Poxviruses capture host genes by LINE-1 retrotransposition

  1. Sarah M Fixsen
  2. Kelsey R Cone
  3. Stephen A Goldstein
  4. Thomas A Sasani
  5. Aaron R Quinlan
  6. Stefan Rothenburg
  7. Nels C Elde  Is a corresponding author
  1. Department of Human Genetics, University of Utah, United States
  2. Department of Medical Microbiology and Immunology, University of California, Davis, United States
  3. Howard Hughes Medical Institute, United States
5 figures and 2 additional files


Figure 1 with 1 supplement
Retrotransposon-mediated transfer of GAAP into poxvirus genomes.

(A) The poxvirus gene vGAAP (orange) is encoded near the inverted terminal repeat (ITR; gray) of cowpox genomes. Signatures of retrotransposition include TSDs (blue), a LINE-1-like endonuclease site (EN; underlined), and a partially degraded poly(A) tail (pink). Some cowpox genomes (CPXV_5′; top line) contain a pseudo-empty site, with a single copy of the TSD sequence. (B) Phylogeny of TMBIM proteins, including TMBIM4, or GAAP, and virus-encoded TMBIM4s (red). Bootstrap values >50 are indicated. TSD, target site duplication.

Figure 1—figure supplement 1
Genetic features of vGAAP in poxvirus genomes.

(A) v-GAAP (orange bar) is encoded by many orthopox species, including most camelpox strains, many cowpox strains, and at least one vaccinia strain. In camelpox viruses, v-GAAP is encoded on the left end of the genome; in all other strains, v-GAAP is on the right end. In all strains, v-GAAP is flanked by CrmE and vaccinia-B22R. In vaccinia strains that do not encode v-GAAP, CrmE is also missing, suggesting recombination of the region between B22R and B25R, which is truncated in the Lister strain. (B) Alignment of target site duplications (blue) and flanking sequence in 21 v-GAAP-encoding poxvirus strains. CMLV, camelpox; CPXV, cowpox; MPXV, monkeypox; VACV, vaccinia. Asterisks denote gene truncations.

Figure 2 with 1 supplement
Experimental capture of K3L by horizontal gene transfer.

(A) Viruses lacking K3L (VCR2) replicated in RK13 cells expressing mCherry-K3L and were transferred to BHK cells lacking K3L to select for capture of mCherry-K3L. (B) Replication of VCR2 in RK13-K3L cells and BHK cells compared to VCR2+mCherry-K3L (see Materials and methods). (C) Replication of recovered isolates in BHK cells. Three biological replicates of each strain/isolate with mean titer and standard error bars are shown in (B) and (C). (D) Vaccinia genome illustrating K3L integrations in recovered isolates indicated by colored triangles. Endogenous K3L location is shown. The central region of the genome is highlighted in yellow. Triangles above and below the genome denote positive and negative sense orientation, respectively. Virus genes interrupted by K3L integrations are indicated. The asterisk denotes a genomic rearrangement in isolate 10 (see Figure 3A).

Figure 2—figure supplement 1
Protein Kinase R (PKR) is activated by binding dsRNA which is made in the cytoplasm during a viral infection.

When activated, PKR binds and phosphorylates the eukaryotic initiation factor, eIF2α, leading to a block in translation and preventing viral replication. K3L acts as a pseudo-substrate of PKR, preventing activated PKR from binding eIF2α, which is then free to initiate translation. In the complementing RK13-K3L cells, an mCherry-K3L fusion gene was integrated into cellular chromosomes. The cellular mCherry-K3L expression construct is shown below. The fusion gene is driven by a strong EF1a promoter, but the construct also includes a poxvirus-specific promoter, the SLP, so that mCherry-K3L could be immediately expressed if transferred.

Figure 3 with 2 supplements
Each virus isolate exhibits signatures of LINE-1-mediated retrotransposition.

(A) A detailed view of the region K3L inserted in each experimentally captured virus isolate. Arrows above cartoons indicate reading frame orientation. Flanking and/or interrupted (*) viral genes (blue/purple boxes), 3′/5′ untranslated regions (UTRs; gray), and poly(A) tails (An; white) are shown. Genomic rearrangements included in isolate 10 are also shown. (B) Schematic of construct integrated in RK13-K3L cells (top) compared to K3L integrations in recovered viruses (bottom). Shared features include spliced introns, 5′ and 3′ UTRs, and poly(A) tails (An). Seven isolates have target site duplications (TSDs, blue) with LINE-1 endonuclease cut sites (EN, cyan). Six isolates encode a guanine (G cap) adjacent to the 5′UTR, indicative of 7-methylguanylate mRNA capping. LINE-1, Long Interspersed Nuclear Element-1.

Figure 3—figure supplement 1
Details of the insertions for each virus isolate that acquired K3L.

(A) Genetic features of each isolate recovered in the screen, including endonuclease (EN) cut site, target site duplication (TSD) length, presence or absence of transcript features: 5′-guanosine triphosphate (G) cap, 5′ untranslated region (UTR), splicing, 3-UTR, and poly adenosine (A) tail, and whether there is evidence of a dsDNA break. ND (not determined). (B) A sequence logo plot of nucleotide preference from EN sites detected among all virus isolates.

Figure 3—figure supplement 2
An Alu-Neo reporter (cartoon above) was used to measure endogenous LINE-1 activity of the RK13-K3L cells in comparison to HeLa-HA cells.

Representative 100 mm dishes from several experiments show visible Neo-resistant colonies. 4× magnification of dishes shown to the right.

Figure 4 with 1 supplement
Homology-driven gene duplication of K3L and eIF2α.

(A) Schematics of mCherry-K3L duplications in isolates 4 and 5. (B) Virus titers after serial infections of eIF2α viruses in HeLa cells (see Materials and methods). (C) Schematics of eIF2α duplications following virus passaging. Asterisks denote gene truncations.

Figure 4—figure supplement 1
Diagrams of the genomic region encompassing K3L in wildtype (WT) vaccinia virus, VCR2 strain, and viruses engineered to express eIF2α and eIF2α S51A.

Arrows indicate reading frame orientation.

Figure 5 with 1 supplement
Model for horizontal transfer of host genes to poxvirus genomes.

The schematic highlights how host genes (purple) are retrotransposed by LINE-1 reverse transcriptase (RT, blue), into virus genomes (yellow box). Following horizontal transfer, gene duplication is facilitated by unequal crossover recombination between TSDs (blue) or flanking repeat sequence (gray). LINE-1, Long Interspersed Nuclear Element-1; TSD, target site duplication.

Figure 5—figure supplement 1
Models of evolution of newly acquired virus genes.

(A) Genes that land in the repetitive region of the ITR are likely to be duplicated by both replication mechanisms (to the other ITR) and recombination mechanisms (tandem duplications). Duplications provide more chances for advantageous mutations to arise, increasing the likelihood of fixation. (B) Genes acquired outside the ITR may still be duplicated via TSD-mediated recombination. (C) Acquired genes that interrupt essential genes are unlikely to be maintained.

Additional files

Supplementary file 1

Gene and protein sequence information.

Accession numbers for sequences used in phylogenetic analysis shown in Figure 1B. Amino acid alignment used in phylogenetic analysis shown in Figure 1B. Supplementary sequences.

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  1. Sarah M Fixsen
  2. Kelsey R Cone
  3. Stephen A Goldstein
  4. Thomas A Sasani
  5. Aaron R Quinlan
  6. Stefan Rothenburg
  7. Nels C Elde
Poxviruses capture host genes by LINE-1 retrotransposition
eLife 11:e63332.