 Cell-attachment protein, hemagglutinin, primary serotype determinant, apoptosis S4 σ 3 41 V Sensitive to protease degradation, binds dsRNA, zinc metalloprotein, effects on cellular translation aV, virion; I, ISVP; C, core.

Outercapsid structural proteins μ1C is proteolytically cleaved from μ1. Glycoprotein. In cytoplasm of virus-infected cells, 95% of protein complexed with σ3 is μ 1C (not μ1). Cleavage of μ1 may be linked to the formation of a complex with σ3. μ1C/σ3 is 1/1 in vivion. Sequences are highly conserved between viruses and serve an structural constraints on the function. Cleavage site located at 42 and 43 AA of μ1 from N-terminus.

μ1C protein cleavage

Digestion of μ1C protein

Site for μ1c protein digestion

σ1 protein Proximately close to the λ2 Is tetramer N-terminus is helix (Fiber), located in a λ2 channel. Has an α-helix coiled-coil structure

Primary structure of σ1 protein

λ2 protein Viral core: 60 nm Icosahedron: 12 verticles, 20 faces, and 30 edges Care spike: 12 Each spike consists of 5 λ2 protein, projects out 5-6 nm above the surface of the core. Identified by mAb to λ2, has a hole or channel formation under EM

Non-structural of reovirus Non-fusogenic mammalian reovirus μNS σNS σ1S Fusogenic reovirus μNS σNS P10: induces cell fusion P17: contains nuclear leader signal (NLS)

Viral replication cycle

Absorption Receptor: sialic acid (some intergrins may involve) Penetration Uncoating incomplete uncoating Virion—ISVP—Core Transcription Newly synthesized +strand mRNA within Core and using conservative transcription model Early transcripts with 5’ capped mRNA Late transcripts without 5’ capped mRNA Early transcripts: L1, M3, S3 and S4 gene segments All ten genes are transcribed at the same time in vitro May have a cellular repressor prevent late transcription and a protein encoded by early gene results in derepression

Replication of viral RNA genome Using conservative transcription model mRNA used as templates for “-” strand RNA synthesis (no 5’ capping) Single round for “-” RNA synthesis σNS assorts 10 segment ssRNA for packaging

Late transcription of virus Transcribed to be a non 5’ capped mRNA for late protein synthesis Appears at 4-6 h after infection, reaches to be maximum at 12 h and then decrease after 12 h Assembly and release σ3 is the last one to the complete particle Once σ3 is assembly, transcriptional activities are terminated

Important Terminology Reassortment Cell fusion from within Cell fusion from without Persistent infection

Protein interactions in viral factories