The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types. IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

The fiber knob protein of human adenovirus type 49 mediates highly efficient and promiscuous infection of cancer cell lines using a novel cell entry mechanism

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied.Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, whilst potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumour types.ImportanceAdenoviruses are powerful tools experimentally and clinically. To maximise efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focussed on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism.We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

Rho GTPases Modulate Entry of Ebola Virus and Vesicular Stomatitis Virus Pseudotyped Vectors

ABSTRACT To explore mechanisms of entry for Ebola virus (EBOV) glycoprotein (GP) pseudotyped virions, we used comparative gene analysis to identify genes whose expression correlated with viral transduction. Candidate genes were identified by using EBOV GP pseudotyped virions to transduce human tumor cell lines that had previously been characterized by cDNA microarray. Transduction profiles for each of these cell lines were generated, and a significant positive correlation was observed between RhoC expression and permissivity for EBOV vector transduction. This correlation was not specific for EBOV vector alone as RhoC also correlated highly with transduction of vesicular stomatitis virus GP (VSVG) pseudotyped vector. Levels of RhoC protein in EBOV and VSV permissive and nonpermissive cells were consistent with the cDNA gene array findings. Additionally, vector transduction was elevated in cells that expressed high levels of endogenous RhoC but not RhoA. RhoB and RhoC overexpression significantly increased EBOV GP and VSVG pseudotyped vector transduction but had minimal effect on human immunodeficiency virus (HIV) GP pseudotyped HIV or adeno-associated virus 2 vector entry, indicating that not all virus uptake was enhanced by expression of these molecules. RhoB and RhoC overexpression also significantly enhanced VSV infection. Similarly, overexpression of RhoC led to a significant increase in fusion of EBOV virus-like particles. Finally, ectopic expression of RhoC resulted in increased nonspecific endocytosis of fluorescent dextran and in formation of increased actin stress fibers compared to RhoA-transfected cells, suggesting that RhoC is enhancing macropinocytosis. In total, our studies implicate RhoB and RhoC in enhanced productive entry of some pseudovirions and suggest the involvement of actin-mediated macropinocytosis as a mechanism of uptake of EBOV GP and VSVG pseudotyped viral particles.

In Vivo Gene Transfer Using a Nonprimate Lentiviral Vector Pseudotyped with Ross River Virus Glycoproteins

ABSTRACT Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 × 108 TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.

Gene Transfer into Marrow Repopulating Cells: Comparison Between Amphotropic and Gibbon Ape Leukemia Virus Pseudotyped Retroviral Vectors in a Competitive Repopulation Assay in Baboons

Many diseases might be treated by gene therapy targeted to the hematopoietic system, but low rates of gene transfer achieved in humans and large animals have limited the application of this technique. We have developed a competitive hematopoietic repopulation assay in baboons to evaluate methods for improving gene transfer and have used this method to compare gene transfer rates for retroviral vectors having an envelope protein (pseudotype) from amphotropic murine retrovirus with similar vectors having an envelope protein derived from gibbon ape leukemia virus (GALV). We hypothesized that vectors with a GALV pseudotype might perform better based on our previous work with cultured human hematopoietic cells. CD34+ marrow cells from each of four untreated baboons were divided into two equal portions that were cocultivated for 48 hours with packaging cells producing equivalent titers of either amphotropic or GALV pseudotyped vectors containing the neo gene. The vectors contained small sequence differences to allow differentiation of cells genetically marked by the different vectors. Nonadherent and adherent cells from the cultures were infused into animals after they received a myeloablative dose of total body irradiation. Polymerase chain reaction (PCR) analysis for neo gene-specific sequences in colony-forming unit–granulocyte-macrophage from cell populations used for transplant showed gene transfer rates of 2.7%, 7.1%, <15%, and 3.9% with the amphotropic vectors and 7.1%, 11.3%, <15%, and 26.4% with the GALV pseudotyped vector. PCR analysis of peripheral blood and marrow cells after engraftment showed the neo gene to be present in all four animals analyzed at levels between 0.1% and 5%. Overall gene transfer efficiency was higher with the GALVpseudotyped vector than with the amphotropic vectors. Southern blot analysis in one animal confirmed a gene transfer efficiency of between 1% and 5%. The higher gene transfer efficiency with the GALV-pseudotyped vector correlated with higher levels of GALV receptor RNA compared with the amphotropic receptor in CD34+ hematopoietic cells. These results show that GALV-pseudotyped vectors are capable of transducing baboon marrow repopulating cells and may allow more efficient gene transfer rates for human gene therapy directed at hematopoietic cells. In addition, our data show considerable differences in gene transfer efficiency between individual baboons, suggesting that a competitive repopulation assay will be critical for evaluation of methods designed to improve gene transfer into hematopoietic stem cells.