Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions.

The development of tissue-selective virus-based vectors requires a better understanding of the role of receptors in gene transfer in vivo, both to rid the vectors of their native tropism and to introduce new specificity. CAR and alphav integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. We have constructed a set of four vectors, which individually retain the wild-type cell interactions, lack CAR binding, lack alphav integrin binding, or lack both CAR and alphav integrin binding. These vectors have been used to examine the roles of CAR and alphav integrin in determining the tropism of Ad vectors in a mouse model following intrajugular or intramuscular injection. CAR was found to play a significant role in liver transduction. The absence of CAR binding alone, however, had little effect on the low level of expression from Ad in other tissues. Binding of alphav integrins appeared to have more influence than did binding of CAR in promoting the expression in these tissues and was also found to be important in liver transduction by Ad vectors. An effect of the penton base modification was a reduction in the number of vector genomes that could be detected in several tissues. In the liver, where CAR binding is important, combining defects in CAR and alphav integrin binding was essential to effectively reduce the high level of expression from Ad vectors. While there may be differences in Ad vector tropism among species, our results indicate that both CAR and alphav integrins can impact vector distribution in vivo. Disruption of both CAR and alphav integrin interactions may be critical for effectively reducing native tropism and enhancing the efficacy of specific targeting ligands in redirecting Ad vectors to target tissues.

Identification of a conserved receptor-binding site on the fiber proteins of CAR-recognizing adenoviridae.

The human adenovirus serotype 5 (Ad5) is used widely for applications in human gene therapy. Cellular attachment of Ad5 is mediated by binding of the carboxyl-terminal knob of its fiber coat protein to the Coxsackie adenovirus receptor (CAR) protein. However, Ad5 binding to CAR hampers the development of adenovirus vectors capable of specifically targeting (diseased) tissues or organs. Through sequence analysis and mutagenesis, a conserved receptor-binding region was identified on the side of three divergent CAR-binding knobs. The feasibility of simultaneous CAR ablation and redirection of an adenovirus to a new receptor is demonstrated.

Construction of a pseudoreceptor that mediates transduction by adenoviruses expressing a ligand in fiber or penton base.

Modification of adenovirus to achieve tissue specific targeting for the delivery of therapeutic genes requires both the ablation of its native tropism and the introduction of specific, novel interactions. Inactivation of the native receptor interactions, however, would cripple the virus for growth in production cells. We have developed an alternative receptor, or pseudoreceptor, for the virus which might allow propagation of viruses with modified fiber proteins that no longer bind to the native adenovirus receptor (coxsackievirus/adenovirus receptor [CAR]). We have constructed a membrane-anchored single-chain antibody [m-scFv(HA)] which recognizes a linear peptide epitope (hemagglutinin [HA]). Incorporation of HA within the HI loop of the fiber protein enabled the modified virus to transduce pseudoreceptor expressing cells under conditions where fiber-CAR interaction was blocked or absent. The pseudoreceptor mediated virus transduction with an efficiency similar to that of CAR. In addition, the HA epitope mediated virus transduction through interaction with the m-scFv(HA) when it was introduced into penton base. These findings indicate that cells expressing the pseudoreceptor should support production of HA-tagged adenoviruses independent of retaining the fiber-CAR interaction. Moreover, they demonstrate that high-affinity targeting ligands may function following insertion into either penton base or fiber.

Mutational analysis of the oligomer assembly domain in the transmembrane subunit of the Rous sarcoma virus glycoprotein.

The transmembrane (TM) subunits of retroviral envelope glycoproteins appear to direct the assembly of the glycoprotein precursor into a discrete oligomeric structure. We have examined mutant Rous sarcoma virus envelope proteins with truncations or deletions within the ectodomain of TM for their ability to oligomerize in a functional manner. Envelope proteins containing an intact surface (SU) domain and a TM domain truncated after residue 120 or 129 formed intracellular trimers in a manner similar to that of proteins that had an intact ectodomain and were efficiently secreted. Whereas independent expression of the SU domain yielded an efficiently transported molecule, proteins containing SU and 17, 29, 37, 59, 73, 88, and 105 residues of TM were defective in intracellular transport. With the exception of a protein truncated after residue 88 of TM, the truncated proteins were also defective in formation of stable trimers that could be detected on sucrose gradients. Deletion mutations within the N-terminal 120 amino acids of TM also disrupted transport to the Golgi complex, but a majority of these mutant glycoproteins were still able to assemble trimers. Deletion of residues 60 to 74 of TM caused the protein to remain monomeric, while a deletion C terminal of residue 88 that removed two cysteine residues resulted in nonspecific aggregation. Thus, it appears that amino acids throughout the N-terminal 120 residues of TM contribute to assembly of a transport-competent trimer. This region of TM contains two amino acid domains capable of forming alpha helices, separated by a potential disulfide-bonded loop. While the N-terminal helical sequence, which extends to residue 85 of TM, may be capable of mediating the formation of Env trimers if C-terminal sequences are deleted, our results show that the putative disulfide-linked loop and C-terminal alpha-helical sequence play a key role in directing the formation of a stable trimer that is competent for intracellular transport.

Expression of the TM protein of Rous sarcoma virus in the absence of SU shows that this domain is capable of oligomerization and intracellular transport.

The SU and TM subunits of the Rous sarcoma virus glycoprotein, which are derived from a single polypeptide precursor, have been expressed independently with a simian virus 40 vector. The TM protein retains the ability to form an oligomer which resembles the TM oligomer derived from the wild-type glycoprotein complex present in virions. Oligomerization of the recombinant TM protein is more rapid than that observed for the intact glycoprotein expressed from the simian virus 40 vector and is required for its transport out of the endoplasmic reticulum. Oligomeric TM is terminally glycosylated in the Golgi complex but is less stable than the intact wild-type protein and does not accumulate at the cell surface. The SU protein, in contrast, does not form detectable oligomers but is efficiently secreted into the culture medium. These observations suggest that the oligomerization domain of the Rous sarcoma virus glycoprotein lies in the TM protein and that it can function independently of SU.

Oligomeric structure of a prototype retrovirus glycoprotein.

The structure of the Rous sarcoma virus envelope glycoprotein complex was studied by sedimentation gradient centrifugation analyses of detergent-solubilized wild-type and mutant envelope (env) gene products. These studies show that the envelope glycoprotein forms an oligomer during biosynthesis, which is most likely a trimer, and that this is the form of the complex found in virions. Our results are consistent with oligomer formation and transport out of the endoplasmic reticulum being closely linked. From analyses of mutant envelope proteins we conclude that the extracellular domain of the glycoprotein is sufficient for oligomer formation but that the transmembrane domain is required to stabilize this complex. Additional experiments suggest that interactions between external domains of the membrane-spanning, gp37 polypeptides are those most important for the formation of trimers. The significance of these observations to retroviral replication and implications for antiviral drug development are discussed.

Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.

CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20-specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2-terminal signal peptide and contains a highly charged COOH-terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).

Identification and characterization of a major surface antigen of Giardia lamblia.

The surface antigens of Giardia lamblia trophozoites were characterized by crossed immunoelectrophoresis, radioiodination, and immunoprecipitation. Crossed immunoelectrophoretic analysis of trophozoites with hyperimmune rabbit anti-trophozoite antiserum revealed a prominent precipitin peak that disappeared upon adsorption of the antiserum with live or formaldehyde-fixed trophozoites. This peak was intensely labeled when the antigen was derived from surface-radioiodinated trophozoites. An antiserum monospecific for the antigen contained in this precipitin peak was prepared. The precipitin peak was shown to contain an antigen with an apparent molecular weight of 82,000 by Western blotting. The antiserum also detected this 82,000-molecular-weight antigen on nitrocellulose blots of trophozoites analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On radioiodination of live trophozoites, an iodinated molecule of 82,000 apparent molecular weight was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was immunoprecipitated by the monospecific antiserum. Preliminary characterization of this antigen with the monospecific antiserum in crossed immunoelectrophoresis revealed that the surface antigen is hydrophobic and thus may be anchored in the plasma membrane, and that it is heat sensitive, but only partially sensitive to pronase or periodate. This antigen was shared by the four G. lamblia strains examined.