Stable retrovirus and methods of use

Abstract

The present invention is a mutant retroviral protease which confers an increase in retroviral stability. Retroviruses expressing the instant mutant retroviral protease exhibit at least a 2-fold increase in infectivity half-life as compared to wild-type retrovirus. Unexpectedly, a Gly119Glu mutation in the protease enhances retroviral stability in the presence of various wild-type envelope proteins including wild-type amphotropic, ecotropic and 10A1 murine leukemia viruses. The improved stability of the mutant retrovirus leads to more facile virus production and enhanced infection efficiency.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a consensus sequence (SEQ ID NO:1) of 4070A wild-type protease (wt-PR; SEQ ID NO:2) and wild-type Moloney ecotropic protease (MoMLV PR; SEQ ID NO:3) and amino acid sequence alignment with Gly119Glu protease (G119E-PR; SEQ ID NO:4). The Gly119Glu mutation is in bold, and differences between 4070A and MoMLV proteases are indicated by an asterisk.

FIG. 2 is an amino acid sequence alignment of retroviral proteases (PRs) based on the solved three-dimensional structures of HIV-1 (human immunodeficiency virus type 1), HIV-2 (human immunodeficiency virus type 2), SIV (simian immunodeficiency virus), FIV (feline immunodeficiency virus), EIAV (equine infectious anemia virus), and RSV (Rous sarcoma virus) PRs. See Wlodawer and Gustchina (2000) Biochim. Biophys. Acta 1477:16-34. The MLV PR sequence was aligned by sequence similarity. Identical residues are bold and underlined, and conserved residues are in bold. The location of the Gly119 residue in the MLV PR is indicated by an asterisk. Secondary structure elements from RSV sequence are represented over the alignment. βBeta strand elements; α, alpha helix. The triangle indicates that location of the active site. See also Marmey, et al. (2005) J. Virol. 2:33.

FIG. 3 shows the decay of wild-type (wt) and Gly119Glu-PR MLV infectivity (n=3; error bars represent standard deviation). The wild-type half-life was 6.8 hours (r²=0.958), while that of Gly119Glu-PR MLV was 13.9 hours (r=0.967).

FIG. 4 shows the effect of Gly119Glu-PR substitution on various pseudotypes and strains of MLV, determined as fraction of virus titer remaining after incubation of virus supernatant for 24 hours at 37° C. (n=3; error bars represent standard deviation). Viruses were generated with 4070A, Moloney (MoMLV), 10A1 or VSV-G Env proteins and with gag-pro-pol genes originating from strain 4070A or MoMLV, as shown. Virus displaying 4070A, 10A1 and VSV-G Env were tittered on HEK-293 cells; viruses displaying MoMLV Env were tittered on NIH3T3 cells.

FIG. 5 shows the production of wild-type (wt) and Gly119Glu-PR MLV. Virus was produced in HEK-293T cells after transfection with pMDM(4070A), pNCgag-pol or pNCgag-pol-Gly119Glu, and pLZCX2. The supernatant was collected every 24 hours after transfection and replaced with fresh growth medium. Virus titer in the supernatant was determined by limiting dilution (n=2; bars represent average titer).

FIG. 6 shows a comparison of transduction efficiency for wild-type and mutant PR MLVs. Wild-type (wt) and Gly119Pro or Gly119Arg mutant virus transduction efficiency was compared in HEK-293 cells after 24-hour and 48-hour infection times. Fresh virus was added after 24 hours for the 48-hour infection. A brief 30 minute pulse infection measured an initial titer that minimized decay for both the wild-type and mutant MLV. In both cases, the mutant MLV maintained higher transduction levels than the wild-type virus (n=3; error bars represent standard deviation).

FIG. 7 shows saturation mutagenesis of residue 119 in MLV PR. Each amino acid was substituted in place of wild-type Gly at position 119 in the PR, and all mutants were found to possess higher thermostability than Gly at this site.

FIG. 8 shows that mutagenesis of residues at positions 15, 37, 39, and 57 of MLV PR increases the thermostability of MLV as compared to wild-type (wt).

FIG. 9 shows virus stability of double mutants by plotting the fraction of virus titer remaining after incubation of virus supernatant for 24 hours at 37° C. The wild-type (wt) virus provided the most drastic reductions in titer compared to all single PR mutants and all but one of the double mutants (n≧3; error bars represent standard deviation).

FIG. 10 shows a comparison of virus production for wild-type and mutant PR MLVs. Virus was produced in HEK-293T cells after transfection with wild-type (wt) or mutant virus DNA. Retroviral supernatant was collected every 24 hours and replaced with fresh growth media. Virus titer was determined by transduction of luc with the luciferase assay (n=3; error bars represent standard deviation).

FIG. 11 shows a comparison of virus decay for wild-type and mutant PR MLVs. Viruses in conditioned medium were incubated at 37° C. for the times shown, and virus activity was measured by luciferase assay and normalized to the 0 hour sample with no incubation. The wild-type (wt) virus half-life was ˜8 hours, while those of the improved mutants (Gly119Pro, Gly119Arg, Val39Ile/Gly119Pro, Ala57Ile/Gly119Arg) were ˜24 hours. The Ala57Ile/Gly119Pro mutant exhibited a half-life of ˜40 hours at 37° C. (n=3; error bars represent standard deviation).

Claims

1. A mutant retroviral protease comprising an amino acid substitution at position 119 of SEQ ID NO:1, or a structural and functional homolog thereof, wherein said mutant retroviral protease increases the stability of a retrovirus.

2. A stable mutant virus expressing the mutant retroviral protease of claim 1.

3. A method for generating a stable mutant retrovirus comprising generating a mutant library of a parental retrovirus and selecting for a stable mutant virus comprising a mutant protease thereby generating a stable mutant retrovirus.

4. A stable mutant retrovirus generated by the method of claim 3.

5. A method for increasing viral production, titer and infection efficiency of a retrovirus comprising expressing a mutant retroviral protease of claim 1 in a retrovirus thereby increasing viral production, titer and infection efficiency.