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Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.

InterProScan. The human and finch sequences used for this comparison are available in supplementary Dataset 1. We note that several of the high quality assemblies Pacbio-based assemblies analyzed were made available through the Vertebrate Genome Project (VGP), as described in (20).
Animal and tissue preparation: Zebra finch (Taeniopygia guttata) blood and tissue samples were obtained from birds reared in our breeding colonies and used in brain studies. Embryos for LV injections and primary cell cultures were obtained from eggs laid in our breeding colonies. Chicken (Gallus gallus) eggs and blood samples were obtained from poultry sources (OR, USA and RN, Brazil). Animal protocols required for this work were approved by OHSU, Caltech and UFRN IACUC's internal review boards and are in accordance with NIH Guidelines.
Primary cultures: Zebra finch and chicken fibroblasts were dissociated from 3-day-old embryos and cultured in DMEM plus 10% FBS. These primary cultures were passed every 48 hr. Once established, fibroblasts were subjected to multiple rounds of infection by lentivirus expressing human LDLR (hLDLR, NCBI number Y114155.1) fused to GFP under the control of the ubiquitous promoter RSV. After 4-5 days, monolayers of wild type (wt) or hLDLR-expressing fibroblasts were incubated with VSV G LV particles containing red fluorescent protein with a nuclear localization signal (NLS-RFP) under RSV, and after a few days were examined for nuclear red fluorescence. VSV-G LV infectivity (infective units/µL) was measured by conducting a serial endpoint dilution assay. Data were analyzed by t-test (n=3 serial dilution plates per group, the experiment was repeated 3 times).
Embryo injections: Embryo injections were performed as previously described (11). Briefly, a small window was opened in the shell of freshly laid, fertilized eggs, and the embryo was visually located. Injections were performed with a hydraulic system using pulled glass pipettes with beveled tips to facilitate penetration. Embryos of both species (7 chicken and 8 finch embryos) were injected with 500 nL of the same viral preparation (LV-RSV-GFP at 10 8 IFU/µL). At the end of the injections, the egg window was sealed and the eggs placed back in an artificial incubator at 37°C and 50% humidity (Grumback, Germany). Embryos were collected 72 hr after injections and inspected for GFP expression under a fluorescence microscope.
Lipids assessment: Blood from normally reared adult male zebra finches was collected in the morning right after lights ON and before birds were allowed to feed (fasting; n=7), or after allowing a 30 min period of ad lib feeding (sated; n= 7). Blood was also collected from poultry roosters before or after they were allowed to feed in the morning (n= 3 each). For quantification of total serum cholesterol and triglyceride concentrations, fasting and sated serum samples were analyzed in duplicate using cholesterol liquid reagents (Pointe Scientific, Canton, MI) and triglycerides liquid reagents (Pointe Scientific, Canton, MI), respectively. For cholesterol, absorbance was measured at 490 nm, and for triglycerides, absorbance was measured at 540 nm using a microplate reader (SpectraMax iD3, Molecular Devices, San Jose, CA). Cholesterol and glycerol standards were used to determine serum concentrations (Pointe Scientific, Canton, MI). The serum lipoprotein distribution was determined by size exclusion chromatography [fast protein liquid chromatography (FPLC)] as previously described (3). Fractions obtained from FPLC were subjected to cholesterol and triglyceride analyses via the same colorimetric assays described above for total cholesterol and triglycerides. We note that for the FPLC analysis, the serum samples from sated and fasting zebra finches were combined into 2 pooled samples (from 3 and 4 birds per group). The separate data from sated and fasting groups are shown in Figs. S3A-B, and the combined data for all finches (4 pooled samples) are shown in Fig. 2D (means +/-sem). All lipid-related analyses were performed at the Lipoprotein Analytical Core of OHSU's Knight Cardiovascular Institute.
Protein identification by Liquid Chromatography-Mass Spectrometry Pooled zebra finch serum was subjected to FPLC as described in Lipids assessment above, fractions of interest were combined, and 10 µg of protein was separated by 4-12% Bis-Tris Novex NuPage gel, with molecular weight assessed by SeeBlue Plus Pre-stained protein standard (Thermo Fisher). Coomassie staining was performed with the Power Blot system according to the manufacturer's protocol (Thermo Fisher). Bands of interest were excised and cut into 1 mm 3 cubes. In-gel tryptic digestion was carried out as previously described (4). In brief, gel pieces were rehydrated in 100 mM ammonium bicarbonate, and protein reduced with 10 mM dithiothreitol, followed by cysteine alkylation with 55 mM iodoacetamide, and digested overnight at 37°C with 1 µg trypsin (Thermo Fisher). Peptides were extracted in 5% formic acid followed by equal volume 100% acetonitrile and dried. Peptides were resuspended in 0.1% trifluoroacetic acid (TFA) and separated by a gradient on PepMap RSLC C18, 2 μm, 75 μm x 25 cm EasySpray column (Thermo Scientific). Data was collected on an Orbitrap Fusion Tribrid instrument configured with an EasySpray NanoSource (Thermo Scientific). RAW files were searched against a Uniprot database for Taeniopygia guttata (downloaded March 2017) with the SEQUEST HT search engine in Proteome Discoverer. Searches were configured with static modifications for carbamidomethyl (+57.021 Da) on cysteines, dynamic modifications for oxidation of methionine residues (+15.9949 Da), parent ion tolerance of 1.25 Da, fragment mass tolerance of 1.0005 Da, monoisotopic masses, and trypsin cleavage (max 2 missed cleavages). The large parent ion tolerance was used to increase the number of peptides being scored to improve discrimination of true versus false identifications. Searches used a reversed sequence decoy strategy to control peptide false discovery and identifications were validated by Percolator software (5). Only peptides with q scores < 0.05 were accepted, and only 1 unique peptide was required for matching a protein entry for its identification. Uncharacterized entries were further characterized by sequence homology using BLASTP with a threshold of 0.001.