High Attenuation and Immunogenicity of a Simian Immunodeficiency Virus Expressing a Proteolysis-resistant Inhibitor of NF-κB

Abstract

NF-B/I B proteins play a major role in the transcriptional regulation of human immunodeficiency virus, type-1 (HIV-1). In the case of simian immunodeficiency virus (SIV) the cellular factors required for the viral transcriptional activation and replication in vivo remain undefined. Here, we demonstrate that the p50/p65 NF-B transcription factors enhanced the Tat-mediated transcriptional activation of SIVmac239. In addition, I B-␣S32/36A, a proteolysis-resistant inhibitor of NF-B, strongly inhibited the Tat-mediated transactivation of SIVmac239. Based on this evidence, we have generated a self-regulatory virus by endowing the genome of SIVmac239 with I B-␣S32/36A; the resulting virus, SIVI B-␣S32/36A, was nef-deleted and expressed the NF-B inhibitor. We show that SIVI B-␣S32/36A was highly and stably attenuated both in cell cultures and in vivo in rhesus macaque as compared with a nef-deleted control virus. Moreover, the high attenuation was associated with a robust immune response as measured by SIVspecific antibody production, tetramer, and intracellular IFN-␥ staining of SIV gag-specific T cells. These results underscore the crucial role of NF-B/I B proteins in the regulation of SIV replication both in cell cultures and in monkeys. Thus, inhibitors of NF-B could efficiently counteract the SIV/HIV replication in vivo and may assist in developing novel approaches for AIDS vaccine and therapy.

Inhibition of HIV-1 replication in primary human monocytes by the IκB-αS32/36A repressor of NF-κB

“…2), indicating that in the setting of HIV infection of primary monocytes NF-κB plays a non-redundant role. These results are in agreement with the evidence that IκB-αS32/36A negatively affected the replication of HIV and SIV in PBMC cultures and in monkeys [16,17]. …”

“…These findings supported an interpretation that in these cellular contexts NF-κB is required for efficient viral replication. We also showed that a recombinant SIV which expressed IκB-αS32/36A inhibitor was also highly replication attenuated in vivo in rhesus macaque [17]. Here, we have extended our analysis of IκB-αS32/36A function in HIV-1 replication to primary monocytes.…”

“…IκB-αS32/36A acts as a potent inhibitor of the NF-κB-dependent gene transcription, including those from the HIV-1 genome [15]. To verify the requirement of NF-κB in the replication of HIV-1 in primary cells, we previously designed HIV-1 and SIV molecular clones containing the IκB-αS32/36A cDNA positioned into the nef region of the respective viral genome [16,17]. We found that these recombinant viruses were highly attenuated for replication in T cell lines as well as in human and simian PHA-activated peripheral blood mononuclear cells, PBMCs [16,17].…”

“…To verify the requirement of NF-κB in the replication of HIV-1 in primary cells, we previously designed HIV-1 and SIV molecular clones containing the IκB-αS32/36A cDNA positioned into the nef region of the respective viral genome [16,17]. We found that these recombinant viruses were highly attenuated for replication in T cell lines as well as in human and simian PHA-activated peripheral blood mononuclear cells, PBMCs [16,17]. These findings supported an interpretation that in these cellular contexts NF-κB is required for efficient viral replication.…”

IκB-α Represses the Transcriptional Activity of the HIV-1 Tat Transactivator by Promoting Its Nuclear Export

“…To generate p3XFLAG-CMV-Tat, p3XFLAG-CMV-Tat C (22,25,27)A, and p3XFLAG-CMV-Tat R(49 -57)A, the sequence of Tat was amplified from the pGEX-2T-Tat expressing vectors (43) and ligated to EcoRI/XbaI-digested p3XFLAG-CMV-7.1 (Sigma). pRc/CMV-HA-I B-␣S32/36A was previously described (44). The plasmids expressing the I B-␣ mutants 120 -317, 1-280, 1-269, 1-242, 72-287, and 72-269 were generated by PCRmediated amplification of the I B-␣ sequence from pCMV4-HA-I B-␣ with appropriate forward and reverse primers followed by ligation to the HindIII/XbaI-digested pCMV4-HA.…”