Alistair H. Kidd

Adenovirus Type 37 Uses Sialic Acid as a Cellular Receptor

ABSTRACT Two cellular receptors for adenovirus, coxsackievirus-adenovirus receptor (CAR) and major histocompatibility complex class I (MHC-I) α2, have recently been identified. In the absence of CAR, MHC-I α2 has been suggested to serve as a cellular attachment protein for subgenus C adenoviruses, while members from all subgenera except subgenus B have been shown to interact with CAR. We have found that adenovirus type 37 (Ad37) attachment to CAR-expressing CHO cells was no better than that to CHO cells lacking CAR expression, suggesting that CAR is not used by Ad37 during attachment. Instead, we have identified sialic acid as a third adenovirus receptor moiety. First, Ad37 attachment to both CAR-expresing CHO cells and MHC-I α2-expressing Daudi cells was sensitive to neuraminidase treatment, which eliminates sialic acid on the cell surface. Second, Ad37 attachment to sialic acid-expressing Pro-5 cells was more than 10-fold stronger than that to the Pro-5 subline Lec2, which is deficient in sialic acid expression. Third, neuraminidase treatment of A549 cells caused a 60% decrease in Ad37 replication in a fluorescent-focus assay. Moreover, the receptor sialoconjugate is most probably a glycoprotein rather than a ganglioside, since Ad37 attachment to sialic acid-expressing Pro-5 cells was sensitive to protease treatment. Ad37 attachment to Pro-5 cells occurs via α(2→3)-linked sialic acid saccharides rather than α(2→6)-linked ones, since (i) α(2→3)-specific but not α(2→6)-specific lectins blocked Ad37 attachment to Pro-5 cells and (ii) pretreatment of Pro-5 cells with α(2→3)-specific neuraminidase resulted in decreased Ad37 binding. Taken together, these results suggest that, unlike Ad5, Ad37 makes use of α(2→3)-linked sialic acid saccharides on glycoproteins for entry instead of using CAR or MHC-I α2.

Initial interactions of subgenus D adenoviruses with A549 cellular receptors : sialic acid versus alpha(v) integrins

ABSTRACT Selected members of the adenovirus family have been shown to interact with the coxsackie adenovirus receptor, αvintegrins, and sialic acid on target cells. Initial interactions of subgenus D adenoviruses with target cells have until now been poorly characterized. Here, we demonstrate that adenovirus type 8 (Ad8), Ad19a, and Ad37 use sialic acid as a functional cellular receptor, whereas the Ad9 and Ad19 prototypes do not.

Adenovirus type 41 lacks an RGD αv-integrin binding motif on the penton base and undergoes delayed uptake in A549 cells

Human adenovirus (Ad) types 2, 3 and 12 are known to interact with cell surface integrins alpha(v)beta(3) and alpha(v)beta(5) through an RGD motif carried by the penton base. This interaction is thought to augment virus entry after initial contact between the fiber and specific receptor(s). Ad40 and Ad41 are the only members of the human subgroup F adenoviruses. The penton base protein sequence of one Ad40 strain is known to carry the motif RGAD rather than RGD, suggesting that not all human adenoviruses use the above integrins for cell entry. We confirmed that different genomic variants of Ad40 all carry an RGAD motif on the penton base, and found that the Ad41 prototype and several other genomic variants of Ad41 carry the motif IGDD in place of RGAD or RGD. This region is most likely exposed on the Ad41 particle, but attempts to block Ad41 infectivity using a homologous peptide were unsuccessful. Infectivity of an Ad41 preparation as measured by fluorescent focus assay in A549 cells was highly dependent on the length of the adsorption period, indicating that fiber-mediated attachment is inefficient in these cells. Moreover, Ad41 virions adsorbed for 1 h were internalized in a semi-linear fashion over 8 h. This inefficient uptake may be a direct consequence of independence of subgroup F adenoviruses from alpha(v)beta(3) and alpha(v)beta(5) integrin-mediated endocytosis. Ad40 and Ad41 may thus have lost or may never have developed a dependence on the penton base RGD motif for entry.

Characterization of fastidious adenovirus types 40 and 41 by DNA restriction enzyme analysis and by neutralizing monoclonal antibodies.

The DNA of 48 strains of adenovirus type 40 (Ad40) and of 128 strains of adenovirus type 41 (Ad41), isolated between 1971 and 1986 from various countries, was characterized by restriction enzyme analysis using nine and ten restriction endonucleases respectively. Five new DNA variants of Ad40 and 18 new DNA variants of Ad41 were detected. Most of the restriction sites which differed among the various DNA variants appeared to be distributed at random over the entire length of the viral genomes of the two serotypes. The number of restriction sites by which two DNA variants differed from each other was used as a measure of their relatedness. Several clusters of closely related DNA variants were observed for each of the two serotypes. The 35 DNA variants of Ad40 and Ad41 were used to test monoclonal antibody preparations for their range of reactivity in a neutralization assay. One monoclonal antibody (5-8), raised against Ad40 strain Dugan, showed type-specific neutralization of all 11 Ad40 DNA variants tested. Six monoclonal antibodies, raised against Ad41 strain Tak, neutralized different proportions of the variants of Ad41. Two of these preparations (1-21 and 3-19) neutralized all 24 Ad41 DNA variants, while a third (1-23) reacted with only 12 Ad41 variants. Three other monoclonal antibody preparations (3-10, 3-18, 7-14) reacted specifically with only 6 of these 12 variants. The patterns of reactivity with the monoclonal antibody preparations correlated with the presence or absence of a HindIII restriction site at 56 map units and of an EcoRI restriction site at 52 map units on the Ad41 DNA. This region of the adenovirus DNA codes for the hexon protein, which is known to contain the type-specific neutralizing antigenic determinants.

The Hepatitis B Virus Pregenome: Prediction of RNA Structure and Implications for the Emergence of Deletions

The terminally redundant pregenomic RNA of human hepatitis B virus (HBV) comprises some 3,330 nucleotides and is a replicative intermediate in the production of the circular DNA genome. Deletions are known to arise in the HBV genome during the course of chronic infection and are sometimes associated with interferon therapy. These deletions are limited to small parts of the genome such as the 357-nucleotide pre-S1 region. Long RNA molecules such as the HBV pregenome have considerable structural flexibility and will undergo secondary structure shifts between energetically favourable states in a continuous and semi-random fashion. Since prediction of structure elements that are highly conserved in different forms of one RNA molecule is now feasible by computer modelling, we have analysed the whole HBV pregenome by two different RNA structure prediction algorithms and by new methods that exploit these algorithms. Significantly, the ends of pregenomic RNA were predicted to undergo both short-range and long-range interactions, which has relevance to our knowledge of the virus replicative cycle. By incorporating phylogenetic information relating to the 6 recognised genotypes of HBV, it was possible to highlight short secondary structures that may be common to all HBV strains. For example, although the pre-S1 region was predicted to undergo local folding of a loosely defined nature, most observed pre-S1 deletions mapped to all or part of an arm carrying a better-defined structure. The loss of such sequences may be mechanistically attributable to polymerase skipping during reverse transcription, and the possible advantages of such deletions are considered.

Genetic conservation within subtypes in the hepatitis B virus pre-S2 region

The antigenic determinants for the main hepatitis B virus (HBV) subtypes adw, adr, ayw and ayr lie in the S (surface) polypeptide. Two amino acid residues in particular, encoded by the S gene at codon positions 122 and 160, have been postulated to determine the different antigenic subtypes. In contrast, the 165 nucleotide pre-S2 gene encodes an immunodominant region common to all subtypes that can give rise to neutralizing antibodies. We have characterized the pre-S2 gene sequences of 29 HBV strains of the three main subtypes, adw, ayw and adr. Seven base positions showed variation that was entirely subtype-specific, with six of these variations leading to subtype-specific amino acid differences. This finding affords the possibility of using pre-S2 sequences for genetic subtyping. Two ayw strains from unrelated patients infected in the Middle East had identical pre-S2 sequences with a block of 12 nucleotides deleted. A geographical correlation with subtype observed from serological results was also apparent from phylogenetic analysis of DNA identities within the pre-S2 region. The results support the concept that the main HBV subtypes truly represent families of phylogenetically different strains.