Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

We have constructed a replication-competent gammaretrovirus (SL3-AP) capable of using the human G-protein-coupled receptor hAPJ as its entry receptor. The envelope protein of the virus was made by insertion of the 13-amino-acid peptide ligand for hAPJ, flanked by linker sequences, into one of the variable loops of the receptor binding domain of SL3-2, a murine leukemia virus (MLV) that uses the xenotropic-polytropic virus receptor Xpr1 and which has a host range limited to murine cells. This envelope protein can utilize hAPJ as well as murine Xpr1 for entry into host cells with equal efficiencies. In addition, the SL3-AP virus replicates in cells expressing either of its receptors, hAPJ and murine Xpr1, and causes resistance to superinfection and downregulation of hAPJ in infected cells. Thus, SL3-AP is the first example of a retargeted replication-competent retrovirus, with replication characteristics and receptor interference properties similar to those of natural isolates.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Construction of a Gammaretrovirus with a Novel Tropism and Wild-Type Replication Kinetics Capable of Using Human APJ as Entry Receptor

Bahrami

Pagh

Ejegod

et al. 2012

“…In addition, Li and colleagues have successfully replaced the receptor-binding domain of Mo-MLV with the ligand of the somatostatin receptor and by this inducing viral infection through the somatostatin receptor with titers equal to that of the wild-type virus (14). However, a later report showed that postbinding restrictions existed, depending on the somatostatin receptor used (15).…”

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confidence: 99%

Directed Molecular Evolution of an Engineered Gammaretroviral Envelope Protein with Dual Receptor Use Shows Stable Maintenance of Both Receptor Specificities

Friis

Iturrioz

Thomsen

et al. 2016

We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization. IMPORTANCEFew successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry. F usion of lipid membranes is not a spontaneous event and thus requires a supply of energy through an active fusion mechanism. Retroviruses are enveloped viruses and hence are strictly dependent on their fusion machinery for entry into host cells. In retroviruses, including murine leukemia virus (MLV), the fusion energy is stored in the envelope (Env) protein that is present in the membrane envelope surrounding the viral particle. Env consists of trimers of two subunits, the N-terminal surface (SU) subunit and the C-terminal transmembrane (TM) subunit. SU is responsible for recognizing and binding cellular receptor proteins, which differ among virus types and define their tropism. The SU and TM subunits remain associated through noncovalent interactions or, as for MLV, through a disulfide bridge that is broken as a result of disulfide isomerization following receptor binding (1). Here, the fusion energy stored in TM and suppressed by SU is released by conformational changes allowing the formation of an elongated triple helix in TM. This leads to insertion of the fusion peptide into the membrane of the host cell and hence induces membrane fusion (2), as reviewed in references 3 and 4. Other reports have suggested a need for proteolytic cleavage of SU in e...

“…Its parent glycoprotein initiates infection by binding to the ecotropic MLV receptor, mCAT-1 (mouse cationic amino acid transporter 1) (14-16), forming MLV-mCAT-1 complexes that are internalized (17). The modifications that created Sst-RBS resulted in a switch from use of mCAT-1 to the use of somatostatin receptors (13), a family of G protein-coupled receptors that are not entry receptors for any known virus and ordinarily recognize SST-14 and its peptide mimetics. SST-14 is an agonist to five somatostatin receptors (SSTR), subtypes 1 through 5, each encoded by a distinct gene (18).…”

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confidence: 99%

Characteristics of the Cellular Receptor Influence the Intracellular Fate and Efficiency of Virus Infection

Krueger

Albritton

2013

Self Cite

The intracellular fate of internalized virus-receptor complexes is suspected of influencing the efficiency of virus infection. However, direct evidence of a link between infection and the fate of internalized virus has been difficult to obtain. To directly address this question, we generated human 293 cell lines stably expressing comparable cell surface levels of three different members of the somatostatin receptor family (SSTR) which have natural differences in intracellular trafficking. Utilizing a glycoprotein that recognizes SSTR, we found that distinctive receptor subtype-specific destinations correlated with observable differences in the level of infection. Infection via SSTR-2 and -3 is restricted at a point after receptor binding and endocytosis but prior to penetration into the host cytoplasm. In contrast, entry via SSTR-5 featured a slower internalization with greater dependence on cholesterol. Quantitative real-time PCR showed that virus bound to SSTR-5 was directed to an intracellular environment that allowed near-wild-type (WT) levels of penetration, possibly due to a more favorable complement of host cell proteases, whereas SSTR-2 and -3 directed virions to a degradative compartment in which cytosol penetration was less efficient. Taken together, the results support that the superior receptor capacity of SSTR-5 results from its internalization into a cellular compartment that is more favorable to the cytoplasmic penetration of viral cores and reverse transcription. They suggest that the intracellular destination of internalized complexes is an important characteristic of a virus receptor and may have exerted a selective pressure on the choice of an entry receptor during evolution of viral glycoproteins.