Rescue of synthetic RNAs into thogoto and influenza A virus particles using core proteins purified from Thogoto virus.
The ribonucleoprotein (RNP) complexes of Thogoto virus (THOV), a tick-borne orthomyxovirus, have been purified from detergent-lysed virions. The purified RNPs were then disrupted by centrifugation through a CsCl-glycerol gradient to obtain fractions highly enriched in nucleoprotein (NP) and virtually devoid of viral genomic RNA. When these NP-enriched fractions were incubated with a synthetic THOV-like RNA, and the mixtures were transfected into THOV-infected cells, the synthetic RNA was expressed and packaged into THOV particles. Similarly, hybrid mixtures containing purified THOV NP and influenza A virus synthetic RNAs (either a model CAT RNA or a gene encoding the viral neuraminidase), were prepared and transfected into influenza A virus-infected cells. The synthetic CAT RNA, was shown to be expressed and packaged into virus particles, and the neuraminidase gene was rescued into influenza virions. These data are discussed in terms of the similarities observed between THOV and influenza A virus and the potential application of the THOV purified proteins for rescuing synthetic genes into infectious viruses.
Gómez-Puertas
P
P
Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda 28220, Madrid, Spain.
Leahy
M B
MB
Nuttall
P A
PA
Portela
A
A
D000818:Animals; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D006224:Cricetinae; D005814:Genes, Viral / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D018117:Thogotovirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014758:Viral Core Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis; D019065:Virus Assembly / Q000502:physiology
D012367:RNA, Viral; D014758:Viral Core Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D009439:Neuraminidase