Klaus Cichutek

Adherens junction protein nectin-4 is the epithelial receptor for measles virus

Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

APOBEC3 Proteins Inhibit Human LINE-1 Retrotransposition

The human cytidine deaminase family APOBEC3 represents a novel group of proteins in the field of innate defense mechanisms that has been shown to be active against a variety of retroviruses. Here we examined whether members of the APO-BEC3 family have an impact on retrotransposition of human long interspersed nuclear elements (LINE-1s or L1s). Using a retrotransposition reporter assay in HeLa cells, we demonstrate that in the presence of transiently transfected APOBEC3A, L1 retrotransposition frequency was reduced by up to 85%. Although APOBEC3G and -3H did not influence L1 retrotransposition notably, expression of APOBEC3B, -3C, and -3F inhibited transposition by ∼75%. Although reverse transcription of L1s occurs in the nucleus and APOBEC3 proteins are believed to act via DNA deamination during reverse transcription, activity against L1 retrotransposition was not correlated with nuclear localization of APOBEC3s. We demonstrate that APOBEC3C and APOBEC3B were endogenously expressed in HeLa cells. Accordingly, down-regulation of APOBEC3C by RNA interference enhanced L1 retrotransposition by ∼78%. Sequence analyses of de novo L1 retrotransposition events that occurred in the presence of overexpressed APOBEC3 proteins as well as the analyses of pre-existing endogenous L1 elements did not reveal an enhanced rate of G-to-A transitions, pointing to a mechanism independent of DNA deamination. This study presents evidence for a role of host-encoded APOBEC3 proteins in the regulation of L1 retrotransposition.

The antiretroviral activity of APOBEC3 is inhibited by the foamy virus accessory Bet protein

Genome hypermutation of different orthoretroviruses by cellular cytidine deaminases of the APOBEC3 family during reverse transcription has recently been observed. Lentiviruses like HIV-1 have acquired proteins preventing genome editing in the newly infected cell. Here we show that feline foamy virus (FFV), a typical member of the foamy retrovirus subfamily Spumaretrovirinae, is also refractory to genome deamination. APOBEC3-like FFV genome editing in APOBEC3-positive feline CRFK cells only occurs when the accessory FFV Bet protein is functionally inactivated. Editing of bet-deficient FFV genomes is paralleled by a strong decrease in FFV titer. In contrast to lentiviruses, cytidine deamination already takes place in APOBEC3-positive FFV-producing cells, because edited proviral DNA genomes are consistently present in released particles. By cloning the feline APOBEC3 orthologue, we found that its homology to the second domain of human APOBEC3F is 48%. Expression of feline APOBEC3 in APOBEC3-negative human 293T cells reproduced the effects seen in homologous CRFK cells: Bet-deficient FFV displayed severely reduced titers, high-level genome editing, reduced particle release, and suppressed Gag processing. Although WT Bet efficiently preserved FFV infectivity and genome integrity, it sustained particle release and Gag processing only when fe3 was moderately expressed. Similar to lentiviral Vif proteins, FFV Bet specifically bound feline APOBEC3. In particles from Bet-deficient FFV, feline APOBEC3 was clearly present, whereas its foamy viral antagonist Bet was undetectable in purified WT particles. This is the first report that, in addition to lentiviruses, the foamy viruses also developed APOBEC3-counter-acting proteins.

Targeted Cell Entry of Lentiviral Vectors

Retargeting of lentiviral vector entry to cell types of interest is a key factor in improving the safety and efficacy of gene transfer. In this study we show that the retargetable envelope glycoproteins of measles virus (MV), namely, the hemagglutinin (H) responsible for receptor recognition and the fusion protein (F), can pseudotype human immunodeficiency virus 1 (HIV-1) vectors when their cytoplasmic tails are truncated. We then pseudotyped HIV-1 vectors with MV glycoproteins displaying on H either the epidermal growth factor or a single-chain antibody directed against CD20, but without the ability to recognize their native receptors. Gene transfer into cells that expressed the targeted receptor was several orders of magnitude more efficient than into cells that did not. High-target versus nontarget cell discrimination was demonstrated in mixed cell populations, where the targeting vector selectively eliminated CD20-positive cells after suicide gene transfer. Remarkably, primary human CD20-positive B lymphocytes were transduced more efficiently by the CD20-targeted vector than by a vector pseudotyped with the vesicular stomatitis virus G (VSV-G) protein. In addition, the CD20-targeted vector was able to transduce even unstimulated primary B cells, whereas VSV-G pseudotyped vectors were unable to do so. Because MV enters cells through direct fusion at the cell membrane, this novel targeting system should be widely applicable.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105+ endothelial cells, human CD133+ hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133+ cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

Functions, Structure, and Read-Through Alternative Splicing of Feline APOBEC3 Genes

BackgroundOver the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene.ResultsHere we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by read-through alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection.ConclusionOur data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher.

DARPins: An Efficient Targeting Domain for Lentiviral Vectors

We have recently developed a retargeting system for lentiviral vectors (LVs) that relies on the pseudotyping of LVs with engineered measles virus (MV) glycoproteins (hemagglutinin (H) and fusion protein (F)). Specificity is provided through display of a single-chain antibody (scFv) as targeting domain by fusion to the MV-H protein. As an alternative to scFv, designed ankyrin repeat proteins (DARPins) can be selected to become high-affinity binders to any kind of target molecule. In this study six HER2/neu-specific DARPins exhibiting different affinities and binding to different HER2/neu epitopes were applied as targeting domains. All H-DARPin fusion proteins were efficiently expressed on the cell surface. Upon coexpression with F, syncytia formation was observed in HER2/neu positive cells only and correlated directly with the HER2/neu receptor density. All H-DARPin proteins incorporated into LVs, albeit at different levels. The vectors only transduced HER2/neu-positive cells, while HER2/neu-negative cells remained untransduced. Highest titers were observed with one particular DARPin binding to the membrane distal domain of HER2/neu with medium affinity. When applied in vivo systemically, HER2/neu-targeted LVs showed exclusive gene expression in HER2/neu positive tumor tissue, while vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped vectors mainly transduced cells in spleen and liver. Thus, DARPins are a promising alternative to scFvs for retargeting of LVs.

Lentiviral vectors with measles virus glycoproteins - dream team for gene transfer?

Lentiviral vectors are potent gene transfer vehicles frequently applied in research and lately also in clinical applications. Recent improvements have come from combining lentiviral vectors with engineered envelope proteins, which now allow targeting of cell entry to any cell population of interest, as well as the transduction of quiescent cells of the haematopoietic system. We propose that measles virus envelope glycoproteins are especially well suited for this purpose because they can mediate pH-independent cell entry at the cell surface membrane and can induce cytoskeleton rearrangements that facilitate the transport of lentiviral core particles to the cell nucleus. Lentiviral vectors pseudotyped with measles virus glycoproteins are expected to improve the safety and efficacy of gene transfer to human cells.

Species-specific Inhibition of APOBEC3C by the Prototype Foamy Virus Protein Bet

The APOBEC3 cytidine deaminases are part of the intrinsic defense of cells against retroviruses. Lentiviruses and spumaviruses have evolved essential accessory proteins, Vif and Bet, respectively, which counteract the APOBEC3 proteins. We show here that Bet of the Prototype foamy virus inhibits the antiviral APOBEC3C activity by a mechanism distinct to Vif: Bet forms a complex with APOBEC3C without inducing its degradation. Bet abolished APOBEC3C dimerization as shown by coimmunoprecipitation and cross-linking experiments. These findings implicate a physical interaction between Bet and the APOBEC3C. Subsequently, we identified the Bet interaction domain in human APOBEC3C in the predicted APOBEC3C dimerization site. Taken together, these data support the hypothesis that Bet inhibits incorporation of APOBEC3Cs into retroviral particles. Bet likely achieves this by trapping APOBEC3C protein in complexes rendering them unavailable for newly generated viruses due to direct immobilization.

Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity

We pseudotyped HIV-1 vectors with cytoplasmic tail-truncated envelope glycoproteins of a wild-type (WT) measles virus (MV). The particles entered the lymphatic cells exclusively through the signaling lymphocyte activation molecule (SLAM, CD150), whereas particles pseudotyped with the MV vaccine strain glycoproteins also recognized the ubiquitous membrane cofactor protein (CD46) as receptor and had less specific cell entry. MVWT-HIV vectors reached titers of 108 t.u. ml−1, which were up to 10-fold higher than those of MVVac-HIV vectors, and discriminated between SLAM-positive and SLAM-negative cells, also in mixed cell cultures. As these vectors transduce primary human cells more efficiently than vesicular stomatitis virus-G pseudotyped vectors do, they are promising candidates for gene transfer to human lymphocytes and certain epithelial cells.