Nonreciprocal pseudotyping: murine leukemia virus proteins cannot efficiently package spleen necrosis virus-based vector RNA

Abstract

It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Psi) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Psi, but MLV can recognize only MLV Psi. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

FIG. 1

Structures of retroviral vectors used to study pseudotyping. pJS12 and pJS14 are SNV-based retroviral vectors, whereas pGA1 is an MLV-based retroviral vector. SNV vectors pJS12 and pJS14 each contain β-gal expressed from the U3 promoter and hygro expressed from an internal SV40 promoter (SV). The 3′ LTR of pJS12 contains MLV U3 instead of SNV U3. pGA1, an MLV-based vector, also contains β-gal and a neo gene that is expressed by an encephalomyocarditis virus IRES. Open boxes represent SNV LTR sequences, and hatched boxes denote MLV LTR sequences. The SNV packaging signal (E) is designated by a thick line, whereas the MLV packaging signal (Ψ) is designated by a thin line.

FIG. 2

Experimental protocol to study whether MLV proteins can pseudotype SNV vectors. Plasmid DNAs were used to separately transfect C3A2 (pJS12 and pJS14) or PA317 (pGA1) helper cells. JS12, JS14, and GA1 virus stocks were harvested separately from transfected helper cells and used to infect PG13 helper cells. Infected PG13 cells were selected with the appropriate drugs, pooled, and expanded. Viruses harvested from the pools were used for either RNA isolation or infection of D17 target cells to determine viral titers. DNA and RNA were also isolated from the infected PG13 cells for structural and expression analysis.

FIG. 3

(A) Predicted proviral structures of JS14, JS12, and GA-1. Zigzag lines, host DNA sequences; cross-hatched box labeled Probe, DNA fragment used to generate the radioactively labeled probe; RV, EcoRV restriction enzyme site. All the other abbreviations are identical to those in Fig. 1. (B) Southern analysis of genomic DNAs isolated from pools of PG13 cells infected with JS12, JS14, or GA1. The probe was a radioactively labeled β-gal DNA fragment (as shown in panel A). JS12 DNA produced 4.1- and 2-kb bands. JS14 DNA produced a smear because a pool of infected PG13 cells was used. GA1 containing genomic DNA produced 3.9- and 2.5-kb bands.

FIG. 4

RNA hybridization studies with cellular and viral RNA from infected PG13 cell pools. (A) RNA analysis of PG13 cellular RNA hybridized with the β-gal probe (Fig. 3A). The dilutions of RNA are shown above the blot. A 7-μg portion of total cellular RNA was used in the 1× dilution. (B) RNA analysis of viral RNA with the β-gal probe. (C) RNA analysis of spiked wild-type SNV RNA with the REV-A env probe. Wild-type SNV was added to the supernatant before the isolation of viral RNA to adjust for the possible loss of RNA during the purification procedures.

FIG. 5

RNA hybridization studies with cellular and viral RNA from infected C3A2 cells. (A) RNA analysis of C3A2 cellular RNA hybridized with the β-gal probe. The dilutions are shown above the blot. A 7-μg portion of total cellular RNA was used in the 1× dilution. (B) RNA analysis of viral RNA with the β-gal probe. (C) RNA analysis of spiked wild-type SNV viral RNA with the REV-A env probe.