Tremendous progress has been made in understanding the molecular basis of the antiviral actions of interferons (IFNs), as well as strategies evolved by viruses to antagonize the actions of IFNs. Furthermore, advances made while elucidating the IFN system have contributed significantly to our understanding in multiple areas of virology and molecular cell biology, ranging from pathways of signal transduction to the biochemical mechanisms of transcriptional and translational control to the molecular basis of viral pathogenesis. IFNs are approved therapeutics and have moved from the basic research laboratory to the clinic. Among the IFN-induced proteins important in the antiviral actions of IFNs are the RNA-dependent protein kinase (PKR), the 2',5'-oligoadenylate synthetase (OAS) and RNase L, and the Mx protein GTPases. Double-stranded RNA plays a central role in modulating protein phosphorylation and RNA degradation catalyzed by the IFN-inducible PKR kinase and the 2'-5'-oligoadenylate-dependent RNase L, respectively, and also in RNA editing by the IFN-inducible RNA-specific adenosine deaminase (ADAR1). IFN also induces a form of inducible nitric oxide synthase (iNOS2) and the major histocompatibility complex class I and II proteins, all of which play important roles in immune response to infections. Several additional genes whose expression profiles are altered in response to IFN treatment and virus infection have been identified by microarray analyses. The availability of cDNA and genomic clones for many of the components of the IFN system, including IFN-alpha, IFN-beta, and IFN-gamma, their receptors, Jak and Stat and IRF signal transduction components, and proteins such as PKR, 2',5'-OAS, Mx, and ADAR, whose expression is regulated by IFNs, has permitted the generation of mutant proteins, cells that overexpress different forms of the proteins, and animals in which their expression has been disrupted by targeted gene disruption. The use of these IFN system reagents, both in cell culture and in whole animals, continues to provide important contributions to our understanding of the virus-host interaction and cellular antiviral response.

Antiviral Actions of Interferons

We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.

Rescue of Influenza A Virus from Recombinant DNA

Characterization of temperature sensitive influenza virus mutants defective in neuraminidase.

https://www.cell.com/cell/pdf/0092-8674(85)90103-5.pdf

Cytotoxic T cells recognize fragments of the influenza nucleoprotein.

Influenza A and B viruses carry two surface glycoproteins, the haemagglutinin (HA) and the neuraminidase (NA). Both proteins have been found to recognise the same host cell molecule, sialic acid. HA binds to sialic acid-containing receptors on target cells to initiate virus infection, whereas NA cleaves sialic acids from cellular receptors and extracellular inhibitors to facilitate progeny virus release and to promote the spread of the infection to neighbouring cells. Numerous studies performed recently have revealed that an optimal interplay between these receptor-binding and receptor-destroying activities of the surface glycoproteins is required for efficient virus replication. An existing balance between the antagonistic HA and NA functions of individual viruses can be disturbed by various events, such as reassortment, virus transmission to a new host, or therapeutic inhibition of neuraminidase. The resulting decrease in the viral replicative fitness is usually overcome by restoration of the functional balance due to compensatory mutations in HA, NA or both proteins.

Functional balance between haemagglutinin and neuraminidase in influenza virus infections

Antiserum to a recombinant between an Ao and an A2 influenza virus had no detectable antibody against an A2 virus in standard hemagglutination-inhibition tests, and inhibited 95% of viral neuraminidase activity at a 1 to 400 dilution. However, on mixing virus with antiserum, a drop of up to 90% in hemagglutinin titer was observed. The effects of ultrasonication and direct electron microscopic examination indicated that the antiserum caused aggregation of virus particles. When antiserum was added to A2 virus-infected chick embryo fibroblasts, release of virus appeared markedly inhibited. After ultrasonication to disrupt aggregates, an increase in released hemagglutinin was observed, but the resulting level was considerably lower than that in control cultures containing normal rabbit serum. In thin sections of infected cells, similar numbers of virus profiles were observed in control and antiserum-treated cultures. A marked increase in release of hemagglutinin was noted if receptor-destroying enzyme was added to antiserum-treated cultures. The results indicate that antibody to neuraminidase does not exert a direct effect on viral maturation, but inhibits the detachment of viral progeny from cell surface receptors.

Effect of Antibody to Neuraminidase on the Maturation and Hemagglutinating Activity of an Influenza A2 Virus

A precursor glycoprotein in influenza C virus.

The synthesis of viral polypeptides was analyzed in BHK-21-F cells infected with the WSN strain of influenza virus at various times in the growth cycle. The relative amounts of polypeptides P, HA, NP, and NS did not change markedly between early and late times in the growth cycle; however, there was a progressive increase in the relative amount of the M polypeptide at later time points. In cell fractionation experiments, the patterns of newly synthesized polypeptides associated with various cytoplasmic fractions remained similar throughout the growth cycle except for an increase in polypeptide M in all fractions late in the growth cycle. The HA polypeptide was chased out of cytoplasmic membranes completely 6 h after synthesis, whereas the M polypeptide was not chased effectively from such membranes. Marked differences were found in the incorporation into mature virions of polypeptides synthesized at different times in the growth cycle. Polypeptides P and NP synthesized at early times were incorporated preferentially, whereas M was synthesized and incorporated predominantly late in the growth cycle. The fact that the rates of incorporation of polypeptides into virions differed significantly from their rates of synthesis indicates that different polypeptides were assembled into virions by distinct pathways.

Time Course of Synthesis and Assembly of Influenza Virus Proteins

Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion

Enveloped viruses which form by budding at the cell surface possess a membrane consisting of a lipid bilayer and a small number of virus-coded polypeptides. Since viral polypeptides become integral components of the plasma membrane during assembly, the process of synthesis and incorporation into membranes of these proteins may reflect the pathway of plasma membrane assembly. Electron microscopic studies have suggested that viral envelope proteins are incorporated into discrete, localized regions of the plasma membrane which serve as recognition sites for the viral nucleocapsid. In influenza virus-infected cells, viral polypeptides are associated with cytoplasmic membranes as well as the plasma membrane. The major envelope glycoprotein appears to be synthesized in rough endoplasmic reticulum, and to migrate to smooth membranes after synthesis. Glycosylation is initiated in rough membranes and progresses further in smooth membranes. Unlike the glycoproteins, the major nonglycosylated polypeptide appears to be inserted directly into the plasma membrane. In the presence of 2-deoxyglucose or high concentrations of glucosamine, aberrant viral glycoproteins are detected which appear to be unglycosylated or partially glycosylated; these are associated with membranes and incorporated into virus particles of reduced infectivity. Therefore normal glycosylation is not essential for incorporation of viral glycoproteins into cellular membranes or virus particles, but is required for normal biological activity. The role of the viral neuraminidase in assembly and release has been studied using mutants defective in neuraminidase at restrictive temperature. Under these conditions virus formation occurs, but the progeny form large aggregates at the cell surface. Colloidal iron hydroxide staining indicates that such virus particles contain neuraminic acid, and these residues appear to serve as receptors leading to the extensive aggregation.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/jss.400020234

Assembly of lipid‐containing viruses

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