Date Published: June 9, 2016
Publisher: Public Library of Science
Author(s): Ruben R. Bender, Anke Muth, Irene C. Schneider, Thorsten Friedel, Jessica Hartmann, Andreas Plückthun, Andrea Maisner, Christian J. Buchholz, Richard K Plemper.
Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.
Cell entry as first step in the viral replication cycle is initiated by the attachment of virus particles to distinct cell surface proteins. While many viral receptors have been identified, there is only limited knowledge available about the molecular requirements that cell surface proteins have to fulfill to act as entry receptors and why they have been chosen during viral evolution . Paramyxoviruses encode two envelope proteins required for cell entry, the receptor attachment protein and the fusion protein (F) which mediates fusion of the viral and cellular membranes upon receptor contact. Three types of attachment proteins can be distinguished, the hemagglutinin-neuraminidase (HN), the hemagglutinin (H) and the glycoprotein (G), which in contrast to the others has no hemagglutinating function. All attachment proteins are type II membrane proteins with a membrane proximal stalk domain and a propeller-like head domain . While HN proteins use sialic acid as receptor, morbillivirus H and henipavirus G recognize proteinaceous receptors. Due to this and its separated attachment and fusion functions, the measles virus (MV) H protein has been the first viral attachment protein that was successfully engineered to use a cell surface protein of choice for entry instead of its natural receptor .
Here we describe successful engineering of the NiV glycoproteins for LV pseudotyping and receptor targeting, which allowed us to rapidly generate a large series of glycoprotein variants attaching to a variety of cell surface proteins and assessing cell entry. For pseudotyping, distinct truncations in G (Gc∆33 and Gc∆34) and F protein (Fc∆22) were found to be optimal. Our data are thus in line with those of Witting et al (2013) for G protein, and with those of Palomares et al. (2012) for F protein. For both, F and G, the enhanced titers correlated well to an enhanced incorporation into LV particles, suggesting steric hindrance as likely explanation for the need for cytoplasmic tail truncations.