Orthomyxoviridae Molecular Virology

Orthomyxoviridae Myxo means mucus Myxovirus (old name): particularly affinity to mucopolysaccharide and glycoprotein, in particular, for sialic acid-containing receptor on cell surface Sialic acid: neuraminic acid Myxovirus divided into Orthomyxoviride and paramyxoviridae In term of Genetic organization Enveloped glycoprotein Replication strategy

Classification On the basis of antigenic cross-reactivity of RNP between members of each type, but not between types Orthomyxoviridae has 5 genera The influenza virus A, B and C Thogotovirus (ticks) Isavirus (infectious salmon anemia virus) Virus is named influenza A/chicken/Hong Kong/220/97(H5N1) (species origin omitted if human) Hemagglutinin (HA): H1-H16 for type A Neuraminidase (NA): NA1-NA9 for type A Hemagglutinin-esterase-fusion (HEF): type C

Virion structure Influenza B: HA, NA, NB, and BM2 Influenza C: has hexagonal reticular structure on the particle surface. No NA, but has HEF

Internal structure of influenza virus under EM

Genome structure and organization Types A and B have 8 segments of (-)ssRNA, encoded 11 viral protein Types C has 7 segments of (-)ssRNA, NA is missed, but has hemaglutinin-esterase-fusion (HEF) protein

Genome structure and protein function of type A

Genome structure and protein function of type B

Genome structure and protein function of type C

Virus replication cycle

Attachment HA of virus has specificity forward sialic acid with different linkages Mutation of HA amino acid may make virus adapt to different species

Entry and uncoating Mediated by clathrin-endocytosis for virus entry Required a low pH to initiate membrane fusion of endolysosome and envelope Low pH induces conformational change of HA (HA1 and HA2) and fusion peptide of HA2 was exposed. Can be blocked with weakly bases (NH4Cl, chloroquine) or ionophores (monensin). HA sequences at HA cleavage site determine pathogenicity Also, M2-mediated release of the RNA results in appearance of free RNA complexes in cytoplasm M2 allows the influx of H+ ions from endosome into the virus particle and disrupts protein-protein interaction and results in the release RNP from M1 protein. Can be blocked by amantadine or rimantadine which block the ion channel activity of M2 protein (inhibited uncoating). BM2 and CM2 have similar functions to M2 (type A)

Hemaglutinin (HA)

Hemaglutinin (HA)

Hemaglutinin (HA)

Hemaglutinin (HA)

M2 protein Is a tetrameric type III integral membrane protein Has ion channel activeity Involves in capturing the RNPs at the assembly site and be a role in virus assembly and budding

Nuclear import of ribonucleoproteins One unusual characteristics of influenza virus in its replication cycle is its dependence on nuclear functions Four proteins (NP, PB2, PB1, and PA) associated with vRNA to form ribonucleoprotein complexes (RNA) and create a panhandle structure. These proteins possess nuclear localization signals (NLS) that mediated their interaction with nuclear import machinery.

Viral RNA synthesis mRNA is made by a primer-dependent mechanism, is capped and polyadenylated Replication of vRNA occurs via a two-step process. A full length, (+) copy of vRNA(cRNA) is first made and used as the template to make more (-)vRNA They use the same viral polymerase complex

Viral RNA promoter

mRNA synthesis of viral RNA

mRNA synthesis of viral RNA Needs cellular mRNA producing enzyme(RNA polymerase II) Actinomycin D inhibits virus replication Cellular DNA destroyed by UV α-amanitin inhibits RNA poly II Nucleus removing PB2 recognizes cap structure of cellular mRNA PB1 also has endonuclease, resulting in cap-snatching phenomenon to supply capped primer for mRNA synthesis Polyadenylation by stutter mechanism Viral splicing is using the cellular splicing machinery

Switch from mRNA to cRNA synthesis Be structurally different in the replication-competent pol from the transscription-competent pol. Due to different domains of pB1 are involved in binding vRNA or cRNA Involves in newly synthesized cRNA and vRNA by soluble NP protein Cap structure may have an effect on RNA synthesis

Regulation of viral gene expression Early expression genes: NP, NS1 mRNA and protein Late expression in genes: HA, NA, M1 and protein NP is required for replication NS1 is a factor in combating the host immune response M1 inhibits viral replication, involves in RNP export from nucleus, only occurs once replication is complete.

RNP export from nucleus Proteins M1 and NS2 involve in directing the nuclear export of RNP. M1 binds RNA (vRNA and NP), also binds to nucleosome, suggesting that M1 interactions cause the dissociation of RNP from nuclear matrix. NS2 interacts with the cellular export machinery

Virus assembly and release Assemble and bud from the apical plasma membrane of polarized cells, is an asymmetric procession Important in viral pathogenesis and tissue tropism Basolateral procession tends to cause systemic disease. HA or NA transmembrane domain is replaced to contain a basoleteral targeting signal and virions still bud from apical plasma membrane Matrix (M1) protein is absolutely required for assembly of virus particles M2 captures RNP at the assembly site

Budding of influenza virus

Packaging of eight RNA segments Mechanism is not well known Random incorporation model Selective incorporation model

Release Neuraminidase (NA) involves virus release by removing the sialic acid NA activity is also required to remove sialic acid from the carbohydrates present the viral glycoproteins themselves so that the individual virus particles do not aggregate on cell surface

Reservoir of virus

Reassortment of virus

Reverse genetics

Antigenic drift Antigenic shift Induces by point or accumulative point mutation of HA gene, resulting in less antigenic change, no new type formed Antigenic shift Induces by gene reassortment of HA gene, resulting in a significant antigenic change, new types of virus formed Defective interference(DI) particle (von Magnus phenomenon)

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