Dual-color HIV reporter system for the detection of latently-infected cells
The present disclosure provides for recombinant nucleic acids, and cells and virions comprising the recombinant nucleic acids, that can be used to identify, isolate, and/or purify cells latently infected with immunodeficiency virus. A subject recombinant nucleic acid includes (a) a first nucleotide sequence encoding a first reporter polypeptide that produces a first detectable signal, where the first nucleotide sequence is operably linked to an immunodeficiency virus promoter and is translated as an early gene; and (b) a second nucleotide sequence encoding a second reporter polypeptide that produces a second detectable signal that is distinguishable from the first detectable signal, where the second nucleotide sequence is operably linked to a non-immunodeficiency virus promoter. In some aspects, the first and second nucleotide sequences are both positioned between a shared 5′ long terminal repeat (LTR) and a shared 3′ LTR. Also provided are related methods.
1. A recombinant nucleic acid, comprising:
(a) a first nucleotide sequence encoding a first reporter polypeptide that produces a first detectable signal, wherein the first nucleotide sequence is operably linked to an immunodeficiency virus promoter and is translated as an early gene; and
(b) a second nucleotide sequence encoding a second reporter polypeptide that produces a second detectable signal that is distinguishable from the first detectable signal, wherein the second nucleotide sequence is operably linked to a non-immunodeficiency virus promoter,
wherein the first and second nucleotide sequences are both positioned between a shared 5′ long terminal repeat (LTR) and a shared 3′ LTR.
2. The recombinant nucleic acid according to claim 1 , wherein the recombinant nucleic acid encodes a transcription-competent immunodeficiency virus and further comprises:
a functional transactivation response element (TAR); and
a third nucleotide sequence, wherein the third nucleotide sequence encodes a functional transactivator protein (Tat) and is positioned between the shared 5′ long terminal repeat (LTR) and the shared 3′ LTR.
3. The recombinant nucleic acid according to claim 2 , wherein the immunodeficiency virus is a human immunodeficiency virus (HIV).
4. The recombinant nucleic acid according to claim 3 , wherein the HIV is HIV-1 or HIV-2.
5. The recombinant nucleic acid according to claim 1 , wherein the first nucleotide sequence is inserted into the locus of an early gene.
6. The recombinant nucleic acid according to claim 5 , wherein the early gene is selected from: nef, tat, and rev.
7. The recombinant nucleic acid according to claim 6 , wherein the early gene is nef.
8. The recombinant nucleic acid according to claim 5 , wherein the early gene is the early gene that is most proximal to the 3′ LTR.
9. The recombinant nucleic acid according to claim 5 , wherein the first nucleotide sequence is inserted in a manner such that the protein encoded by the early gene is not functional or is not produced.
10. The recombinant nucleic acid according to claim 5 , wherein the first nucleotide sequence is inserted in a manner such that a protein encoded by the early gene is a fusion protein comprising the protein encoded by the early gene and the first reporter polypeptide.
11. The recombinant nucleic acid of claim 1 , wherein at least one of the first and second reporter polypeptides is a fluorescent protein.
12. The recombinant nucleic acid of claim 11 , wherein one of the first and second reporter polypeptides is a red fluorescent protein, and the other is a green fluorescent protein.
13. The recombinant nucleic acid of claim 1 , wherein the non-immunodeficiency virus promoter is selected from a group consisting of: an EF1α promoter, a Spleen Focus Forming Virus promoter, a ubiquitin promoter, and a cytomegalovirus (CMV) promoter.
14. The recombinant nucleic acid of claim 1 , wherein the 5′ LTR comprises the immunodeficiency virus promoter.
15. A virion comprising a recombinant nucleic acid according to claim 1 .