Construction and testing of orfA +/- FIV reporter viruses

Abstract

Single cycle reporter viruses that preserve the majority of the HIV-1 genome, long terminal repeat-promoted transcription and Rev-dependent structural protein expression are useful for investigating the viral life cycle. Reporter viruses that encode the viral proteins in cis in this way have been lacking for feline immunodeficiency virus (FIV), where the field has used genetically minimized transfer vectors with viral proteins supplied in trans. Here we report construction and use of a panel of single cycle FIV reporter viruses that express fluorescent protein markers. The viruses can be produced to high titer using human cell transfection and can transduce diverse target cells. To illustrate utility, we tested versions that are (+) and (-) for OrfA, an FIV accessory protein required for replication in primary lymphocytes and previously implicated in down-regulation of the primary FIV entry receptor CD134. We observed CD134 down-regulation after infection with or without OrfA, and equivalent virion production as well. These results suggest a role for FIV proteins besides Env or OrfA in CD134 down-regulation.

Keywords: FIV, lentivirus, reporter virus, OrfA.

Figure 1

Virus design and testing of biological activity of restored OrfA in replicating FIV. (A) Virus genome arrangements. U3: 3’ unique element. U5: 5’ unique element. R: repeat element. PPT: polypurine tract. 2A: porcine teschovirus 2A peptide. CMV: human cytomegalovirus immediate early gene promoter. efs: envelope frame shift (black arrowhead). CT5efs has a frameshift in env, which was used as the insertion point for the 2A peptide as well. Env*: N-terminal Env protein fragment resulting from the frame shift. Open arrowheads indicate the GFP and mCherry stop codons (the env mRNA fragment distal to the stop codons is untranslated). (B) Immunoblotting demonstrates that the P2A peptide results in co-translational cleavage and generation of free GPF (left) and mCherry (right). Lane 1: cells expressing eGFP or mCherry; Lane 2: untransfected negative control; Lanes 3 and 4: reporter viruses. 293T cell lysates were harvested 48 hours after transfection and blotted with antibodies to GFP or mCherry. (C-F) Replication of viruses produced from CT5 and CT5^A+ in primary feline PBMC, feline T-cell lines Mya-1 and FetJ, and CrFK cells. Cells were not induced with any additional agents, such as soluble CD134 [34] with the exception that Mya-1 cells were maintained with human IL-2 as described in Section 3. Error bars represent standard deviation of duplicate measurements.

Figure 2

Virus production. Supernatant reverse transcriptase activities of the different reporter viruses. For vector GiNSIN, the source of RT activity is the packaging plasmid pFP93 used to trans-package the transfer vector [6,40]. Background RT activities determined in supernatants of un-transfected control cells, which were between 0.1 - 1 % of transfected cell supernatant values, were subtracted before graphing. Error bars represent standard deviation of duplicate measurements.

Figure 3

Reporter virus titers on (A) CrFK; (B) G355-5; and(C) FetJ cells. Viruses and the GiNSIN vector were pseudotyped here and in following experiments with vesicular stomatitis virus glycoprotein G (VSV-G). Error bars represent standard deviations of duplicate measurements.

Figure 4

Testing of OrfA (+) and (-) reporter virus. (A) CrFK-CD134 cells were derived as described in the Experimental Section and the stable cell line was infected with increasing amounts of each CT5cherry or CT5-cherry^A+ reporter virus. Lentiviral vector TsinCherry [25], which expresses mCherry under control of the hCMV promoter but encodes no viral proteins, was used as a control. Flow cytometry for CD134 surface expression was performed at 72 hours. (B) 30,000 CrFK-CD134 cells were infected with CT5efs and CTefs^A+ viruses at equivalent MOI (0.5 and 2.6). MOI was calculated by pre-titration on CrFK-CD134 cells with the focal infectivity assay of Remington et al. [50]. Flow cytometry for CD134 surface expression was performed at 72 hours. Error bars represent standard deviations of duplicate measurements. (C) Immunoblotting for FIV Gag/Pol (top blot) and tubulin (bottom blot) in lysates of the cells shown in panel B (MOI = 2.6). Numbers indicate MW markers (kDa).