Changes in viral protein function that accompany retroviral endogenization

Abstract

Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells and have been maintained in whole or part as heritable genomic elements. The last known endogenization events occurred several million years ago, and therefore stepwise analysis of retroviral endogenization has not been possible. A unique opportunity to study this process became available when a full-length ERV isolated from koalas (KoRV) was shown to have integrated into their germ line within the past 100 years. Even though KoRV shares 78% nucleotide identity with the exogenous and highly infectious gibbon ape leukemia virus (GALV), the infectivity of KoRV, like that of other ERVs, is substantially lower than that of GALV. Differences in the protein coding regions of KoRV that distinguish it from GALV were introduced into the GALV genome, and their functional consequences were assessed. We identified a KoRV gagpol L domain mutation as well as five residues present in the KoRV envelope (env) that, when substituted for the corresponding residues of GALV, resulted in vectors exhibiting substantially reduced titers similar to those observed with KoRV vectors. In addition, KoRV env protein lacks an intact CETTG motif that we have identified as invariant among highly infectious gammaretroviruses. Disruption of this motif in GALV results in vectors with reduced syncytia forming capabilities. Functional assessment of specific sequences that contribute to KoRV's attenuation from a highly infectious GALV-like progenitor virus has allowed the identification of specific modifications in the KoRV genome that correlate with its endogenization.

Fig. 1.

Infectivity studies of GALV and KoRV retroviral vectors. (A) Titers of GALV and KoRV enveloped vectors containing different gagpol proteins. MDTFPiT1 cells were exposed to GALV or KoRV enveloped vectors assembled with either GALV or KoRV gagpol proteins. Titers were normalized to GALV env with GALV gagpol vectors (titer, average ± SEM, 2.6 × 10⁶ ± 9.3 × 10⁵) and were determined by averaging the number of blue-forming units (bfu) per milliliter of vector supernatant from at least three independent experiments ± SEM. (B) Alignment of part of GALV gagpol and KoRV gagpol with two L domains shown in boxes. The two amino acids as encoded by KoRV gagpol and targeted for mutagenesis in GALV gagpol are highlighted in yellow. Residue numbers for GALV gagpol. (C) MDTFPiT1 cells were exposed to GALV or KoRV enveloped vectors assembled with mutant GALV/SRLPIY gagpol proteins. Titers were normalized as described in A.

Fig. 2.

Disruption of the conserved CETTG motif in GALV envelope results in reduced cytopathogenicity in target cells. (A) MDTFPiT1 cells were exposed to vectors composed of GALV gagpol and GALV env or mutant GALV/CETAG or GALV/CGTAG env. Seventy-two hours after the cells were exposed to vectors, syncytia were determined by counting the number of cells containing four or more nuclei. Data were derived from five independent experiments and are normalized to the number of syncytia observed on MDTFPiT1 cells exposed to GALV enveloped particles ± SEM. (B) Structural model of Friend MLV RBD (Protein Data Bank ID code 1AOL). The CETTG motif is shown as spheres. (C) CETTG and mutant CETAG and CGTAG motifs shown in spheres. The CETAG T135A and CGTAG E133G and T135A mutations are shown element color mode. Mutations are indicated with arrowheads. Depictions of structural models were made by using the PyMol (DeLano Scientific, Palo Alto, CA) software program.

Fig. 3.

Infectivity studies of mutated KoRV and GALV envelope retroviral vectors. (A) Partial alignment of individual GALV envelope proteins GALV SEATO, GALV SF, GALV Brain, and GALV HI (Hall's Island). Residues highlighted in yellow are those that vary among the four proteins. (B) Partial alignment of KoRV and GALV SEATO mature env proteins. Schematic representation of the mature envelope protein is shown above alignment to designate where the aligned residues are found. Residue numbers are for GALV mature envelope. Highlighted residues represent groups of mutations made in the respective wild-type envs. The mutants are color coded as follows: GTDV residues 59–62 (orange); SKRV residues 64, 66–68 (yellow); DTAK residues 74, 76, 77, 80 (blue); AI residues 86, 87 (black); MST residues 98, 100, 102 (purple); LSK residues 141–142 (pink). (C) Titers on MDTFPiT1 cells exposed to GALV (black), KoRV (gray), or KoRV/AI (white) enveloped vectors. Normalized titers of KoRV/AI enveloped vectors containing one additional group of mutations are color coded to correspond to the mutations shown in Fig. 3B. All vectors were assembled with GALV gagpol. Vectors bearing KoRV/AI envelopes wherein the GALV DTAK (blue) residues were substituted failed to assemble into infectious particles (data not shown). Titers were normalized to that of GALV env with GALV gagpol and determined as described in Fig. 1. (D) Vector titers of GALV, mutant GALV/QPR, or GALV/TL/QPR env assembled with GALV gagpol. Vectors bearing GALV/TL+QPR env and GALV gagpol failed to assemble into infectious particles. (E) Vector titers on MDTFPiT1 cells exposed to GALV env or GALV/TL env with GALV gagpol or mutant GALV/PRLPIY gagpol vectors. Titers were normalized to that of GALV env with GALV gagpol and determined as described for Fig. 1. (F) Alignment of KoRV and GALV envelope residues 51–151 with residues in the KoRV envelope that have been shown to be critical for the attenuation of envelope functions are highlighted in yellow.