J Virol 77(12): 6709-6719

Mechanisms of Avian Retroviral Host Range Extension

Department of Biochemistry and Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111

^{Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-6528. Fax: (617) 636-4086. E-mail:}ude.stfut@niffoc.nhoj.

^{Present address: McArdle Laboratory for Cancer Research, University of Wisconsin—Madison, Madison, WI 53706.}

Received 2002 Nov 15; Accepted 2003 Mar 21.

Abstract

Alpharetroviruses provide a useful system for the study of the molecular mechanisms of host range and receptor interaction. These viruses can be divided into subgroups based on diverse receptor usage due to variability within the two host range determining regions, hr1 and hr2, in their envelope glycoprotein SU (gp85). In previous work, our laboratory described selection from a subgroup B avian sarcoma-leukosis virus of an extended-host-range variant (LT/SI) with two adjacent amino acid substitutions in hr1. This virus retains its ability to use the subgroup BD receptor but can also infect QT6/BD cells, which bear a related subgroup E receptor (R. A. Taplitz and J. M. Coffin, J. Virol 71:7814-7819, 1997). Here, we report further analysis of this unusual variant. First, one (L154S) of the two substitutions is sufficient for host range extension, while the other (T155I) does not alter host range. Second, these mutations extend host range to non-avian cell types, including human, dog, cat, mouse, rat, and hamster. Third, interference experiments imply that the mutants interact efficiently with the subgroup BD receptor and possibly the related subgroup E receptor, but they have another means of entry that is not dependent on these interactions. Fourth, binding studies indicate that the mutant SU proteins retain the ability to interact as monomers with subgroup BD and BDE receptors but only bind the subgroup E receptor in the context of an Env trimer. Further, the mutant SU proteins bind well to chicken cells but do not bind any better than wild-type subgroup B to QT6 or human cells, even though the corresponding viruses are capable of infecting these cells.

Abstract

Alpharetroviruses, or avian sarcoma-leukosis viruses (ASLVs), display a great deal of diversity in their envelope glycoprotein (Env) sequences leading to diverse host range but, with the exception of the long terminal repeat (LTR), are nearly identical throughout the remainder of their genomes (11, 15, 16, 25-27, 29, 42). This pattern suggests a response to selective pressures to replicate in a variety of hosts. Alpharetroviruses are divided into subgroups (A to J) depending on host range, superinfection resistance patterns, and neutralizing antibody cross-reactivity. The surface (SU) subunit of the envelope glycoprotein is responsible for receptor recognition. Through a poorly characterized process, probably requiring a low-pH step (34), the SU-receptor interaction triggers the transmembrane (TM) subunit to mediate fusion between the viral envelope and the target cell membrane (10, 26, 48).

Receptors for ASLV of subgroups A, B, D, and E have been cloned. The receptor for subgroup A viruses is a low-density lipoprotein receptor-like protein and is unrelated to any other known retroviral receptor (5, 51). The receptors for B, D, and E are encoded by orthologous genes in the tumor necrosis factor receptor family (2, 3, 8, 40). Chickens have two alleles capable of acting as the receptor for these viruses. The tv-b^s1 allele can serve as a receptor for all three subgroups. Infection with virus of subgroups B or D blocks superinfection by all three of these subgroups. Infection with subgroup E virus blocks superinfection by virus of subgroup E but allows subsequent infection by B or D virus (26, 48). This nonreciprocal interference probably reflects the presence of two different conformational forms of the receptor on the cell surface. Subgroup B and D viruses can recognize both forms, while subgroup E viruses can only recognize one (1). The second allele, tv-b^s3, can serve as a receptor for subgroups B and D only. Quail, turkey, and some related birds have an allele for a third type of receptor, tv-b^q or tv-b^t, conferring susceptibility to infection by subgroup E but not subgroup B and D viruses (2, 13, 14, 26, 35, 48). Cells used for these studies are designated by the first letter of the species from which they are derived and the classical alpharetrovirus subgroups (A to E) to which the cells are resistant. For example, C/E are chicken cells that are resistant to infection by subgroup B alpharetroviruses and therefore susceptible to infection by subgroup A, B, C, and D alpharetroviruses.

Our laboratory has previously shown that determinants of host range and receptor recognition lie predominantly within two short stretches of gp85 SU called hr1 and hr2 (15, 16) (Fig. (Fig.1).1). We have described a chimeric ASLV, NTRE4, which has a recombinant envelope that has a subgroup E hr2 but otherwise consists of subgroup B sequences. Unlike either parent, this virus can infect both C/E and Q/BD cells. It exhibits reciprocal interference with subgroup B viruses on C/E chicken embryo fibroblasts (CEF) and with subgroup E viruses on quail and turkey cells, indicating that it makes use of both the subgroup B and subgroup E receptors (47). Our laboratory has also described a mutant virus, LT154/155SI (S20) (43), that is derived from a subgroup B virus selected for host range extension to quail cells. It contains two amino acid substitutions in hr1 that are sufficient for this host range extension (Fig. (Fig.1).1). In the study reported here, we have examined these two substitutions individually for host range extension and measured binding and interference patterns among these mutants, wild-type subgroup B and E viruses, and NTRE4. Finally, we examined the ability of these viruses to infect a panel of cell lines from diverse species. We found that the L154S mutation alone suffices for the extension of host range and that virus containing this mutation is also capable of infecting cell lines of human, dog, cat, and to a lesser extent mouse, rat, and hamster origin. Surprisingly, the large increase in the ability of the mutant viruses to infect these cells was not accompanied by a detectable increase in binding of their SU proteins. However, binding to one candidate receptor, TVB-T, was detectable when the envelope glycoproteins were expressed on the cell surface. These findings suggest novel modes of interaction with receptors and perhaps entry pathways that are receptor independent.

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FIG. 1.

Schematic representation of the alpharetrovirus gp85 SU. Sequences shown to be important for host range determination are represented by boxes (black for subgroup B and hashed for subgroup E). vr1, vr2, and vr3 are variable regions, and hr1 and hr2 are highly variable host-range-determining regions. NTRE4 is a chimeric virus resulting in recombination between td-PrRSV-B and RAV-0. The location of the point mutations studied here is indicated at the bottom in the hr1 rectangle. WT subgroup E (RAV-0), subgroup B (td-PrRSV-B), and the mutant amino acid sequences are listed below. The host range phenotypes are listed on the right (43, 47).

Acknowledgments

We are grateful to J. A. T. Young, J. Brojatsch, and H. Adkins for cells, receptors, immunoadhesins, and helpful discussions. We thank C. Bencsics for extensive discussions and M. Bostic-Fitzgerald for administrative and editorial assistance.

This work was supported by grant R35CA 44385 from the National Cancer Institute. J.M.C. is a research professor of the American Cancer Society.

Acknowledgments

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