Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

Abstract

The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the aminoterminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the aminoterminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.Cell entry by retroviruses is mediated by the virally encoded Env glycoprotein on the virus surface. The Env protein of Moloney murine leukemia virus (MoMLV), the best-characterized mammalian type C retrovirus, is synthesized as a single 85-kDa precursor that includes an amino-terminal signal sequence that directs its insertion into the lumen of the endoplasmic reticulum (ER) (61). During transport to the cell surface, the Env precursor undergoes a series of posttranslational modifications, including glycosylation, oligomerization, disulfide bond formation and cleavage into surface (SU) and transmembrane (TM) subunits (19,46,47,48). At the cell surface, the Env trimer is incorporated into the membrane of the budding virion, where it is further modified by the virally encoded protease, which removes the carboxy-terminal 16 amino acids (the R-peptide) of TM to yield the entry-competent glycoprotein (22, 50, 51, 52).The entry process is initiated by the specific interaction of the Env protein with the receptor protein MCAT-1, which is located on the surface of a susceptible cell (1). Receptorbound virion particles are then internalized, probably by clathrin-independent endocytosis (38). By analogy with the influenza hemagglutinin (HA) fusion protein, the reduced pH within the endosome mediates a conformational change in the Moloney Env protein that exposes the fusion peptide at the amino terminus of TM (55). Fusion, initiated by the insertion of the fusion peptide into the endosomal membrane, permits ...

Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting

“…We have used this technique for high-resolution functional mapping of the bacterial supF gene (3) and HIV coreceptor CCR5 (4). It has also been used to analyze the 5Ј end of HIV (5) and MoMLV envelope sequences (6).…”

Stoichiometry of Murine Leukemia Virus Envelope Protein-Mediated Fusion and Its Neutralization

“…Moloney MLV Env constructs expressing Env with a 2F5 epitope inserted in SU or TM or an HA epitope in SU have been described previously (25). Briefly, an extended 2F5 epitope (or an HA epitope where it applies) was inserted at a unique SgrAI site in the SU gene, in a region that encodes a proline-rich segment previously shown to tolerate small insertions (15,34,42), or inserted 14 amino acids upstream of the transmembrane domain of TM, which is the location of the natural 2F5 epitope in HIV-1. Cells expressing these Envs specifically fuse with target cells expressing the ecotropic MLV receptor mCAT1 (mouse cationic amino acid transporter 1) (1).…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…Both rational ligand insertion and random peptide display require knowledge of insertion sites that do not compromise envelope function and are exposed on the protein surface for receptor binding Roth, 2002, 2003;Bupp et al, 2005Bupp et al, , 2006. However, if information on such permissive insertion sites is not available, it can be also newly obtained by transposon-based scanning of envelope proteins (Rothenberg et al, 2001;Yu and Schaffer, 2006b). The insertion and excision of a bacteriophage Mu transposase cassette into cDNA encoding the Moloney murine leukemia virus (MoMLV) envelope protein resulted in the duplication of five amino acids at a random insertion site.…”

“…The insertion and excision of a bacteriophage Mu transposase cassette into cDNA encoding the Moloney murine leukemia virus (MoMLV) envelope protein resulted in the duplication of five amino acids at a random insertion site. Subsequent selection of the resulting mutant library for the ability to mediate viral replication in culture led to the identification of multiple sites permissive for the five amino acid insertion (Rothenberg et al, 2001). More recently, a 13 amino acid sequence, including a 6 histidine peptide, was randomly inserted into envelope proteins of vesicular stomatitis virus (VSVG), and the selection of the resulting library for the ability to mediate viral replication in culture and for the capacity to bind an immobilized metal column led to the identification of several permissive insertion sites on the outer surface of VSVG (Yu and Schaffer, 2006b).…”