In the past decade, adenovirus vectors have generated tremendous interest, especially in gene therapy applications. In the so-called 'first generation' adenovirus vectors, the transgenes are inserted in place of the E1 region, or less often the E3 region. Although second-generation and helperdependent adenovirus vectors will probably prevail in the future in applications that require long-term gene expression, first generation adenovirus vectors will remain very useful in

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA.Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region. In contrast, the STNV-1 and -2 trailers represent half of the genome and share 64% nucleotide sequence conservation (4). STNV RNA is characterized by the absence of both a cap structure at the 5' end and a poly(A) tail at the 3' end (11). This peculiarity has two implications with regard to translation.First, STNV RNA apparently uses a translational initiation mechanism that bypasses the common cap recognition step. This mechanism may be quite different from that used by other uncapped viral RNAs like those of picornaviruses, comoviruses, and potyviruses (3, 27, 28). Picornaviral RNAs have long 5' untranslated sequences ranging from 600 to 1,200 nt, which carry multiple noninitiating AUG codons and which can fold into highly conserved secondary structures involved in translation initiation (reference 27 and references therein). In contrast, the STNV leader sequence is very short, and its predicted hairpin structure is not as extensive and stable (4). Interestingly, translation of STNV RNA in vitro, as of poliovirus, is stimulated by the cap binding factor eukaryotic initiation factor 4F (eIF-4F) (1, 6).Second, the absence of a poly(A) tail poses the question of how STNV RNA substitutes for the major cytoplasmic functions associated with this structure, i.e., the control of mRNA stability and the modulation of translation efficiency. The poly(A) tail, complexed with poly(A)-binding protein, is believed to enhance the formation of 80S translational initi-* Corresponding author. ation complexes, presumably through promoting some sort of interaction between 5'-and 3'-proximal elements of the mRNA (20). The exact role of the STNV trailer in viral replication and translation is not known, but its regulatory function is suggested by its sequence conservation, which exceeds that of the coat protein coding region. Phylogenetic comparison between the STNV-1 and STNV-2 trailer sequences revealed the presence of three pseudoknotted structures which are also found in several other nonpolyadenylated plant viral RNAs such as that of tobacco mosaic virus (TMV) (4, 23). In the latter case, it was shown that the pseudoknot domain can ...

Background: Although recombinant adenovirus vectors are attractive for use in gene expression studies and therapeutic applications, the construction of these vectors remains relatively time-consumning. We report here a strategy that simplifies the production of adenoviruses using the Cre-loxP system. Materials and Methods: Full-length recombinant adenovirus DNA was generated in vitro by Cre-mediated recombination between loxP sites in a linearized shuttle

SummaryThe RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5Ј cap and a 3Ј poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5Ј-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5Ј region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5Ј 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5Ј 38 nt.

A robust immune response is generated against components of the adenovirus capsid. In particular, a potent and long-lived humoral response is elicited against the hexon protein. This is due to the efficient presentation of adenovirus capsid proteins to CD4 ؉ T cells by antigen-presenting cells, in addition to the highly repetitive structure of the adenovirus capsids, which can efficiently stimulate B-cell proliferation. In the present study, we take advantage of this immune response by inserting epitopes against which an antibody response is desired into the adenovirus hexon. We use a B-cell epitope from Bacillus anthracis protective antigen (PA) as a model antigen to characterize hypervariable region 5 (HVR5) of hexon as a site for peptide insertion. We demonstrate that HVR5 can accommodate a peptide of up to 36 amino acids without adversely affecting virus infectivity, growth, or stability. Viruses containing chimeric hexons elicited antibodies against PA in mice, with total immunoglobulin G (IgG) titers reaching approximately 1 ؋ 10 3 after two injections. The antibody response contained both IgG1 and IgG2a subtypes, suggesting that Th1 and Th2 immunity had been stimulated. Coinjection of wild-type adenovirus and a synthetic peptide from PA produced no detectable antibodies, indicating that incorporation of the epitope into the capsid was crucial for immune stimulation. Together, these results indicate that the adenovirus capsid is an efficient vehicle for presenting B-cell epitopes to the immune system, making this a useful approach for the design of epitope-based vaccines.