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Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians - PubMed

Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians

Sandra Blaise et al. J Virol. 2004 Jan.

Abstract

A member of the HERV-W family of human endogenous retroviruses (HERV) had previously been demonstrated to encode a functional envelope which can form pseudotypes with human immunodeficiency virus type 1 virions and confer infectivity on the resulting retrovirus particles. Here we show that a second envelope protein sorted out by a systematic search for fusogenic proteins that we made among all the HERV coding envelope genes and belonging to the HERV-FRD family can also make pseudotypes and confer infectivity. We further show that the orthologous envelope genes that were isolated from simians-from New World monkeys to humans-are also functional in the infectivity assay, with one singular exception for the gibbon HERV-FRD gene, which is found to be fusogenic in a cell-cell fusion assay, as observed for the other simian envelopes, but which is not infectious. Sequence comparison of the FRD envelopes revealed a limited number of mutations among simians, and one point mutation-located in the TM subunit-was shown to be responsible for the loss of infectivity of the gibbon envelope. The functional characterization of the identified envelopes is strongly indicative of an ancestral retrovirus infection and endogenization, with some of the envelope functions subsequently retained in evolution.

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Figures

FIG. 1.
FIG. 1.

Infection assays using retroviral particles pseudotyped with the HERV-FRD and control envelopes. (A) Rationale of the assay. To determine whether the HERV-FRD envelope could confer infectivity on MLV, HIV, or SIV Env-defective pseudoparticles, corresponding pseudotype viruses were produced by cotransfecting 7.5 × 105 293T cells with 0.55 μg of the phCMV-HERV-FRD vector expressing the HERV-FRD envelope (or pcDNA3 for the negative control and phCMVampho for the amphotropic MLV envelope positive control); 1.75 μg of a vector encoding the retroviral proteins (except the envelope) of MLV (phCMVintron) (13), HIV (pCMVΔR8.91) (20), or SIV (pSIV3+) (13); and 1.75 μg of the corresponding defective retroviral vectors marked with a β-galactosidase reporter gene (pMFGsnlslacZ [7], pHR′CMVlacZ [20], and R9SA [14]), by calcium phosphate precipitation (Invitrogen). At 36 h posttransfection, viral supernatants were collected and filtered through 0.45-μm-pore-size membranes. Target cells (murine 3T3; feline G355-5; or human TE671, HeLa, and 293T cells) were seeded in 24-well plates at a density of 104 cells per well and incubated overnight at 37°C. Five to 500 μl of virus samples containing 4 μg of Polybrene per ml was added to the cells and centrifuged for spinoculation at 1,200 × g for 2 h 30 min at 25°C (10). After removal of the supernatants, the cells were incubated in regular medium for 60 h at 37°C. Viral titers were then measured by X-Gal (5-bromo-4-chloro-3-indolylphosphate) staining of the cells and expressed as lacZ CFU per milliliter of viral supernatant. (B) Infectivity of SIV-based retroviral particles pseudotyped with the HERV-FRD envelope (or with no protein in the “SIV-None” control), as assayed using human 293T cells as target cells and X-Gal staining, following the protocol described for panel A.

FIG. 2.
FIG. 2.

Infection and cell-cell fusion activities and host range of the HERV-FRD and simian orthologous envelopes. Upper panel: infection of feline (G355-5), human (293T and HeLa), and murine (3T3) cells by SIV-based particles pseudotyped with the indicated ERV-FRD envelopes. Experimental conditions were the same as in Fig. 1, with negative and positive control values as in Table 1. NWM, New World monkey (Callithrix jacchus marmoset). Lower panel: cell-cell fusion assay for the ERV-FRD envelopes and cells as in the upper panel. The G355-5 and HeLa cells were transfected using Lipofectamine (Invitrogen; 2 μg of DNA for 5 × 105 cells), and the 293T and TE671 cells were transfected using calcium phosphate precipitation (Invitrogen; 5 μg of DNA for 5 × 105 cells). Fusion activities were measured 12 to 36 h posttransfection of the corresponding expression vectors. To visualize syncytia, cells were fixed in methanol and stained by adding May-Grünwald and Giemsa solutions (Sigma) according to the manufacturer's instructions. The fusion index, which represents the percentage of fusion events in a cell population, is defined as [(N − S)/T] × 100, where N is the number of nuclei in the syncytia, S is the number of syncytia, and T is the total number of nuclei counted.

FIG. 3.
FIG. 3.

Comparison of the simian ERV-FRD envelopes and identification of a unique point mutation responsible for the gibbon envelope loss of function. (A) Aligned primary sequences of the HERV-FRD and simian orthologous envelopes. Abbreviations: Hu, Homo sapiens; Cpz, chimpanzee; Go, gorilla; Oo, orangutan; Gib, gibbon; Mcq, macaque; Nwm, New World monkey. Accession numbers are AL136139 for the human gene and AJ577595 to AJ577600 for the simian genes. The RVRR canonical motif for SU-TM cleavage is highlighted, and the predicted transmembrane hydrophobic domain is boxed. Amino acids differing between the human and gibbon proteins are indicated with an asterisk, among which those unique to the gibbonsequence are shaded. The two amino acids in the TM moiety of the gibbon protein that were mutated and assayed for reversion to the infectious phenotype are indicated with an arrow. (B) Infection of feline (G355-5) and human (293T) cells by SIV-based particles pseudotyped with the wild-type (wt) and mutant (F490→L or T532→N) gibbon FRD envelopes, with the human FRD envelope as a control. The indicated amino acid changes in the gibbon envelope were performed by a three-fragment ligation, using PCR fragments generated with oligonucleotides containing the gibbon-to-human single-base mutations (TTT→CTT and AAT→ACT for amino acid positions 490 and 532, respectively), and subsequent sequencing of the resulting constructs. Infection assays were performed as described for Fig. 1. The viral titers are averages from two independent experiments.

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