TITLE: MH-Gag plasmid (Chimeric MLV/HIV-Gag) aka pSR389
CASE NUMBER: C13554
ABSTRACT
JHU researchers made use of a chimeric Moloney murine leukemia virus (MMLV)-HIV-1 Gag in which the p17 matrix domain of HIV-1 was replaced with the p15 matrix and p12 domains from MMLV. Murine cells expressing this construct released significant amounts of VLPs. The construct preserved H-2d-restricted antigenic determinants in the remaining portion of HIV-1 Gag, allowing immunogenicity studies to be performed with mice.
DESCRIPTION DETAIL
Plasmid DNA constructs.
MMLV-HIV-1 Gag (MHGag) was created by replacing the p17 matrix domain of HIV-1 Gag with the homologous p15 matrix domain from MMLV. In addition, the p12 domain from MMLV Gag, which has no homolog in HIV-1 Gag, was included in the construct. The MMLV p15-p12 segment was amplified with the sense PCR primer 5′-ATTATAGGTACCATGGGCCAGACTGTTACCACTC-3′, antisense primer 5′-GACTCCACTACCTCGCAGGCATTCCCCATCGTGCAGAACATCCAGG-3′, and plasmid pMMLVgag-pol carrying the MMLV gag-pol gene as a template. Myristoylation of the glycine residue at the amino termini of both MMLV p15 and HIV-1 p17 proteins is essential for membrane association by Gag and virion budding. Deletion of this residue eliminates the ability of Gag to form VLPs. To create a version of MHGag with this glycine deleted (abbreviated ΔG-MHGag), the sense primer 5′-ATTATAGGTACCATGCAGACTGTTACCACTCCCTTAAGTTTG-3′ was used instead for PCR, which was otherwise performed as described above. A segment of HIV-1 Gag downstream of matrix was amplified with the sense primer 5′-CCCATCGTGCAGAACATCCAGG-3′, the antisense primer 5′-GCCTGAACAAGATCGTGCG-3′, and plasmid pVRC3900 carrying codon-optimized HXB2 HIV-1 gag as a template. The MMLV matrix p12 and HIV-1 Gag PCR products contained complementary regions corresponding to the 5′ end of HIV-1 p24 capsid. These two PCR products were annealed and extended off each other and then digested with Acc65I and SbfI. The plasmid pCiCagPRE was created by replacing non-codon-optimized gag in pCigagPRE with gag from pVRC3900. pCiCagPRE was digested with Acc65I and SbfI to create the vector into which the MHGag construct was ligated, creating pCiMHGag. In a similar manner, pCiΔG-MHGag was made. In summary, MHGag comprises an amino-terminal component derived from MMLV Gag starting with the amino acid sequence MGQTVTTPLS from p15 and ending with the sequence VADSTTSQAF from p12. This is fused in frame to a carboxy-terminal component derived from codon-optimized HXB2 HIV-1 Gag starting with the amino acid sequence PIVQNIQGQM from p24 capsid and ending with the sequence SLFGSDPSSQ from p6, preserving the H-2Kd-restricted epitope, AMQMLKETI, found in HIV-1 p24.
ASSOCIATED PUBLICATIONS: J Virol. 2005 Feb;79(3):1701-12.
