Kristina Lindman

There Are Two Different Species B Adenovirus Receptors: sBAR, Common to Species B1 and B2 Adenoviruses, and sB2AR, Exclusively Used by Species B2 Adenoviruses

ABSTRACT Unlike most adenovirus (Ad) serotypes, the species B Ads do not use the coxsackie-adenovirus receptor as an attachment receptor. The species B attachment receptor(s) has not yet been identified and is also poorly characterized. Species B Ads can be further divided into species B1 and B2 Ads, and these display different organ tropisms, suggesting a difference in receptor usage. We have studied the receptor interactions of the species B1 serotypes 3p and 7p and the species B2 serotypes 11p and 35 and characterized the properties of the species B receptor(s). Reciprocal blocking experiments using unlabeled Ad11p or Ad3p virions to block the binding to A549 cells of 35S-labeled 3p, 7p, 11p, and 35 showed that only Ad11p virions efficiently blocked the binding of all the species B Ads studied (≥70%). Thus, there is apparently a common species B Ad receptor (sBAR). However, Ad3p virions only partially (≤30%) blocked the binding of Ad11p and Ad35 to A549 cells. Binding experiments after trypsin treatment of the cells confirmed that the species B2 serotypes address at least two different receptors on A549 and J82 cells, since sBAR is trypsin sensitive but the species B2 Ad receptor (sB2AR) is not. Both receptors are proteins or glycoproteins, since binding of all species B serotypes was abolished after proteinase K or subtilisin treatment of A549 or J82 cells. Furthermore, binding of the species B serotypes to sBAR was abolished with EDTA and restored with Ca2+, whereas the binding of Ad11p and Ad35 to SB2AR was independent of divalent cations.

The Arg279Gln Substitution in the Adenovirus Type 11p (Ad11p) Fiber Knob Abolishes EDTA-Resistant Binding to A549 and CHO-CD46 Cells, Converting the Phenotype to That of Ad7p.

ABSTRACT The major determinant of adenovirus (Ad) attachment to host cells is the C-terminal knob domain of the trimeric fiber protein. Ad type 11p (Ad11p; species B2) in contrast to Ad7p (species B1) utilizes at least two different cellular attachment receptors, designated sBAR (species B adenovirus receptor) and sB2AR (species B2 adenovirus receptor). CD46 has recently been identified as one of the Ad11p attachment receptors. However, CD46 did not seem to constitute a functional receptor for Ad7p. Although Ad7p shares high knob amino acid identity with Ad11p, Ad7p is deficient in binding to both sB2AR and CD46. To determine what regions of the Ad11p fiber knob are necessary for sB2AR-CD46 interaction, we constructed recombinant fiber knobs (rFK) with Ad11p/Ad7p chimeras and Ad11p sequences having a single amino acid substitution from Ad7p. Binding of the constructs to A549 and CHO-CD46 BC1 isoform-expressing cells was analyzed by flow cytometry. Our results indicate that an Arg279Glu substitution is sufficient to convert the Ad11p receptor-interaction phenotype to that of Ad7p and abolish sB2AR and CD46 interaction. Also a Glu279Arg substitution in Ad7p rFKs increases CD46 binding. Thus, the lateral HI loop of the Ad11p fiber knob seems to be the key determinant for Ad11p sB2AR-CD46 interaction. This result is comparable to another non-coxsackie-adenovirus receptor binding Ad (Ad37p), where substitution of one amino acid abolishes virus-cell interaction. In conjunction with previous results, our findings also strongly suggest that sB2AR is equivalent to CD46.

Adenovirus 11p downregulates CD46 early in infection

Adenovirus 11 prototype (Ad11p), belonging to species B, uses CD46 as an attachment receptor. CD46, a complement regulatory molecule, is expressed on all human nucleated cells. We show here that Ad11p virions downregulate CD46 on the surface of K562 cells as early as 5min p.i. Specific binding to CD46 by the Ad11p fiber knob was required to mediate downregulation. The complement regulatory factors CD55 and CD59 were also reduced to a significant extent as a consequence of Ad11p binding to K562 cells. In contrast, binding of Ad7p did not result in downregulation of CD46 early in infection. Thus, the presumed interaction between Ad7p and CD46 did not have the same consequences as the Ad11p-CD46 interaction, the latter virus (Ad11p) being a promising gene therapy vector candidate. These findings may lead to a better understanding of the pathogenesis of species B adenovirus infections.

Small-Molecule Screening Using a Whole-Cell Viral Replication Reporter Gene Assay Identifies 2-{[2-(Benzoylamino)Benzoyl]Amino}-Benzoic Acid as a Novel Antiadenoviral Compound

ABSTRACT Adenovirus infections are widespread in society and are occasionally associated with severe, but rarely with life-threatening, disease in otherwise healthy individuals. In contrast, adenovirus infections present a real threat to immunocompromised individuals and can result in disseminated and fatal disease. The number of patients undergoing immunosuppressive therapy for solid organ or hematopoietic stem cell transplantation is steadily increasing, as is the number of AIDS patients, and this makes the problem of adenovirus infections even more urgent to solve. There is no formally approved treatment of adenovirus infections today, and existing antiviral agents evaluated for their antiadenoviral effect give inconsistent results. We have developed a whole cell-based assay for high-throughput screening of potential antiadenoviral compounds. The assay is unique in that it is based on a replication-competent adenovirus type 11p green fluorescent protein (GFP)-expressing vector (RCAd11pGFP). This allows measurement of fluorescence changes as a direct result of RCAd11pGFP genome expression. Using this assay, we have screened 9,800 commercially available small organic compounds. Initially, we observed approximately 400 compounds that inhibited adenovirus expression in vitro by ≥80%, but only 24 were later confirmed as dose-dependent inhibitors of adenovirus. One compound in particular, 2-{[2-(benzoylamino)benzoyl]amino}-benzoic acid, turned out to be a potent inhibitor of adenovirus replication.