Molecular Biology and Application of Recombinant Foamy Viruses

Overview

The goal of the research group “Molecular Biology and Application of Recombinant Foamy Viruses” is to increase understanding of foamy virus (FV) replication, host-virus interactions, potential disease association, and molecular biology. We will exploit these insights towards the construction and optimization of FV-based vectors for targeted gene delivery and vaccination. Our research also aims to understand potential detrimental effects of persistent antiviral restriction factor activation leading to genetic instability and cancer.

FVs are retroviruses whose specific molecular biology led to their placement into the distinct subfamily of Spumaretrovirinae. FVs are much less studied than many other human viruses since they are considered apathogenic, although the few studies that have addressed this issue draw inconsistent conclusions. However, due to their unique molecular biology, ability to infect humans across species barriers (zoonosis), and their potential use as vehicles for gene transfer or vaccine antigen delivery, FVs have garnered increasing attention. In addition, host-virus interactions at the molecular, cellular, and organismal levels and their consequences on virus-host co-evolution, e.g. the oncogenic spill-over of host-mediated restriction factor activation, are studied. The projects highlighted below and schematically presented in Fig. 1 are explored by our laboratory both as individual modules and in conjunction with other projects.

Figure 1. Project overview.
© dkfz.de

Present Research Projects

1. Molecular biology of foamy viruses.

Current studies revealed that budding of FV particles depends on a highly specific interaction between Gag and an N-terminal motif in the FV Env leader protein (Elp). However, Env-independent Gag budding can be achieved by adding myristic acid membrane-targeting signals to the N-terminus of Gag (Liu et al., 2011). N-terminal residues of Gag are not only essential for interaction with Elp, but could also be involved in driving cytosolic capsid assembly. In order to identify functional relevant modules in FV proteins and/or to increase the function or efficacy of engineered mutants, in vitro evolution and selection of FV stocks displaying desirable phenotypes is a highly potent experimental tool.

  • We are currently exploring the contribution of individual amino acids in this region in driving cytosolic capsid assembly.
  • We are also working to identify the mechanisms by which Elp retargets the capsid for budding and particle release.
  • In addition, we showed that full-length Env is released as Env-only sub-viral particles (SVPs), which could be exploited for antigen presentation. The Env SVPs and Elp fusion proteins will be exploited for chimeric vaccine antigen presentation on FV particle surfaces to generate improved immune responses (see below).
  • Using in vitro evolution and selection, cell-free infectivity of a highly cell-associated FV isolate has been enhanced by several logs. Selection for quickly replicating FVs has led to surprising new insights into virus replication, particle release or cellular non-permissiveness/restriction.

2. Construction, optimization, and evaluation of FV-based gene delivery and vaccination vectors.

Safe and efficient replication-deficient feline FV (FFV) vectors for gene delivery have previously been generated and tested in cell cultures, with special emphasis on fine-mapping of essential cis-acting sequences to increase vector packaging size and biological safety (Liu et al., 2008). Based on new findings on FV particle assembly and release, our focus has shifted towards the construction of replication-competent FFV vectors, Env-only SVPs, or Gag-Env virus-like particles (VLP) for antigen delivery. The FV vaccine vector studies focus on presenting vaccine antigens on SVPs to illicit effective immune responses.

  • We are currently testing whether B- and T-cell vaccine epitopes selected solely on sequence homology with the FV proteome can be inserted into FV proteins. This strategy to “silently” insert epitopes into viral proteins aims to preserve protein function and viral replication of FV vectors. This could allow long-term antigen presentation and T- and B-cell response maturation. Initial testing of viral, tumor (melanoma), and model epitopes suggests that this strategy is feasible. In parallel, epitopes have been inserted into protein sequences known to tolerate sequence changes. If the initial vaccine antigen delivery concept turns out to be viable, next-generation vectors will be further optimized and expanded by inserting multiple epitopes of a given tumor entity/virus into a single FV vector.
  • Vector application in small animal models will be conducted with our partners to test immunogenicity (efficacy) and safety of the vectors in vivo. In such animal studies we also aim at gaining a better understanding of FV replication in vivo.
  • In a project on FV-based HIV vaccine vectors (Mühle et al., 2012), HIV epitopes known to be targeted by broadly neutralizing antibodies were engineered into the corresponding FFV Env TM sequence (to be published). We found that full-length Env can be utilized as scaffold for vaccine antigen presentation. HIV epitope-carrying Env SVPs can be enriched and are currently being evaluated as an HIV vaccine in mice.

3. FV-host interactions at the molecular level are important for vector function and safety.

Past studies on APOBEC3 (A3) restriction and viral counter-defense (Chareza et al., 2012; LaRue et al., 2009; Münk et al., 2010; 2008; Perkovic et al., 2010; Zielonka et al, 2010) have significantly contributed to the overall understanding of the molecular mechanisms and evolutionary dynamics of these virus-host systems (La Rue et al., 2009). We have shown that the mechanisms of HIV Vif and FV Bet-dependent A3 inactivation are fundamentally different (Chareza et al., 2012). FFV Bet can be replaced by feline immunodeficiency virus Vif in genetically stable and fully replication-competent FFV chimeras. Because Vif now prevents host APOBEC3- mediated restriction, a role normally played by FV Bet, there is strong pressure to maintain this heterologous antigen. By in vitro evolution, we even selected vector variants with almost wild-type replication characteristics (unpublished).

  • Mapping experiments revealed that the bel2 exon of Bet is required to counteract A3, while the bel1 exon and C-terminal sequences regulate protein stability and steady-state levels (Slavkovic Lukic et al., 2013). Importantly, PFV Bet cannot replace FFV Bet and domain swapping does not generate functional proteins. FV Bet-A3 binding and A3 inactivation cannot be dissociated by site-specific mutagenesis, suggesting that Bet-A3 is sufficient for inactivation (Slavkovic Lukic et al., 2013). Further studies will focus on whether sequestration, mis-localization or shielding of binding sites determines A3 inactivation. Characterization of this mechanism is essential in generating Bet-based broadly-acting A3 suppressors.

4. Infection and inflammation-associated genotoxicity and oncogenicity.

In order to explain inflammation-associated cancer development, we are currently studying whether excess, untimely and/or prolonged expression of A3 cytidine deaminase restriction factors affects host genome integrity through DNA editing.

  • We are currently characterizing the genome editing activity of human A3 proteins by using Tet-regulated huA3 expressing cells and an HSV thymidine kinase pro-drug toxicity screen. Our results indicate that A3 proteins have mutagenic/oncogenic potential. We will determine whether FV Bet proteins are suitable antagonists to target the mutagenic function of A3. In addition, we will identify A3 residues critical for Bet binding and investigate whether FV Bet is the ancestor of different A3-interacting proteins.
  • A3 expression in malignant and non-malignant tissues is being examined by immunohistochemistry in hepatitis C virus-induced hepatocellular carcinoma. These studies will be extended to other tumor entities. In addition, a newly-established quantitative RT-PCR will be used for A3 expression profiling.

5. FV–host interactions at the organismal level: search for zoonosis-associated disease and malignancy.

Primate FVs have the highest zoonotic potential among retroviruses and thus may pose a health threat in particular geographical regions or person groups. We are studying whether non-primate FVs from livestock, companion and wild animals may be a relevant source of zoonosis. To analyze the repertoire of non-primates FVs to which humans are exposed in developed countries, we use serological and genome-based detection systems (Bleiholder et al., 2011; Mühle et al., 2011). Together with our cooperation partners, we have identified and characterized FVs in wild cats that are highly related to FFV (Kehl et al., 2013) and detected serological markers of a bovine FV-like sheep virus (unpublished). FVs isolated from different cat species or geographically distant animals show remarkably high relatedness (Kehl et al., 2013; Materniak et al., 2013). This and the absence of adaptive genetic changes in BFV upon long-term experimental infection of sheep (Materniak et al., 2013) point to an extreme conservation of the FV genome.

The high sequence conservation shown in our studies indicates low FV turnover and cell-mediated persistence and possible transmission. In this respect, BFV is an ideal model due to its high cell association and apparent transmission through direct contact with infected cells (Romen et al., 2007).

  • Together with our cooperation partners, we will study the effects of FVs on the host cell miRNA repertoire by overall gene expression profiling of FV infected cells and animals. We expect to identify FV genes that, due to extensive FV-host coevolution, have the ability to modulate innate and adaptive immune functions necessary to allow long-term survival and immune evasion by infected cells. We also predict that these functions play a role in promoting the apparently disease-free equilibrium between host and virus. These studies are of prime importance for the development and application of replicating FV vectors.
  • In animal studies, we will also identify target cells/organs of FV replication, persistence, and spread, and address issues related to virus/vector pathogenicity. Epidemiology of FVs in wild hosts may provide new insights into FV pathogenicity, since we expect, to find disease association(s) only in the wild and not in domestic pets or livestock, an observation already made for chimpanzee immunodeficiency viruses that only in the wild is associated with increased mortality of infected animals.

to top