Murine Leukemia Virus

Total RNA was used for cDNA synthesis essentially as described [15 ]. Briefly, 100 ng of RNA in a final volume of 10 μl including 1 μl of 10x poly(A) polymerase buffer, 0.1 mM of ATP, 1 μM of RT-primer, 0.1 mM of each deoxynucleotide (dATP, dCTP, dGTP and dTTP), 100 units of MuLV reverse transcriptase (New England Biolabs, USA) and 1 unit of poly(A) polymerase (New England Biolabs, USA) was incubated at 42°C for 1 hour followed by enzyme inactivation at 95°C for 5 minutes. The sequence of the RT-primer was 5'-CAGGTCCAGTTTTTTTTTTTTTTTVN, where V is A, C and G and N is A, C, G and T. The primer was purchased from TAG Copenhagen (Denmark).
For the microRNA LNA™ PCR kit from Exiqon (Denmark) cDNA synthesis was done according to the manufacturer's instructions.

The heat capacity curve of DPPC/POPG MLVs was recorded in the absence and presence of both peptides, using a high sensitivity Nano DSC (TA Instruments, New Castle, DE, USA) equipped with 300 µL twin gold capillary cells, pressurized to 3 atm. Each sample was scanned from 20 to 55 °C at least four times, using a scanning rate of 1 °C/min. For each thermogram, a background blank thermogram was subtracted. Data were analyzed using the Nano Analyze software, provided by the manufacturer. Enthalpy values were obtained by direct integration of the normalized baseline subtracted peaks.

C57BL/6 (WT) or Rag deficient mice on the C57BL/6 background were purchased from Jackson Laboratories and E_VH1^−/− mice were constructed using genomic editing and then maintained in colonies at the University of Illinois College of Medicine, or Scripps Research. All procedures involving mice were approved by the Institutional Animal Care Committee of the University of Illinois College of Medicine, and the Scripps Research Institute, in accordance with protocols approved by the UIC and Scripps Research Institute Institutional Animal Care and Use Committees. Mice were housed in sterile static microisolator cages on autoclaved corncob bedding with water bottles. Food was irradiated (Envigo 7912), water was autoclaved and both were provided ad libitum. The standard photoperiod is 14 hours of light and 10 hours of darkness for mouse rooms. Mice receive autoclaved nesting material to enrich their environments. Cage bedding is changed in either a biosafety cabinet or a HEPA-filtered animal transfer station at least weekly. Housing density and cage size are consistent with the recommendations of the Guide for the Care and Use of Laboratory Animals. The ambient temperature and humidity of the rodent housing rooms are consistent with the recommendations of the Guide for the Care and Use of Laboratory Animals.
Bone marrow (BM) was collected from humerus, tibia and femur bones of WT and E_VH1^−/− mice. Rag deficient CD19 + pro-B cells were isolated from BM using anti-CD19 coupled magnetic beads (Miltenyi) and cultured in the presence of IL7 (1% vol/vol supernatant of a J558L cell line stably expressing IL7) for 4 days. The Abelson-MuLV transformed (Abl-t) pro-B cell line, 445.3 (Rag1−/−) on the C57Bl/6 background was kindly provided by Dr. B. Sleckman (University of Alabama at Birmingham)^{57 (link)}. A newly derived subclone, 445.3.11 from the Abl-t 445.3 line were cultured in RPMI 1640 (Cellgro), 10% (v/v) FBS, 4 mM glutamine (Gibco), 1 mM sodium pyruvate (Gibco), 1X nonessential amino acid (Gibco), 5000 units/ml Penicillin and 5000 mg/ml Streptomycin (Gibco), 50 mM β-mercaptoethanol (Sigma) and maintained at approximately 5x10e5 cells/ml. Splenic T cells were enriched using Mouse T Cell Enrichment Columns (MTCC-5; R&D Systems) and cultured at a density of 5 × 10⁵ to 1 × 10⁶, stimulated in RPMI 1640 and glutamine (4 mM) and penicillin-streptomycin supplemented with FCS (10% v/v), and activated with Con A (5 ng/ml; 15324505; MP Biomedicals).

Virus neutralization assays were performed to test plasma and MAb neutralization of PSVs. Plasma samples were prepared by heat inactivation at 56°C for 45 min, followed by 5-min centrifugation at 2,000 rpm to remove particulates. Plasma samples were used at a starting dilution of 1:20 in supplemented DMEM; MAbs were used at a starting concentration of 25 μg/mL in supplemented DMEM. Viruses were diluted to a concentration to yield luminescence at least three times above the cell-only controls, as determined by the PSV titration. Neutralization reagents were serially diluted 1:4 in independent duplicates per plate and incubated with equal volumes of virus for 1 h at 37°C before adding TZM-bl cells with 40 μg/mL of DEAE-dextran (Sigma, St. Louis, MO, USA). The plates were incubated at 37°C for 48 h before reading the luminescence. Nonspecific neutralization by plasma was evaluated using MuLV env DNA pseudotyped with the pSG3Δenv backbone DNA. Uninfected normal human serum was also tested against all the PSVs as a negative control.