Polyadenylation of genomic RNA and initiation of antigenomic RNA in a positive-strand RNA virus are controlled by the same cis-element.

Genomes and antigenomes of many positive-strand RNA viruses contain 3'-poly(A) and 5'-poly(U) tracts, respectively, serving as mutual templates. Mechanism(s) controlling the length of these homopolymeric stretches are not well understood. Here, we show that in coxsackievirus B3 (CVB3) and three other enteroviruses the poly(A) tract is approximately 80-90 and the poly(U) tract is approximately 20 nt-long. Mutagenesis analysis indicate that the length of the CVB3 3'-poly(A) is determined by the oriR, a cis-element in the 3'-noncoding region of viral RNA. In contrast, while mutations of the oriR inhibit initiation of (-) RNA synthesis, they do not affect the 5'-poly(U) length. Poly(A)-lacking genomes are able to acquire genetically unstable AU-rich poly(A)-terminated 3'-tails, which may be generated by a mechanism distinct from the cognate viral RNA polyadenylation. The aberrant tails ensure only inefficient replication. The possibility of RNA replication independent of oriR and poly(A) demonstrate that highly debilitated viruses are able to survive by utilizing 'emergence', perhaps atavistic, mechanisms.

Increased immunogenicity of recombinant Ad35-based malaria vaccine through formulation with aluminium phosphate adjuvant.

Previously, we have shown the potency of recombinant Adenovirus serotype 35 viral vaccines (rAd35) to induce strong immune response against the circumsporozoite protein (CS) of the plasmodium parasite. To further optimize immunogenicity of Ad35-based malaria vaccines we formulated rAd35.CS vaccine with aluminium phosphate adjuvant (AlPO(4)). In contrast to the conventional protein based vaccines no absorption to aluminium adjuvant was observed and rAd35 viral in vitro infectivity in mammalian cells was preserved. Immunization with Ad35.CS formulated with AlPO(4) resulted in significantly higher CS specific T and B cell responses in mice upon either single or prime-boost vaccination regimens as compared to rAd35.CS alone. With these results we report for the first time the feasibility of using an AlPO(4) adjuvant to increase the potency of a live adenovirus serotype 35-based vaccine.

Structural and functional integrity of the coxsackievirus B3 oriR: spacing between coaxial RNA helices.

The enterovirus oriR is composed of two helices, X and Y, anchored by a kissing (K) interaction. For proper oriR function, certain areas of these helices should be specifically oriented towards each other. It was hypothesized that the single-stranded nucleotides bridging the coaxial helices (Y-X and K-Y linkers) are important to determine this orientation. Spatial changes were introduced by altering the linker length between the helices of the coxsackievirus B3 oriR. Changing the linker lengths resulted in defective RNA replication, probably because of an altered oriR geometry. The identity of the linker residues also played a role, possibly because of sequence-specific ligand recognition. Although each point mutation altering the primary sequence of the Y-X spacer resulted in defective growth at 36 degrees C, the mutations had a wild-type phenotype at 39 degrees C, indicating a cold-sensitive phenotype. The results show that the intrinsic connection between oriR structure and function is fine-tuned by the spacing between the coaxial RNA helices.

Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis.

A stem-loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a cis-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication in vivo and abolished uridylylation of VPg in vitro. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R(1)NNNAAR(2)NNNNNNR(3), is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an in vitro translation/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered with positive-strand RNA synthesis, but not with negative-strand synthesis. All CRE(2C) point mutants affecting the enteroviral CRE consensus loop, however, showed a marked decrease in efficiency to induce negative-strand synthesis. Moreover, a transition (A(5)G) regarding the first templating adenosine residue in the loop was even unable to initiate complementary negative-strand synthesis above detectable levels. Taken together, these results indicate that the CVB3 CRE(2C) is not only required for the initiation of positive-strand RNA synthesis, but also plays an essential role in the efficient initiation of negative-strand RNA synthesis, a conclusion that has not been reached previously by using the cell-free system.

Biological significance of a human enterovirus B-specific RNA element in the 3' nontranslated region.

The secondary structures predicted for the enteroviral 3' nontranslated region (3'NTR) all seem to indicate a conformation consisting of two (X and Y) hairpin structures. The higher-order RNA structure of the 3'NTR appears to exist as an intramolecular kissing interaction between the loops of these two hairpin structures. The enterovirus B-like subgroup possesses an additional stem-loop structure, domain Z, which is not present in the poliovirus-like enteroviruses. It has been suggested that the Z domain originated from a burst of short sequence repetitions (E. V. Pilipenko, S. V. Maslova, A. N. Sinyakov, and V. I. Agol, Nucleic Acids Res. 20:1739-1745, 1992). However, no functional features have yet been ascribed to this enterovirus B-like-specific RNA element in the 3'NTR. In this study, we tested the functional characteristics and biological significance of domain Z. A mutant of the cardiovirulent coxsackievirus group B3 strain Nancy which completely lacked the Z domain and which therefore acquired enterovirus C-like secondary structures exhibited a wild-type growth phenotype, as determined by single-cycle growth analysis with BGM cells. This result proves that the Z domain is virtually dispensable for viral growth in tissue cultures. Partial distortion of the Z domain structure resulted in a disabled virus with reduced growth kinetics, probably due to alternative conformations of the overall structure of the domain. Infection of mice showed that the recombinant coxsackievirus group B3 mutant which completely lacked the Z domain was less virulent. Pancreatic tissues from mice infected with wild-type virus and recombinant virus were equally affected. However, the heart tissue from mice infected with the recombinant virus showed only slight signs of myocarditis. These results suggest that the enterovirus B-like-specific Z domain plays a role in coxsackievirus-induced pathogenesis.