Antiviral activity of guanylate binding proteins

Viruses modulate numerous cellular pathways and hijack a plethora of host factors to ensure efficient replication and spread. For example, many viral pathogens exploit the cellular serine protease furin for the proteolytic maturation of their envelope glycoproteins. Our lab has recently identified guanylate-binding proteins 2 and 5 (GBP2 and GBP5) as two IFN-inducible host factors that reduce the proteolytic activity of furin (Braun and Hotter et al., 2019). As a result, conversion of the HIV-1 envelope (Env) precursor gp160 into functional mature gp120/gp41 trimers is impaired. Since this process primes HIV-1 Env for membrane fusion, viral particles produced in the presence of GBP2 and GBP5 are only poorly infectious. Similarly, GBP2 and GBP5 also restrict replication of additional furin-dependent viruses, including Zika, measles and highly pathogenic avian influenza A viruses (Fig. 1). In contrast, infectivity of virions carrying the protease-independent glycoprotein of vesicular stomatitis virus is not reduced. Mutational analyses revealed that the antiviral activity of GBP2 and GBP5 requires a C-terminal lipid membrane anchor, but not their GTPase activity. Furthermore, our results show that expression of GBP2 and GBP5 is driven by endogenous retroviral promoters that are activated upon viral infection.

Profilbild von Jun.Prof. Daniel Sauter

Jun.Prof. Daniel Sauter

Group leader

Since furin also cleaves and activates a variety of host cell factors as well as bacterial toxins, its regulation by GBP2 and GBP5 may play an important role beyond viral infections (Braun and Sauter et al., 2019). Indeed, GBP2 and GBP5 also inhibit the proteolytic maturation of cellular proteins, including factors involved in tumor development (Braun and Hotter et al., 2019). To better understand the role of GBP2 and GBP5 in proteolytic protein maturation, we are currently investigating whether these two proteins specifically interfere with the maturation and activity of furin or also target other members of the proprotein convertase family.

Modulation of NF-κB activation by primate lentiviruses

The cellular transcription factor NF-kB regulates the expression of genes involved in inflammation and immunity, including interferon-stimulated genes that protect against viral pathogens. Yet, NF-kB is not only a key mediator of antiviral immune responses but also exploited by many viruses for transcription of viral genes. Replication of HIV, for example, depends on binding of NF-kB p65/p50 heterodimers to the viral LTR promoter.
We have previously shown that primate lentiviruses tightly regulate the activation of this transcription factor throughout their replication cycle. Whereas the early protein Nef of most primate lentiviruses boosts the activation of NF-kB to initiate LTR-dependent viral gene expression, Vpu inhibits the activation of NF‑kB during later stages to limit the induction of an antiviral immune response (Sauter et al., 2015; Heusinger and Kirchhoff, 2017). Transcriptome analyses of HIV-1 infected primary cells revealed that Vpu exerts broad immune-suppressive effects as its ability to inhibit NF-κB activation reduces the expression of pro-inflammatory cytokines, restriction factors and other antiviral genes (Langer, Hammer and Hopfensperger et al., 2019).
Viruses that do not encode a vpu gene may employ alternative strategies to prevent immune activation after NF-kB-dependent initiation of viral gene expression. For example, the Nef proteins of most viruses lacking a vpu gene efficiently down-modulate the T cell receptor CD3 from the cell surface. This Nef function was lost in most vpu-expressing viruses suggesting that the acquisition of Vpu-mediated NF‑kB inhibition may have reduced the selection pressure for suppression of T cell activation by Nef. We have recently identified two viruses, SIVcol and SIVolc, which have evolved yet another strategy, and use their Vpr protein to suppress NF-kB-mediated immune activation (Hotter and Krabbe et al., 2017). Interestingly, these viruses lack a vpu gene and the CD3 down-modulation function of Nef. In a collaborative research project, we also identified HIV-2 Vpx as an inhibitor of NF-κB-driven gene expression (Landsberg et al., 2018). Thus, primate lentiviruses have evolved at least four independent mechanisms to inhibit the expression of cellular NF-kB target genes (Fig. 2).
Since chronic (NF-κB-driven) immune activation is a hallmark of AIDS, our findings will help to better understand the pathophysiology of HIV. Notably, NF-kB also plays also a key role in the reactivation of HIV from latency. Thus, understanding the molecular basis of NF-kB modulation by HIV and related lentiviruses may also lead to novel approaches to reactivate the virus for “kick and kill” cure strategies.

Coevolution of tetherin with enveloped viruses

Tetherin is a cellular restriction factor that inhibits the release of a large variety of human and animal viruses by directly trapping them at the membranes of infected cells. Until recently, only mammalian orthologs of tetherin had been described and the deep evolutionary origins of this antiviral protein had remained obscure.
The antiviral activity of tetherin depends on an unusual topology comprising an N-terminal transmembrane domain, followed by an extracellular coiled-coil region and a C-terminal glycosylphosphatidylinositol (GPI) anchor. One of the two membrane anchors is inserted into assembling virions, while the other remains in the plasma membrane of the infected cell. Thus, tetherin entraps budding viruses by physically bridging viral and cellular membranes. In addition to its ability to restrict virus release, tetherin also acts as an innate sensor inducing NF‑kB-dependent expression of antiviral genes.
To better understand the coevolution of tetherin with primate lentiviruses and other enveloped viruses, we have investigated the evolutionary history of this fascinating protein. Characterizing tetherin orthologs from diverse vertebrate species, we have shown that this antiviral protein emerged more than 450 million years ago (Heusinger et al., 2015) (Fig. 3). Thus, not only mammals, but also reptiles, birds and even fish express this restriction factor. Since efficient tetherin antagonism has been suggested to be a prerequisite for successful spread of HIV-1 in the human population (Sauter, Schindler and Specht et al., 2009; Sauter and Kirchhoff, 2019), we are currently defining the role of tetherin in the cross-species transmission of influenza A viruses and other zoonotic viral pathogens.

Tetherin-driven evolution of HIV

One major research focus of our lab is the coevolution of primate lentiviruses with their respective host species. We are especially interested in the zoonotic transmissions of simian immunodeficiency viruses infecting chimpanzees (SIVcpz) and gorillas (SIVgor) to humans and their subsequent adaptations that contributed to the spread of HIV in the human population. Simian immunodeficiency viruses have been transmitted at least four times independently to humans, giving rise to HIV-1 groups M, N, O and P. Interestingly, only HIV-1 group M strains spread world-wide and are almost entirely responsible for the current AIDS pandemic. In contrast, HIV-1 O is endemic in Western Central Africa, with around 100,000 infected individuals, and groups N and P are very rare with only about 20 and 2 known cases, respectively. In 2009, we identified the antiviral protein tetherin as a key player in the evolution and spread of HIV-1 (Sauter, Schindler and Specht et al., 2009; Sauter and Kirchhoff, 2019). Most simian immunodeficiency viruses – including the direct precursors of HIV-1 – use their accessory protein Nef to counteract tetherin in their respective host species (Fig. 4). Human tetherin, however, is resistant against SIV Nef due to a protective deletion in its cytoplasmic tail and thus poses a significant barrier for successful zoonotic transmission of SIV to humans (Sauter et al., 2010; Sauter, Schindler and Specht et al., 2009).

Interestingly, the four groups of HIV-1 (M, N, O and P) have evolved different mechanisms to overcome this hurdle: Whereas pandemic HIV‑1 group M viruses switched from Nef to Vpu to counteract human tetherin, rare group P and N Vpus do not or only poorly antagonize this restriction factor (Sauter, Schindler and Specht et al., 2009; Sauter et al., 2011; Sauter et al., 2012). Furthermore, we could show that Nef proteins of epidemic HIV‑1 group O viruses evolved the ability to target a region adjacent to the deletion in human tetherin to increase virion release from infected CD4+ T cells (Kluge, Mack and Iyer et al., 2015; Mack et al., 2017). Thus, our data strongly suggest that tetherin counteraction is a prerequisite for the efficient spread of lentiviruses in the human population. This hypothesis is further supported by two collaborative studies with the labs of Kei Sato (Kyoto) and Beatrice Hahn (Philadelphia), in which we analyzed the replicative fitness of HIV and SIV mutants that selectively fail to antagonize tetherin (Kmiec et al., 2016; Yamada et al., 2018). Experiments in primary CD4+ T cells and humanized mice demonstrated that human tetherin-specific adaptations confer a significant selection advantage to the virus as they increase IFN resistance and replication capacity during acute infection.
In summary, our results provide a first plausible explanation why only viruses of HIV‑1 groups M and O spread significantly in the human population, whereas those of groups N and P remain largely restricted to Western Central Africa.
More recently, we showed that SIVsmm, the simian precursor of HIV-2 uses two proteins (Nef and Env) to antagonize tetherin in sooty mangabeys, its natural host species (Heusinger et al., 2018). While human tetherin is resistant to Nef, SIVsmm Env is able to antagonize both monkey and human tetherin. Thus, we identified a phenotypic trait of SIVsmm that may have facilitated its successful zoonotic transmission to humans and helps to explain why SIVsmm has been able to cross the species barrier to humans on at least nine independent occasions (Sauter and Kirchhoff, 2019).

Role of fusion proteins in HIV replication

Pandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. In addition to these canonical proteins, several studies have reported the existence of fusion proteins, most of which are the result of alternative splicing, when exons of regular and/or alternative open reading frames (ORFs) are brought together. Although some of these proteins are expressed at high levels, their role in the viral replication cycle remained poorly understood (Langer and Sauter, 2017).
Notably, alternative splicing is not the only mechanism that can generate unusual fusion proteins in HIV-1. In 2010, Kraus and colleagues reported an HIV-1 gene arrangement in which rev1 and vpu genes were present in the same reading frame without an intervening stop codon. Analysis of the deduced protein sequence of this gene fusion suggests that it spans the plasma membrane like Vpu, but may contain an additional extracellular Rev-derived N-terminal domain.

Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance had remained unclear. Using infectious molecular clones differing only in their ability to express this fusion protein, we could show that Rev1-Vpu does neither affect known Vpu and Rev functions nor enhance viral replication (Fig. 5). Instead, the rev1-vpu fusion gene seems to have a neutral phenotype. We therefore hypothesize that this unusual polymorphism may be the epiphenomenon of other adaptive changes, such as mutations optimizing Env expression (Langer et al., 2015).