Studies on the paramyxovirus accessory genes by reverse genetics in the Sendai virus-mouse system

Abstract

Nucleotide sequencing of the entire genomes was completed in the 1980s for most members of the Paramyxoviridae. It then became a new common task with challenge for researchers in the field to establish a system to recover the virus entirely from cDNA, thereby allowing reverse genetics (free manipulation of the viral genome). Using Sendai virus, we established a system of incomparable virus recovery efficiency early on. This technology was then fully exploited in answering a series of long-held questions. In particular, two accessory genes whose functions had remained enigmatic were demonstrated to encode special functions critical in viral in vivo pathogenesis producing fatal pneumonia in mice, although dispensable in virus replication at the in vitro cellular level. Their in vivo functions were found to counteract the two respective facets of the antiviral state induced by interferons and an interferon regulatory factor 3-dependent but yet unknown effector. These achievements appear to have facilitated a scientific trend where the accessory genes are a focus of active investigation in studies on other paramyxoviruses and opened up a new common ground shared between virology and immunology.

Fig. 1

Schematic diagram of an SeV particle. (Adapted from Ref. with permission.)

Fig. 2

Expression of C and V proteins from SeV P gene and recovery of knockout viruses; V(-) with the entire V protein deleted, VΔC with the V-unique region deleted and 4C(-) with all four C proteins (C′, C, Y1 and Y2) deleted. For details see the text. Deletion of the respective proteins was verified by Western blotting of cells infected with the recovered knockout viruses. Wt, cells infected with wild-type SeV. The dotted line in the insert indicates deletion. (Adapted from Ref. with permission.)

Fig. 4

Growth and pathogenicity of SeV Cm^* in wild-type and STAT1^−/− mice. Five-week-old wild-type STAT1^+/+ 129S6 and STAT1 deficient (STAT1^−/−) 129S6 mice were infected intranasally with 10⁷ CIU of SeV Wt or SeV Cm^*. Two or three mice from each group were sacrificed at the intervals indicated and examined for virus infectivity in the lung (top) and lung consolidation (bottom). The symbol “†” indicates a dead mouse. Actual macroscopic views of the lung corresponding to the consolidation scores of 0 (no lesion) through 4 (maximum) are presented as references (right). When a mouse died, one point was added to the consolidation score (score 5) (Modified from Ref. 26).

Fig. 3

Replication of SeV Cm^* in wild-type cells and in STAT1 deficient cells. Parental SeV Wt (wild type) and SeV Cm^* were inoculated to the parental STAT1^+/+2fTGH cells (left) and to its STAT1 deficient (STAT1^−/−) derivative, U3A cells (right) at an m.o.i. of 5. Cells were collected at the hours indicated. The cell lysates were immunoblotted with a mixture of anti STAT1 and anti STAT2 antibodies (top). Titers of SeV Wt (open circle) and SeV Cm^* (closed circle) in the culture fluid are shown (bottom) (Modified from Ref. 26).

Fig. 5

Replication and pathogenicity of SeV V(-) and SeV VΔC in IRF3^−/− mice. (A) Five-week-old IRF3^+/+ C57BL/6J and IRF3 deficient (IRF3^−/−) C57BL/6J mice were infected intranasally with 10⁷ CIU of SeV Wt, SeV V(-) or SeV VΔC. Two or three mice from each group were sacrificed at the indicated intervals and examined for virus infectivity in the lung and lung consolidation. The symbol “†” indicates a dead mouse. (B) Six- to seven-week-old IFN-α/β R^+/+ A129/Sv and IFN-α/β receptor deficient (IFN-α/β R^−/−) A129/Sv mice were infected intranasally with 10⁷ CIU of SeV Wt or SeV V(-) (left). Five-week-old STAT1^+/+ 129S6 and STAT1 deficient (STAT1^−/−) 129S6 mice were infected intranasally with 10⁷ CIU of SeV Wt or SeV V(-) (right). Viral infectivity in the lung was measured at the indicated time intervals after infection (Modified from Ref. 30).

Fig. 6

(A) Expression of accessory genes from members of the subfamily Paramyxovirinae. The fifth gene encoding the receptor binding protein is named HN for Respiro-, Avula- and Rubulavirus, H for Morbillivirus and G for Henipavirus. Dotted line in the P gene, editing site;•, cap of mRNA; AA, 3′ polyadenylic acid of mRNA. SH, inserted between the F and HN genes (Modified from Ref. 8). (B) The coding assignment of the individual inhibitory functions. –, data not available; W, encoded by the mRNA generated by +2G insertion. For details refer to the text and Refs. , and .