HIV immune activation and morphogenesis

From left to right: Julia Nehls, Kristin Höhne, Karen Bayer, Marcos Gondim, Ute Finkel, Michael Schindler, Herwig Koppensteiner, Stephan Hofmann

The scope of our group is to investigate mechanisms involved in the cellular dysregulation and thereby pathogenesis of HIV-1 and HCV infections. We are mainly interested in three complementary topics:

Generalized immune activation and AIDS progression

HIV-1 has evolved sophisticated mechanism to achieve optimal propagation and to evade the hosts’ innate and adaptive immune response. For one, HIV induces unspecific immune activation, which allows productive gene expression and viral protein production. On the other hand it blocks host cell restriction factors of the innate immunity and subverts multiple mechanisms of the adaptive immune response.

Our lab has a long standing track record in analyzing the manipulation of infected T cells and macrophages by HIV-1 Nef and Vpu (e.g. Kühl et al., 2011; Schindler et al., 2010; Sauter et al., 2009; Schindler et al., 2008; Schindler et al., 2007). However, not only Nef and Vpu, but also the other accessory protein Vpr and the regulatory proteins Tat and Rev contribute to immune evasion and generalized immune activation. Thus, we expanded our research to Vpr and Tat and will also analyze immune-regulatory functions of the Rev protein in cooperation with the group of Prof. Dr. Ruth Brack-Werner who is an expert in Rev biology.

Figure 1: HIV-1 Vpr mediates PARP translocation.

Hela cells were transfected with Vpr-YFP and PARP-CFP expression plasmids. The nucleus was stained with DRAQ5. Translocation of PARP by Vpr could boost HIV LTR transactivation and NFAT (nuclear factor of activated T cells) activity. Samples were embedded and confocal images taken with a Zeiss LSM510.

Imaging HIV-1 and HCV morphogenesis

The role of HIV-containing compartments in macrophages

Macrophages play an important role in HIV-1 transmission, pathogenesis and the establishment of latent reservoirs. Interestingly, the replication cycle of HIV-1 in macrophages differs substantially from that in T cells and it was reported that HIV is found in so called virus containing compartments (VCC) within macrophages. However, the tempo-spatial order of VCC formation and the biological impact of HIV-1 within VCCs was unclear. We generated GFP-tagged HIV-1 that is able to replicate in primary macrophages and monitored the formation of VCCs by time lapse microscopy. These experiments revealed that VCCs are formed within macrophages, are membrane enclosed and contain mature HIV-1 particles. Most importantly we could show that HIV-1 virions within macrophage internal compartments were protected from neutralizing antibodies and could be transferred to adjacent T cells. Thus, storage of HIV-1 in macrophage internal compartments represents an immune evasion and is an obstacle for the elimination or control of HIV-1 by the immune system (Koppensteiner et al., 2012).

Actually, we aim to elucidate the molecular constituents of VCCs and the mechanism of VCC formation. The long-term goal is to find countermeasures against HIV replication and persistence in the macrophage reservoir.

Figure 2: HIV-1 within VCCs is protected from neutralizing antibodies. Macrophages infected with HIV-1 expressing CFP-tagged Gag were either fixed and permeabilized or left untreated and incubated at 37 °C for one hour with anti-CD81, or with a broadly neutralizing antibody targeting the viral gp120. In non-permeabilized macrophages the neutralizing gp120 antibody could not enter VCCs (Koppensteiner et al., 2012).

HCV assembly and egress

In order to study assembly and release of HCV in living cells, we generated fluorescently labelled HCV genomes carrying chromophores in structural as well as non-structural genes. Currently, we extensively characterize these viruses by virological techniques and state-of-the-art combinations of live cell imaging, confocal microscopy and electron microscopy. The goal is to elucidate the as-yet unknown pathway(s) of HCV egress.

Figure 3: Localization of HCV E1, NS5A and Core in HCV expressing Huh7.5 liver cells. Huh7.5 cells were electroporated with HCV that expresses mCherry within E1 and GFP within NS5A. Cells were fixed 72 hours post electroporation, permeabilized and immunostained for HCV Core. Arrows show viral protein accumulation at the surface of lipid droplets. Confocal microscopy was performed with a Zeiss LSM510. Scale bars indicate a distance of 5 µm.

Identification and inhibition of viral protein interactions

HIV and HCV manipulate the infected host cell to allow viral replication and immune evasion with only 10 (HCV) to 15 (HIV-1) viral proteins being expressed. We established a new technique to quantify Foersters resonance energy transfer (FRET) by FACS and which allows us to investigate viral protein interactions at medium- to high-throughput (Banning et al., 2010). FACS-FRET is non-invasive, works in any compartment of living mammalian cells and can be combined with fluorescence microscopy to assess the localization of an interaction. Currently, we generate the intra-protein networks of HIV-1 and HCV by testing all viral proteins for potential interactions against each other. Furthermore, we exploit FACS-FRET high throughput screening in order to identify novel protein interactions of HIV-1 and HCV and set up novel FRET-based screening methods to find compounds for their inhibition. In sum, the major goal of this project is to better understand the basic mechanisms of how HIV-1 and HCV manipulate target host cells and to offer new therapeutic targets and drugs for intervention.

Experimental layout for FRET-based high throughput screening via FACS (Banning et al., 2010)