Analysis of LaCrosse virus S mRNA 5' termini in infected mosquito cells and Aedes triseriatus mosquitoes

Nucleotide sequences were determined for the 5' termini of La Crosse virus (LAC) S segment mRNA from persistently infected mosquito cell cultures (C6/36 from Aedes albopictus) and embryos (Aedes triseriatus). LAC primes transcription of its mRNA with ''scavenged'' 5' caps and adjacent oligonucleotides from host mRNAs, and these non-virus-encoded 5'-terminal extensions are heterogeneous in infected mammalian cells. The nature of mosquito host-derived primers has not been previously investigated. During early C6/36 cell infection, LAC mRNA 5'-terminal sequences were heterogeneous, but variability decreased as infection persisted. One predominant sequence, 5' CCACTCGCCACT (sequence 1), was observed throughout C6/36 tell infection but was more prevalent after 15 days postinfection. This LAC mRNA 5'-terminal sequence comprised 81% of the scavenged host oligonucleotides from vertically infected A. triseriatus eggs during embryogenesis. As these embryos progressed in the dormant overwintering stage (diapause), the predominant scavenged sequence became 5' AGGAAAAGATGGT (sequence 2), and sequence 1 became less prevalent. As the eggs emerged from diapause, the LAC mRNA 5' termini were more variable; 33% had sequence 1, and the remainder were heterogeneous. In post-diapausing eggs, 100% of viral mRNAs had sequence 1 at their 5' termini. Molecular analyses thus revealed continuous but selective LAC cap scavenging during persistent C6/36 cell infection and during embryogenesis and diapause in A. triseriatus eggs. The variety of host-derived sequences was limited in both biosynthetically active (embryonating) and dormant (diapausing) eggs.