Klaus Strebel, Ph.D.

Recent Progress

Mannose receptor inhibits virus release from HIV-infected macrophages

Human mannose receptor 1 (MRC1) is expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. MRC1was reported to serve as entry receptor for invading pathogens such as bacteria, fungi, viruses (incl. HIV-1), and other parasites. Interestingly, the MRC1-mediated uptake of HIV-1 by macrophages does not lead to productive infection but results in the processing in MHC-II-containing compartments for subsequent antigen presentation. Thus, MRC1 constitutes part of a cellular innate host defense mechanism.

It was reported that productive HIV-1 infection of macrophages results in the downmodulation of MRC1. However, nothing was known about the effect of MRC1 on virus replication. Our overall interest in the HIV-induced modulation of cell surface markers such as CD4 or BST-2 and their functional consequences for virus replication prompted us to examine the functional correlation between MRC1 expression on primary human macrophages (MDM) and productive HIV-1 replication.

We found that silencing of MRC1 in MDM resulted in enhanced virus production similar to what was previously observed in response to the HIV-1 Vpu-induced antagonism of BST-2. Conversely, overexpression of MRC1 following transient transfection of HEK293T cells resulted in significant inhibition of particle release (Figure 1). Virions produced in the presence of MRC1 accumulated in clusters at the cell surface. These virions, when detached from the cells, were fully infectious. Despite its phenotypical similarity to BST-2-mediated restriction of virus release, MRC1-dependent inhibition of virus production was BST-2 independent because: (1) it was observed in HEK293T cells, which are inherently BST-2-negative; and (2) the presence of Vpu had no impact on MRC1-dependent inhibition of virus release. Surprisingly, deletion of Env did not alleviate the MRC1-imposed inhibition of virus release, suggesting the involvement of additional cellular factor(s) in the process (Figure 2). Overall, our data suggest that MRC1 represents an antiviral factor affecting virus release in a BST-2-like but mechanistically distinct manner.

Fig. 1: Expression of hMRC1 results in the accumulation of clusters of viral particles at the cell surface. HEK293T cells were transfected with pAD8 together with empty vector (left) or pCMV6-hMRC1 (right) and cultured for 32 hr. Cells were then processed for transmission EM.

Credit: NIAID

Fig. 2: Retention of virus particles by BST-2 (left) and hMRC1 (right). BST-2 tethers virions via its two membrane anchors (red). hMRC1 retains viral particles via its carbohydrate recognition domains (blue) that attach to carbohydrate moieties (yellow) on virus-associated glycoproteins (e. g. HIV-1 Env).

Credit: NIAID

Publications arising from this work

Sukegawa S., Miyagi E., Bouamr F., Farkasova H., and Strebel K. Mannose Receptor 1 restricts HIV virus release from infected macrophages. Cell Reports 22(3):786-795 (2018).

HIV-1 Tat induces transcriptional silencing of the mrc1 promoter.

Some pathogens, including HIV-1, have evolved to antagonize the innate immune function of MRC1 by downregulating mannose receptor from the cell surface. In the case of HIV-1, the mechanism of MRC1 downmodulation is still under debate. Nef, Vpr, and Tat were all implicated in this effect. Nef was reported to induce cell surface downmodulation of MRC1 without affecting its steady-state levels while Vpr was reported to induce degradation of MRC1. Finally, HIV-1 Tat was reported to inhibit transcription from the rat MRC1 promoter, a finding that was not confirmed by others. Thus, the precise mechanism of HIV-induced down-modulation of MRC1 from the surface of productively infected macrophages remained unclear.

Our own experiments suggest inhibition of MRC1 at the transcriptional level. Indeed, individual deletion of any of the viral accessory proteins did not interfere with the HIV-induced downmodulation of MRC1. To directly test the impact of Tat on MRC1 expression, we cloned a 150 bp fragment of the human MRC1 promoter into the promoter trap vector pGL3 (pGL-hMRC1-Luc; Figure 3A). Unexpectedly, transfection of pGL-hMRC1-Luc together with Tat neither activated nor inhibited luciferase expression (not shown). Inspection of the MRC1 promoter sequence revealed the presence of several transcription factor binding sites, including two copies of a putative binding site for the myeloid-specific transcription factor PU.1. Indeed, when co-transfecting PU.1 vector with pGL-hMRC1-Luc, we observed a dose-dependent increase in luciferase activity (Figure 3B). Of note, when increasing amounts of Tat were transfected together with constant amounts of PU.1, we observed a dose-dependent inhibition of luciferase activity (Figure 3C). Thus, HIV-1 Tat acts as a PU.1-dependent transcriptional inhibitor of the MRC1 promoter. The same result was obtained when full-length proviral vectors were tested. The inhibitory effect of Tat on MRC1 promoter activity is contrasted by its well-known activating effect on the HIV-1 LTR promoter. The HIV-1 LTR does not have any classical PU.1 binding sites.

Fig. 3: Tat inhibits the PU.1 dependent activation of the MRC1 promoter. (A) Schematic of the test vector. (B) PU.1 activates the MRC1 promoter in a dose-dependent manner. (C) Expression of various forms of Tat inhibit the PU.1 activated MRC1 promoter. Tat-86 and Tat-101 are two forms of AD8 Tat. pNL-A1 expresses NL4-3 Tat.

Credit: NIAID

We nevertheless tested the effect of PU.1 on the transcriptional activity of the HIV-1 LTR promoter. For that purpose, a roughly 600 bp fragment from the NL4-3 5’-LTR was cloned into the pGL3-Luc vector (Figure 4A). As expected, the LTR-Luc vector had significant basal activity, which was increased by more than 10-fold in the presence of Tat. Intriguingly, co-expression of PU.1 induced a dose-dependent inhibition of the LTR-mediated luciferase expression both in the absence and presence of Tat (Figure 4B). Thus, PU.1 behaved as a transcriptional activator of the MRC1 promoter but acted as a transcriptional inhibitor of the HIV-1 LTR promoter in a Tat independent manner. Importantly, siRNA-mediated silencing of PU.1 in HIV-infected MDM resulted in increased HIV-1 gene expression, indicating that the inhibitory effect of PU.1 on HIV-1 transcription can be observed in HIV-infected primary human macrophages and is therefore functionally relevant. We propose a model of a regulatory circuit, in which PU.1 inhibits the HIV-1 LTR resulting in reduced expression of Tat (Fig. 5:c). However, Tat can increase its own expression by activating the HIV LTR (Fig. 5:d). Increased Tat levels result in reduced levels of PU.1 (Fig. 5:e), which antagonizes its effect on the HIV LTR (Fig. 5:c) and at the same time reduces MRC1 promoter activity (Fig. 5:b). Reduced MRC1 levels then lead to increased viral particle release (Fig. 5:a). Thus, HIV-1 Tat, through its effect on PU.1 increases HIV-1 gene expression and at the same time facilitates the release of viral particles from infected macrophages via inhibition of MRC1 expression.

Fig. 4: PU.1 inhibits the HIV-1 LTR. (A) Diagram of the indicator plasmid used. (B) PU.1 inhibits LTR promoter activity +/- Tat in a dose-dependent manner.

Credit: NIAID

Publications arising from this work

Kao et al 2022 (in press).

Proposed Course of Work

We will continue our work on PU.1 and MRC1 expression. We have mutated or deleted the two putative PU.1 binding sites on the MRC1 promoter element either alone or in combination. Preliminary data indicate that the integrity of one of the two binding sites is sufficient for PU.1 responsiveness. We will perform electrophoretic mobility shift assays (EMSA) to determine binding of PU.1 and Tat to the various promoter elements. In particular, we will investigate if PU.1 binding to the HIV-1 LTR is required and if so, try to identify the binding site. We will also transfer PU.1 binding sites into heterologous, PU.1 unresponsive, promoters to determine the minimal sequences required for PU.1 responsiveness. PU.1 has an N-terminal transcriptional activation domain, a central PEST sequence, and a C-terminal DNA binding domain. We will study PU.1 mutants to determine whether the same domains are required for transcriptional activation and inhibition.

Figure 5: PU.1 inhibits transcription from the HIV-1 LTR but activates the MRC1 promoter. PU.1 is a myeloid-specific transcription factor that regulates expression of multiple genes, including mannose receptor (MRC1). PU.1 mediated upregulation (blue arrow) of MRC1 [a] (see Fig. 3B) accounts for the previously observed inhibition of HIV-1 virus release [b]. This effect is counteracted by the HIV-encoded Tat protein that inhibits PU.1 function [c] (see Fig. 3C). Inhibition of PU.1 function by Tat reduces MRC1 expression, which contributes to enhanced virus release. While PU.1 upregulates MRC1 expression, it has an inhibitory effect on HIV-1 LTR promoter activity [d] (see Fig.4B). This leads to reduced Tat protein synthesis [e], which in turn increases the inhibition of HIV-1 LTR transcription by PU.1 [d]. However, a positive feedback loop ensures continued Tat synthesis via TAR-dependent activation of HIV-1 transcription [f] and thus control of PU.1 expression. Aside from Tat, HIV-1 Vpr and Nef have been implicated in the regulation of MRC1 [g].

Credit: NIAID