Determinants of high titer in recombinant porcine endogenous retroviruses

Abstract

Porcine endogenous retroviruses (PERVs) pose a potential stumbling block for therapeutic xenotransplantation, with the greatest threat coming from viruses generated by recombination between members of the PERV subgroup A (PERV-A) and PERV-C families (PERV-A/C recombinants). PERV-A and PERV-B have been shown to infect human cells in culture, albeit with low titers. PERV-C has a more restricted host range and cannot infect human cells. A recombinant PERV-A/C virus (PERV-A14/220) contains the PERV-A sequence between the end of pol and the middle of the SU region in env. The remaining sequence is derived from PERV-C. PERV-A14/220 is approximately 500-fold more infectious than PERV-A. To determine the molecular basis for the increased infectivity of PERV-A14/220, we have made a series of vector constructs. The primary determinant for the enhanced replicative potential of the recombinant virus appeared to be the env gene. Using a series of chimeric env genes, we could identify two determinants of high infectivity; one was an isoleucine to valine substitution at position 140 between variable regions A and B, and the other lies within the proline rich region. Taken together, these results show that the novel juxtaposition of env gene sequences enhanced the infectivity of PERV-A14/220 for human cells, perhaps by stabilization of the envelope glycoprotein or increased receptor binding.

FIG. 1.

Structure of the PERV-A14/220 genome. PERV-A14/220 is the product of a recombination between PERV-A (striped) and PERV-C (open) that retains the tropism of PERV-A. The PERV-A-derived sequence consists of a maximum of 913 bp corresponding to nucleotides 5374 to 6280 in the PERV-A14/220 sequence. This region encompasses the 3′ end of pol as well as variable regions A and B (VRA and VRB, respectively) but not the PRR of env. Approximate locations of variable regions A and B, the PRR, and the packaging signal (ψ) are indicated.

FIG. 2.

Chronically infected PERV cell lines. (A) Level (cpm) of RT in the supernatant of 293T cells transfected with the following proviral constructs and passaged at 3- to 5-day intervals for approximately 50 days: PERV-A14/220 (A14/220) (1), PERV-Ap60 (A) and PERV-Bp17 (B) (2), and MLV 4070A (5). (B) Infectious titers of chronically infected cell lines, transduced with the p5G2 vector at a multiplicity of infection of >7 at day 40 of culture. Virus-containing supernatant was harvested 2 days later and titrated on human 293 cells. Levels of virus packaging the neomycin resistance gene were measured by G418 selection and represented as infectious per milliliter.

FIG. 3.

Comparison of PERV Gag-Pols. (A) VSV-G pseudotyped particles containing the Gag-Pol cores from PERV-A14/220, PERV-A, PERV-B, or amphotropic MLV were titrated onto human 293 or rat HSN cells. The percentage of transduction was measured by FACS analysis. A representative experiment is shown reflecting transduction with 135 μl of each virus. (B) Ratio of the level of transduction of PERV-A14/220 Gag-Pol to PERV-A Gag-Pol. Results are the means of three independent experiments for 293 and HSN cells.

FIG. 4.

Comparison of PERV Env proteins. Moloney MLV cores were pseudotyped with PERV and amphotropic MLV envelopes. Viruses were titrated on 293 and TE671 cells by G418 selection.

FIG. 5.

Structure and function of chimeric Env proteins. Envelope constructs that mimic the recombination event that occurred in PERV-A14/220 were tested for biological activity by pseudotyping an MLV-based LacZ vector and by titration on 293T cells. Open boxes, PERV-A14/220-derived sequences of PERV-C origin; hashed boxes, PERV-A14/220-derived sequences of PERV-A origin; solid boxes, PERV-A-derived sequences. Constructs are named to identify their plasmids of origin; thus, (A14/220)/(A) env consists of 5′ sequences from a PERV-A14/220-derived sequence and 3′ sequences from PERV-A.

FIG. 6.

Effect of site-directed mutagenesis on residues 48 (D48N) and 140 (I140V) of the chimeric envelopes of PERV-A14/220 and PERV-A. Env function was assayed by LacZ titration. Open boxes, PERV-A14/220-derived sequences of PERV-C origin; hashed boxes, PERV-A14/220-derived sequences of PERV-A origin; solid boxes, PERV-A-derived sequences. Constructs are named as described in the legend of Fig. 5.

FIG. 7.

Analysis of the role of PERV-C-derived sequences in the C-terminal portion of the PERV-A14/220 envelope. Chimeric envelopes were engineered to mimic a recombination event between PERV-A and PERV-C that occurred at the SU/TM boundary or just after the RBD and were tested for function as described in the legend of Fig. 5. The presence of a valine or isoleucine at position 140 is indicated. Open boxes, PERV-A14/220-derived sequences of PERV-C origin; hashed boxes, PERV-A14/220-derived sequences of PERV-A origin; solid boxes, PERV-A-derived sequences. Constructs are named as described in the legend of Fig. 5.

FIG. 8.

Analysis of the role of the PRR. The PCR product between the recombination position in PERV-A14/220 to the end of the PRR was used to produce the PERV-A14/220 and PERV-A envelopes. The 300-bp product was then used as a site-directed mutagenesis primer to produce envelopes with the alternative PRR (indicated by the underscore). The presence of a valine or isoleucine at position 140 is indicated. The titers of the PRR envelopes were determined by LacZ staining on 293 cells. Open boxes, PERV-A14/220-derived sequences of PERV-C origin; hashed boxes, PERV-A14/220-derived sequences of PERV-A origin; solid boxes, PERV-A-derived sequences.

FIG. 9.

Comparison of the PRR region of PERV-A and PERV-A14/220. ClustalW alignment of the section containing the PRR mutated as described in the legend of Fig. 8. Predicted conserved and variable regions of the PRR are shown in the first and second boxes, respectively. Symbols: asterisk, identical; colon, conserved; period, semiconserved.