1. Reoviridae
Molecular Virology

2. 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)

3. 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

4. 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.

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

6. 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

7. 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

8. 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.

9. Ten Segmented dsRNA

10. Gene organization

11. 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).

12. Structure of virus particles

13. Structural composition of orthoreovirus

14. 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

15. 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.

16. 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

17. 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.

18. 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.

19. μ1C protein cleavage

20. Digestion of μ1C protein

21. Site for μ1c protein digestion

22. 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

23. Primary structure of σ1 protein

24. 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

25. 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)

26. Viral replication cycle

27. 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

28. 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

29. 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

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

31. Protein interactions in viral factories