Endogenous retroviruses mobilized during friend murine leukemia virus infection

Abstract

We have demonstrated in a mouse model that infection with a retrovirus can lead not only to the generation of recombinants between exogenous and endogenous gammaretrovirus, but also to the mobilization of endogenous proviruses by pseudotyping entire polytropic proviral transcripts and facilitating their infectious spread to new cells. However, the frequency of this occurrence, the kinetics, and the identity of mobilized endogenous proviruses was unclear. Here we find that these mobilized transcripts are detected after only one day of infection. They predominate over recombinant polytropic viruses early in infection, persist throughout the course of disease and are comprised of multiple different polytropic proviruses. Other endogenous retroviral elements such as intracisternal A particles (IAPs) were not detected. The integration of the endogenous transcripts into new cells could result in loss of transcriptional control and elevated expression which may facilitate pathogenesis, perhaps by contributing to the generation of polytropic recombinant viruses.

Keywords: Endogenous; Mouse; Proviruses; Pseudotyping; Retroviruses.

Figure 1. Mobilization of endogenous mouse polytropic proviruses at early times after infection

Mouse endogenous polytropic viral LTRs transferred to FRE cells after co-cultivation with splenocytes from F-MuLV-infected mice were detected by the PCR. Days post-infection (DPI) are indicated below the lanes. Also included is a negative control (FRE) of a PCR using DNA from FRE cells co-cultivated with splenocytes from an uninfected mouse. A 100-bp DNA ladder is in the left lane. DNA amplicons corresponding to the two structural classes of endogenous polytropic viruses, Modified polytropic proviruses (Mpmv) and Polytropic (Pmv), are indicated.

Figure 2. Incidence of endogenous polytropic MuLV subclasses mobilized during F-MuLV infection

Clonal FRE cell lines were derived from cultures of FRE cells co-cultivated with splenocytes from F-MuLV-infected NFS/N mice at various times after infection. Each clone was tested for the presence of Pmv or Mpmv LTRs, and the percentage of positive clones for each subclass was plotted. All clones that tested positive harbored either a Pmv or Mpmv provirus but not both. The numbers of clones tested for each dpi were: 191 clones at 1 dpi, 77 clones at 7 dpi, 188 clones at 14 dpi, and 43 clones at 34 dpi.

Figure 3. Incidence of mobilized polytropic proviruses and recombinant polytropic MuLVs during infection with F-MuLV

Splenocytes from F-MuLV-infected NFS/N mice infected for various times were co-cultivated with FRE cells. Subsequently, clonal cell lines of the FRE cells were derived and each clone tested for the presence of endogenous polytropic LTR and env sequences. Clones infected with recombinant MuLVs exhibited polytropic env sequences but not polytropic LTR sequences. Clones harboring mobilized mouse proviruses exhibited polytropic LTR sequences. The numbers of clones tested for each dpi were: 191 clones at 1 dpi, 77 clones at 7 dpi, 89 clones at 14 dpi, and 43 clones at 34 dpi.

Figure 4. Pmv and Mpmv regions of mobilized proviruses from NFS/N mice

Three of the mobilized proviruses were found to be recombinants of Pmv and Mpmv sequences. The mobilized proviruses are depicted by bar diagrams determined by alignments with consensus sequences of Pmvs and Mpmvs from C57BL/6 mice. Also shown is a diagram of the C57BL/6 Pmv10 provirus that shares common deletions and a recombination region with some of the mobilized proviruses. The approximate positions of proviral genes are indicated in the diagram above. Small difference in the LTRs and env gene sequences of the recombinant proviruses are not shown. Deletions in the mobilized proviruses are indicated by gaps in the bar diagrams. Clone designations and the duration of F-MuLV infections are indicated to the right of the bar diagrams.

Figure 5. C57BL/6 proviruses closely related to mobilized NFS/N endogenous proviruses

Mobilized endogenous proviruses from NFS/N proviruses were aligned with Pmv or Mpmv consensus sequences. Pmv20 and the mobilized provirus from Clone 15 were aligned with the Pmv consensus sequence (Jern, Stoye et al., 2007). MpmvΔenv and the mobilized proviruses from Clones 47 and 155 were aligned with Mpmv consensus sequences (Jern, Stoye et al., 2007). The proviruses are represented by bar diagrams with deletions indicated by gaps in the bar diagrams. The positions of base mismatches with respect to the consensus sequences are indicated by vertical lines above each bar diagram. The positions of proviral genes on the Pmv consensus sequences are indicated in the diagram above the bar diagrams. The Mpmv consensus sequence is slightly larger than the Pmv consensus sequence, thus the bar diagrams for the Mpmv sequences (crosshatched) do not strictly correspond to the proviral gene positions.