Adenovirus Serotype 7 Retention in a Late Endosomal Compartment prior to Cytosol Escape Is Modulated by Fiber Protein

Abstract

The intracellular trafficking of adenovirus (Ad) subgroup B (e.g., Ad7) differs from that of subgroup C (e.g., Ad5) in that Ad5 rapidly escapes from endocytic compartments following infection whereas Ad7 accumulates in organelles. To assess the hypothesis that Ad7 is targeted to the lysosomal pathway, Ad7 and Ad5 were conjugated with fluorophores and their trafficking in A549 epithelial cells was analyzed by fluorescence microscopy. Within 1 h after infection, Ad7, but not Ad5, accumulated in the cytoplasm of A549 cells. The pH in the environment of Ad5 was nearly neutral (pH 7), while Ad7 occupied acidic compartments (pH 5) over the first 2 h with a gradual shift toward neutrality by 8 h. Ad7 partially colocalized with ␣ 2 -macroglobulin and late endosomal and lysosomal marker proteins, including Rab7, mannose-6-phosphate receptor, and LAMP-1. The pH optimum for membrane lysis by Ad7, as well as a chimeric Ad5 capsid that expressed the Ad7 fiber (Ad5fiber7), was pH 5.5, while that for lysis by Ad5 was pH 6.0. Thus, the native trafficking pathway for Ad7 involves residence in late endosomes and lysosomes, with information encoded in the Ad7 fiber acting as a pH-dependent trigger for membrane lysis and escape to the cytosol.

Employment of microarray analysis to characterize biologic differences associated with tropism-modified adenoviral vectors: utilization of non-native cellular entry pathways

“…Modified vector-tropism alters not only the primary receptor engaged but, in selected cases, also the overall entry pathway of the virion. 21 This consideration dictated our choice of a later time point for analysis. Our observation of clear differences between the tested Ads validates this approach.…”

“…As previously noted, Ad tropism alterations can result not just in differential employment of primary receptor but also can route the virus via an entirely alternate internalization pathway. 21 This consideration may explain the magnitude of genetic changes observed. However, other variables may contribute or be associated with the Figure 2 Venn diagram illustrating the numerical relationship between Ad5luc1, Ad5lucRGD, and Ad5/3luc1 responsive genes in the M21 human melanoma cell line.…”

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…47 In addition to cellular binding, the fiber and its knob domain have been suggested to carry several functions important for intracellular trafficking, membrane lysis and virus maturation. [20][21][22][23][24][25] Fiber content of FibDCAR-HI-Link-ZHZH viruses was 12-29% of WT, which is an improvement from earlier studies where knobless virions have had fiber content of 2-10%. 10 Another improvement of Ad5/FibDCAR-HI-Link-ZHZH was the PP-to-PFU ratio, which was 140, as compared to 70 for the WT virus.…”

“…12,40 These growth defects can be explained by a low-fiber content caused by the lack of the fiber knob hampering virus entry and intracellular trafficking and a loss of other growth -or viral assembly functions carried by the fiber knob. [20][21][22][23][24][25] Only in the de-knobbed virus Ad5/R7-C2C2, containing the Ig binding C2 domain from Streptococcal protein G, we found a normal fiber content indicating that this fiber somehow compensates, as far as fiber content of the virus is concerned, for the loss of the fiber knob. 40 Therefore, a novel approach was also employed in the present study.…”

“…10,12 The poor performance of viruses with de-knobbed fibers is due to the fact that the knob has other important functions in the life cycle of the virus than just binding to the cellular receptor. [20][21][22][23][24][25] These facts make such viruses difficult to use for gene therapeutic purposes.…”