Replication of enhancer-deficient amphotropic murine leukemia virus in human cells

Abstract

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. The generation of replication-competent virus is considered less likely with vectors that delete the viral transcription elements. This conclusion is based on data obtained in rodents, where MLV replication depends on the expression of viral genes under the control of 75-bp enhancer elements in the long terminal repeat. We demonstrate here that in some human cells replication of amphotropic MLV is possible in the absence of these enhancer elements. Replication of the enhancer-deficient virus MLV-(MOA)Delta E is observed in selected human sarcoma and B lymphoma lines and proceeds at a lower rate than that of the intact virus. No insertion of a foreign promoter or enhancer into the long terminal repeat was detected. Our data suggest the presence of a secondary enhancer element within the MLV provirus that can in selected human cells mediate virus transcription and replication in the absence of the 75-bp U3 enhancers.

Figure 1

Replication of amphotropic MLV in human sarcoma and lymphomas. Cell lines infected by either MLV-(MOA) (○) or MLV-(MOA)ΔE (●) or mock-infected (□) were passaged for the indicated time periods, and the RT activity in the supernatants was determined. One representative experiment is shown for each cell line.

Figure 2

Southern blot analysis of infected HT-1080 cells. Proviral expression plasmids (Plasmid), pMLV-(MOA) (MOA), and pMLV-(MOA)ΔE (ΔE) were compared with DNA from mock-infected (Mock), MLV-(MOA)-infected (MOA), or MLV-(MOA)ΔE-infected (ΔE) MCF-7, HT-1080, and ZR-75–1 cells. DNA was either HindIII-digested and analyzed with probe 1 (A), or HindIII and KpnI-digested and tested with either probe 2 (B) or a CMV promoter probe (C). A provirus restriction map is shown below the blots.

Figure 3

Southern blot analysis of infected lymphoma cells. DNA from mock-infected (Mock), MLV-(MOA)-infected (MOA), or MLV-(MOA)ΔE-infected (ΔE) MHH-PREB-1 (MHH) and Ramos cells (Ram) was compared with the corresponding proviral expression plasmids (PLA). DNA was either HindIII-digested and analyzed with probe 1 (A), or HindIII- and KpnI-digested and tested with either probe 2 (B) or a CMV promoter probe (C). For provirus restriction maps and probes see Fig. 2.

Figure 4

RT-PCR analysis of viral RNA. Virus RNA isolated from HT-1080 and NIH/3T3 cells (A) or Ramos and MHH-PREB-1 cells (MHH) (B) that are either mock- (Mock), MLV-(MOA)- (MOA), or MLV-(MOA)ΔE-infected (ΔE) was used for RT-PCR amplification of the U3-R region (see C) with primer sets FS1/FS3 and FS1/HR2. Products were separated on a polyacrylamide gel and silver-stained.

Figure 5

Restriction analysis of PCR-amplified amphotropic MLV provirus. Genome-size provirus fragments from R to R region were PCR-amplified, digested with KpnI (K), XhoI (X), and SspI (Ss) and separated on a 0.8% agarose gel. Template DNA was genomic DNA from HT-1080, Ramos, and MHH-PREB-1 cells (MHH) infected with either MLV-(MOA) or MLV-(MOA)ΔE (MOA, ΔE). The corresponding viral expression plasmids (PLASMID) were used as control templates. A provirus restriction map is shown below the gel.

Figure 6

Infection studies with intact and enhancer-deficient amphotropic MLV. MLV-(MOA)- or MLV-(MOA)ΔE-containing media from infected HT-1080 cells were harvested and filtered, and the RT activities were determined. Virus-containing medium equivalent to 10⁵ cpm of RT activity were used for infection of MDA-MB-435S, NIH 3T3, HT-1080, and MCF-7 cells. Cells were passaged for 60 days, and RT activity was determined.