The nucleocapsid domain is responsible for the ability of spleen necrosis virus (SNV) Gag polyprotein to package both SNV and murine leukemia virus RNA.
Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.
Certo
J L
JL
Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia 26506, USA.
Kabdulov
T O
TO
Paulson
M L
ML
Anderson
J A
JA
Hu
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism; D015683:Gene Products, gag; D005822:Genetic Vectors; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D019251:Nucleocapsid / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D001356:Reticuloendotheliosis virus / Q000235:genetics / Q000502:physiology*; D017422:Sequence Analysis, DNA; D014162:Transfection; D019065:Virus Assembly
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D012367:RNA, Viral