Research Group of T. Gramberg


Head of Institute:
Prof. Dr. med. Klaus Überla

Innate and Intrinsic Immunity in Retroviral Infection

Over the last couple of years the importance of cell intrinsic immune mechanisms in fighting viral infections became more and more evident. A key to efficient viral replication within a host cell is to circumvent these antiviral mechanisms. This includes avoiding being sensed by the cell as a pathogen (“non-self”) and to counteract or evade intrinsic antiviral mechanisms within the host cell. To counteract intrinsic immunity, lentiviruses like HIV-1, have evolved specialized accessory proteins that are essential for viral replication in vivo and have been shown to counteract innate immune mechanisms. The major focus of our research is to understand how innate immune mechanisms restrict retroviral replication and analyzing the role of lentiviral accessory proteins in counteracting cellular defense mechanisms. In addition, we are also interested in understanding how retroviral infections are recognized by different host cells, like macrophages or dendritic cells. In addition to exogenous retroviruses, we are also including endogenous retroviruses and retroelements in our studies to analyze the role of restriction factors, like SAMHD1, or innate sensing molecules on the replication of mobile genetic elements. Comparing the replication of exogenous viruses with their endogenous “cousins” in light of intrinsic restriction and immune sensing will further contribute to a better understanding of antiviral innate immune mechanisms, auto immunity, and genome stability of the host.

The antiretroviral restriction factor SAMHD1

Viruses of the HIV-2/SIVsm lineage encode in addition to Vpr the closely related accessory protein Vpx. Vpx is not required for virus replication in T cells but facilitates the infection of myeloid target cells, like macrophages and dendritic cells. In these cells, Vpx has been shown to neutralize the antiviral activity of the restriction factor SAMHD1. Mutations in SAMHD1 are known to cause the Aicardi-Goutieres syndrome (AGS) in humans. AGS is a hereditary autoimmune disease caused by an immune response to accumulating intracellular nucleic acids. SAMHD1 has been shown to act as a dNTP triphosphohydrolase and it has been suggested that SAMHD1 inhibits retroviral reverse transcription by depleting the intracellular dNTP pool. We were part of a study showing for the first time that SAMHD1 restricts infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs) in primary cells, thereby lowering their concentrations below those required for the viral DNA synthesis. In addition, we also contributed to the recent finding that SAMHD1 is not only active in myeloid cells, but also in resting CD4+ T cells. It is well known that resting CD4+ T cells are highly resistant to productive HIV-1 infection. Our work could demonstrate that SAMHD1 is highly expressed and active in resting T cells, thereby contributing to the restriction of HIV in these important immune cells. Recently, we were able to characterize the antiviral range of the SAMHD1-mediated restriction. We found that not only HIV and SIV are blocked, but many diverse retroviruses belonging to the alpha-, beta-, and gamma-retrovirus genera are affected by the SAMHD1-mediated block to reverse transcription. 

Analyzing the function of SAMHD1 in vivo using knockout mice

To determine the role of SAMHD1 in vivo, we analyzed SAMHD1 knockout mice. In these mice we found that also mouse SAMHD1 reduces cellular dNTP concentrations and restricts retroviral replication in lymphocytes, macrophages, and dendritic cells in vitro and in vivo. Importantly, the absence of SAMHD1 triggered an IFN-dependent transcriptional upregulation of type I IFN-inducible genes in various cell types. A finding that is indicative of a spontaneous IFN production in SAMHD1 knockout mice. Therefore, SAMHD1-deficient mice may be serving as model system to identify the mechanisms that trigger pathogenic type I IFN responses and to analyze the mechanism how SAMHD1 restricts retroviral infection.

To better characterize the antiviral activity of SAMHD1, we employed our knockout mouse model to analyze the mechanism and the regulation of SAMHD1 in greater detail. We found that, similar to the human protein, the antiviral activity of murine SAMHD1 is also regulated by phosphorylation. This phosphorylation takes place at a threonine residue with a CDK1 binding site, is cell-cycle-dependent, and correlates negatively with the antiviral activity of SAMHD1. Since the mechanism of SAMHD1 restriction is controversially discussed, we analyzed the influence of the dNTP hydrolase activity and the potential RNase activity of SAMHD1 on retroviral infection in murine cells. We found that HIV and MLV infection is blocked at the level of reverse transcription but did not observe an effect of SAMHD1 on incoming viral RNA. This finding suggests that the recently suggested RNase activity of SAMHD1 is less important in the mouse.

The proposed mechanisms of the SAMHD1-mediated HIV-1 restriction.
Upon entry of HIV-1 into myeloid cells or resting CD4+ T cells, SAMHD1 blocks viral replication early during the viral lifecycle at or prior to reverse transcription. The dNTP hydrolase activity of SAMHD1 depletes the intracellular dNTP pool below the level required for reverse transcription. Alternatively, SAMHD1 may directly target incoming viral genomic RNA for degradation. In cycling cells, the antiviral activity of SAMHD1 is inactivated by phosphorylation at threonine 592 by CDK1/2 and Cyclin A2. The phosphorylation at T592 is lost in resting cells, activating the SAMHD1-mediated block to infection in a cell-cycle dependent manner.

The antiretroviral activity of members of the TRIM protein family

Another cellular restriction to retroviral infection is mediated by TRIM5α, one of over 70 members of the tripartite motif (TRIM) protein family. It mediates intracellular immunity against a variety of retroviruses in a species-specific manner. Of particular interest, TRIM5α of rhesus macaques potently blocks HIV-1 infection, whereas the human protein is not active against HIV-1. The antiviral function of many closely related TRIM proteins is still ill-described. It has been shown that the expression of many TRIM proteins can be upregulated by type I interferon, which hints towards a possible function as antiviral protein. We are therefore interested in discovering and characterizing the antiretroviral properties of members of the TRIM family.

One TRIM protein we are interested in is TRIM19 (PML). Most PML isoforms are nuclear and in the nucleus they are forming structures called nuclear bodies (NB), or nuclear domains 10 (ND10). NBs are dynamic, spherical, macromolecular structures that represent accumulations of multiple proteins in distinct nuclear foci. We found that the shRNA-mediated knockdown of PML in human foreskin fibroblast (HFF) but not in 293T cells enhances HIV-1 infection up to 4-fold, indicating that PML restricts HIV-1 infection in a cell type-dependent manner. In addition, we are able to show that the knockdown of the PML-NB-associated proteins hDaxx and SP100, which can act as antiviral proteins themselves, did not influence HIV infection. This indicates that the restriction to HIV infection is PML specific. We are currently characterizing this phenotype in more detail, and this study will shed light upon the question whether PML is able to inhibit retroviral infection. Characterizing PML as a new antiretroviral factor will potentially uncover new mechanistic insights in HIV biology, maybe offering new weak spots in HIV replication that could be targeted by antiretroviral drug development.

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