Reconstitution of an infectious human endogenous retrovirus - PubMed (original) (raw)

Reconstitution of an infectious human endogenous retrovirus

Young Nam Lee et al. PLoS Pathog. 2007 Jan.

Abstract

The human genome represents a fossil record of ancient retroviruses that once replicated in the ancestors of contemporary humans. Indeed, approximately 8% of human DNA is composed of sequences that are recognizably retroviral. Despite occasional reports associating human endogenous retrovirus (HERV) expression with human disease, almost all HERV genomes contain obviously inactivating mutations, and none are thought to be capable of replication. Nonetheless, one family of HERVs, namely HERV-K(HML-2), may have replicated in human ancestors less than 1 million years ago. By deriving a consensus sequence, we reconstructed a proviral clone (HERV-KCON) that likely resembles the progenitor of HERV-K(HML-2) variants that entered the human genome within the last few million years. We show that HERV-KCON Gag and protease proteins mediate efficient assembly and processing into retrovirus-like particles. Moreover, reporter genes inserted into the HERV-KCON genome and packaged into HERV-K particles are capable of infectious transfer and stable integration in a manner that requires reverse transcription. Additionally, we show that HERV-KCON Env is capable of pseudotyping HIV-1 particles and mediating entry into human and nonhuman cell lines. Furthermore, we show that HERV-KCON is resistant to inhibition by the human retrovirus restriction factors tripartite motif 5alpha and apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) 3G but is inhibited by APOBEC 3F. Overall, the resurrection of this extinct infectious agent in a functional form from molecular fossils should enable studies of the molecular virology and pathogenic potential of this ancient human retrovirus.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Design and Analysis of a Consensus HERV-K Provirus

(A) Diagram of HERV-K(HML-2) provirus. ORFs are depicted as boxes. Proviruses used in the design of HERV-KCON that contain intact versions of Gag, protease, Pol, and Env are listed under each ORF (*K108 encodes a full-length Pol ORF, but a presumed essential YIDD motif is mutated). (B) Phylogenetic analysis of HERV-KCON and the ten proviruses used to generate it, constructed using Kimura 2-parameter algorithm in the TreeMaker program after gap-stripping the sequence alignment (

http://www.hiv.lanl.gov/content/hiv-db/CONTAM/TreeMaker/TreeMaker.html

). (C) Comparison of HERV-KCON with the ten HERV-K proviruses. Each contributing provirus was compared to HERV-KCON using HYPERMUT, and the number of nucleotide differences for each provirus relative to HERV-KCON is plotted.

Figure 2

Figure 2. Assembly, Processing, and Release of HERV-K Virus-Like Particles

(A–D) 293T cells were transfected with Gag-, Gag-PR–, or Gag-PR-Pol–expressing vectors. (A) Western blot analysis of cell lysates (left) and virions (center and right) using a commercially available antibody to HERV-K Gag. Center shows VLPs from 293T cells transfected with a plasmid-expressing Gag, and right shows VLPs from Gag-PR– and Gag-PR-Pol–expressing 293T cells. Decreasing amounts of virion lysate (0.1, 0.05, or 0.025 μl for Gag; 0.4, 0.2, or 0.1 μl for Gag-PR and Gag-PR-Pol) were loaded to semiquantitatively estimate relative levels of VLP production. (B) Silver stain analysis of a 4% to 20% gradient SDS-PAGE gel loaded with VLPs harvested from 293T cells transfected with plasmids expressing Gag, Gag-PR, Gag-PR-Pol, or empty plasmid control. An asterisk marks a nonspecific 66-kDa protein band, most probably BSA, that is abundant in the culture medium. (C) Silver stain analysis of VLPs harvested from 293T cells containing Gag, Gag-PR, Gag-PR-Pol, or Gag-PR(mut) encoding an active site mutation (DTG-AAA) in protease. An asterisk marks a nonspecific 66-kDa protein band, most probably BSA, that is abundant in the culture medium. (D) Reverse transcriptase activity in culture supernatants of 293T cells transfected with empty pCRV1 (vector) or vectors expressing HERV-KCON Gag, Gag-PR, or Gag-PR-Pol proteins, as indicated. Enzymatic activity was determined relative to a recombinant HIV-1 reverse transcriptase standard and is representative of three experiments. Supernatants from 293T cells transfected with an HIV-1–based proviral plasmid are included for comparison. (E) Two representative 293T cells transfected with HERV-KCON Gag and Gag-GFP expression plasmids. Cells were fixed 18 h post-transfection, and nuclei were stained with DAPI (blue) prior to visualization by deconvolution microscopy. Top, Images acquired at the mid-section of the cell to show localization of Gag-GFP proteins; bottom, focused on the bottom of the cell to show accumulated VLPs at the cell–coverslip interface. (F) Gallery of electron micrographs of 293T cells transfected with a Gag-PR–expressing plasmid. Black scale bars in the upper and middle panels represent 500 nm, while scale bars in the lower two panels represent 100 nm.

Figure 3

Figure 3. Generation of Single Cycle Infectious Virions Containing HERV-KCON Genomes and Gag-PR-Pol

(A) Schematic representation of the HERV-KCON–based CHKCG genome. Changes to HERV-KCON are depicted in white boxes. The 5′ LTR was modified to include the CMV promoter inserted in place of the U3 region. The Env ORF, which now contains CMV-GFP, is disrupted. (B and C) Photomicrographs of 293T cell monolayers after infection with CHKCG-carrying HERV-KCON(VSV-G) pseudotyped virions, which were generated following transfection of 293T cells with the indicated plasmid mixtures. (D and E) Infectious titers of HERV-KCON(VSV-G) pseudotyped virions generated following transfection with the indicated plasmid mixtures in the absence (D) or presence (E) of K-Rev/Rec and using 293T target cells. GFP-positive foci were enumerated visually and expressed as infectious units per milliliter of virion-containing supernatant. (F) Infectious titers of CHKCG containing HERV-KCON(VSV-G) using 293T target cells in the presence or absence of 50 μM AZT. All data are representative of at least three experiments.

Figure 4

Figure 4. Transduction Mediated by HERV-KCON Gag-PR-Pol and Genomes Results in Stable Proviral Integration

(A) Puromycin-resistant colonies of 293T cells infected with either VSV-G pseudotyped (left) or Env defective (right) virions carrying the CHKCP genome. Infected 293T cells were selected in 0.5 μg/ml puromycin for 2 wk and then fixed and stained to reveal colonies of viable cells. Data are representative of at least three experiments. (B) Experimental strategy for detection of HERV-KCON proviruses in CHO745 cells using PCR primers targeted to HERV-KCON Gag and LTR sequences, or flanking hamster DNA sequences. (C) PCR amplification of HERV-KCON gag DNA using Gag-S and Gag-AS primers in four expanded clones of puromycin-resistant CHO745 cells transduced with CHKCP-containing HERV-KCON(VSV-G) particles. (D) Nucleotide sequences at the 5′ and 3′ ends of integrated CHKCP proviral DNA, revealing six nucleotide duplicated sequences at the CHKCP integration sites. (E) Verification of the presence and absence of an integrated provirus and the empty preintegration site in CHKCP-transduced and naïve CHO745 cells using combinations of HERV-K and hamster DNA targeted PCR primers (see [B] for primer design strategy). DNA templates and PCR primer pairs used are indicated above each lane, and the expected PCR product size is given below each lane. A representative analysis of a single CHKCG-carrying CHO745 cell clone is shown; similar results were obtained with two additional clones. Uninfected CHO745 cells and human 293T cells serve as controls.

Figure 5

Figure 5. HERV-KCON Tropism

(A) Human, squirrel monkey, feline, hamster, or murine cells were infected with VSV-G pseudotyped HERV-KCON particles. Two days postinfection, GFP+ foci were quantified microscopically, and titers are expressed as number of infectious units (i.u.) per milliliter of virus-containing supernatant applied. (B) Human, squirrel monkey, feline, or murine cells were infected with HERV-KCON Env pseudotyped HIV-1 particles as in (A). Two days postinfection, GFP-positive foci were quantified. All data are representative of at least three experiments.

Figure 6

Figure 6. Effects of TRIM5 and APOBEC3 Proteins on HERV-KCON Infectivity

(A) Unmanipulated CHO cells or variants stably expressing human TRIM5α, rhesus monkey TRIM5α, or owl monkey TRIM-Cyp were infected with VSV-G pseudotyped retroviral vectors that are sensitive to one or more of the TRIM5 proteins (N-MLV or HIV-1) or TRIM5-resistant controls (B-MLV or HIV-1 carrying SIVmac CA HIV(SCA)), as indicated. Two days postinfection, the percentage of GFP+ cells was determined using FACS. (B) The same panel of CHO-derived TRIM5-expressing CHO cell lines were inoculated with HERV-KCON(VSV-G). Two days postinfection, GFP+ foci were quantified. (C) APOBEC3F and APOBEC3G expression plasmids were cotransfected into 293T cells during generation of CHKCG-containing HERV-KCON(VSV-G) particles. Fresh 293T cells were infected with the resulting viral supernatant, and GFP+ foci were quantified 2 d later. (D) Western blot analysis, using the anti–HERV-K Gag antibody, of cell and HERV-KCON virion lysates generated upon coexpression of APOBEC3F or APOBEC3G, as indicated. All data are representative of at least three experiments.

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