Construction of a doxycycline-dependent simian immunodeficiency virus reveals a nontranscriptional function of tat in viral replication

Abstract

In the quest for an effective vaccine against human immunodeficiency virus (HIV), live attenuated virus vaccines have proven to be very effective in the experimental model system of simian immunodeficiency virus (SIV) in macaques. However, live attenuated HIV vaccines are considered unsafe for use in humans because the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. As an alternative approach, we earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (DOX). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient DOX administration. Since the effectiveness and safety of such a conditionally live AIDS vaccine should be tested in macaques, we constructed a similar DOX-dependent SIVmac239 variant in which the Tat-TAR (trans-acting responsive) transcription control mechanism was functionally replaced by the DOX-inducible Tet-On regulatory mechanism. Moreover, this virus can be used as a tool in SIV biology studies and vaccine research because both the level and duration of replication can be controlled by DOX administration. Unexpectedly, the new SIV variant required a wild-type Tat protein for replication, although gene expression was fully controlled by the incorporated Tet-On system. This result suggests that Tat has a second function in SIV replication in addition to its role in the activation of transcription.

FIG. 1.

Design of the DOX-inducible SIV-rtTA variant. For the construction of a conditionally live SIVmac239 variant, we inactivated the Tat-TAR regulatory mechanism through mutation of TAR (TAR^m; loop and bulge mutations as shown in Fig. 2A) and Tat (Y55A substitution; Fig. 3B) and introduced the Tet-On regulatory mechanism through the insertion of two tetO elements in the U3 promoter region (Fig. 3A and 4C) and the rtTA gene at the site of the nef gene (Fig. 4). In addition, we mutated the original AUG^Nef translation start codon and three downstream AUG codons that precede the new AUG^rtTA start codon on the spliced Nef transcript (AUG^m; Fig. 4B).

FIG. 2.

Inactivation of TAR. (A) The 5′ end of the nascent SIV transcript folds the TAR hairpin structure with three stem-loop regions (SL1 to SL3). Binding of Tat to TAR enhances transcription from the LTR promoter. To abolish Tat responsiveness, we mutated the bulge and loop sequences in SL1 and SL2. (B) Binding of SIV Tat to wild-type (TAR^wt) and mutant (TAR^m) TAR RNA was analyzed in an EMSA. TAR RNA was incubated with 0, 4, or 40 ng Tat protein and analyzed on a nondenaturing gel. The positions of unbound TAR RNA and the TAR-Tat complex are indicated.

FIG. 3.

Expression from the SIV-rtTA LTR promoter is controlled by DOX. (A) To turn the SIV LTR into a DOX-inducible promoter, we mutated TAR and introduced two tetO elements between the NF-κB and Sp1 binding sites. This arrangement had previously been shown to be effective in the DOX-dependent HIV-rtTA variant. (B) Mutation of Tat. The SIVmac239 Tat protein consists of 130 amino acids and has a modular structure similar to that of the HIV-1 Tat protein, which consists of 86 to 101 amino acids (depending on the viral isolate). Both proteins have a transcription activation domain that can be subdivided in an N-terminal acidic domain, a cysteine-rich domain, a central core domain, and an RNA-binding domain consisting of a stretch of positively charged amino acids and therefore termed the basic domain. For the construction of HIV-rtTA, we previously inactivated HIV-1 Tat through the introduction of a tyrosine-to-alanine substitution at position 26 (Y26A) in the cysteine-rich domain. This cysteine-rich domain is highly conserved, and the tyrosine at position 26 in HIV-1 Tat corresponds to the tyrosine at position 55 in SIVmac239 Tat. We therefore introduced the Y55A mutation in the SIVmac239 Tat open reading frame. This mutation did not affect any other gene or known underlying sequence element. (C) C33A cells were transfected with a plasmid in which the expression of firefly luciferase was controlled by the LTR promoter of either SIV-rtTA, SIV-rtTA-TAR^wt, or HIV-rtTA and an rtTA-expressing plasmid. Transfected cells were cultured with 0 to 1,000 ng/ml DOX. (D to F) Cells were transfected with a plasmid in which the expression of firefly luciferase was controlled by the LTR promoter of either SIV-rtTA, SIV-rtTA-TAR^wt, or HIV-1 and 0 to 50 ng of a plasmid expressing wild-type SIV Tat (D), HIV-1 Tat (E), or Y55A-mutated SIV Tat (F). The intracellular luciferase level, which reflects promoter activity, was measured at 2 days after transfection. The error bars represent the standard deviations for two to five experiments (n.d., not determined).

FIG. 4.

Insertion of the rtTA gene and deletion of U3 sequences. In SIVmac239, the 5′ end of the nef open reading frame overlaps the env gene, while the 3′ end overlaps the U-box (U), the PPT, and the U3 region of the LTR promoter (including the attachment sequence required for integration; att). The SIV-rtTA modifications are shown schematically (A) and in detail (B and C). In SIV-rtTA, we replaced the nef sequences downstream of the env gene and upstream of the U-box with the rtTA gene. In addition, we mutated the original AUG^Nef translation start codon and three downstream AUG codons (AUG^II-IV) that precede the new AUG^rtTA start codon on the spliced Nef transcript (AUG codons are underlined in panel B). The four AUG mutations (boxed in gray in panel B) were chosen to be synonymous in the env open reading frame and thus not to affect the Env protein. The SIV-rtTA-Env^wt variant does not carry these four AUG mutations and will produce a Nef-rtTA fusion protein in which 60 Nef amino acids are fused to the N terminus of the rtTA protein. In the SIV-rtTA-Env^wt-stop variant, a translation stop codon was introduced between these Env and rtTA sequences (translation stop codons are indicated with *; mutations are boxed in gray). Accordingly, translation starting at AUG^Nef will result in the production of a 57-amino-acid Nef polypeptide, while the reinitiation of translation at the AUG^rtTA will result in the production of rtTA. In SIV-rtTA-TAR^wt, the wild-type TAR sequence was reintroduced. In the SIV-rtTA-ΔU3 variants, the Nef-U3 sequences present between the att sequence and either the NF-κB site (SIV-rtTA-ΔU3-380) or the tetO elements (SIV-rtTA-ΔU3-405) were deleted. The numbering is according to the SIVmac239 proviral genome sequence in GenBank/EMBL (accession number M33262; gi 334647).

FIG. 5.

Replication of SIV-rtTA variants. PM1 T cells were transfected with plasmids encoding the SIV-rtTA variants SIVmac239 and HIV-rtTA. Cells were cultured in the presence (1 μg/ml) and absence of DOX, and viral replication was monitored by measuring the RT level in the culture supernatant. Similar DOX-dependent replication of SIV-rtTA-Tat^wt and lack of replication of the other SIV-rtTA variants was observed in independent experiments.

FIG. 6.

SIV-rtTA-Tat^wt gene expression is controlled by DOX. C33A cells were transfected with the SIV-rtTA (Tat^m), SIV-rtTA-Tat^wt, or SIVmac239 molecular clone and cultured without and with 1 μg/ml DOX for 2 days. Virus production was monitored by measuring the RT (right) and CA-p27 (left) levels in the culture supernatant. The error bars represent the standard deviations from two experiments (n.d., not determined).