During human adenovirus 5 infection, a temporal cascade of gene expression leads ultimately to the production of large amounts of the proteins needed to construct progeny virions. However, the mechanism for the activation of the major late gene that encodes these viral structural proteins has not been well understood. We show here that two key positive regulators of the major late gene, L4-22K and L4-33K, previously thought to be expressed under the control of the major late promoter itself, initially are expressed from a novel promoter that is embedded within the major late gene and dedicated to their expression. This L4 promoter is required for late gene expression and is activated by a combination of viral protein activators produced during the infection, including E1A, E4 Orf3, and the intermediate-phase protein IVa2, and also by viral genome replication. This new understanding redraws the long-established view of how adenoviral gene expression patterns are controlled and offers new ways to manipulate that gene expression cascade for adenovirus vector applications.Although years of study have produced a detailed understanding of most molecular events during human adenovirus type 5 (Ad5) infection (4, 27), how the transition in viral gene expression from the early to the late phase is controlled has remained poorly defined. This control is crucial, since it determines the activity of the genes that encode virion proteins and, hence, the productivity of the infection. Residual activity from these genes is a confounding factor in the utility of E1-deleted Ad5 vectors for long-term gene delivery (51).The initial expression of E1A from the linear Ad5 genome provides transcriptional activators that, with host proteins, turn on the expression of the remaining early genes E1B, E2, E3, and E4 (Fig. 1A). The major-late transcription unit (MLTU) also is weakly active at this time, but only the most 5Ј-proximal L1 product is produced (1,35,41). Around the time of transition to the late phase of infection, when the replication of the viral genome also begins, the transcription of intermediate genes IX and IVa2 commences (13,39,48,49), while majorlate promoter (MLP) activity greatly increases, and its scope expands to direct the expression of a full set of around 15 MLTU products from regions L1 to L5 via alternative splicing and polyadenylation (35,41). This transition in MLTU activity reflects transcriptional and posttranscriptional changes, both of which require proteins encoded by genes in the MLTU L4 region (Fig. 1B). L4-22K and L4-33K act posttranscriptionally to activate the production of the full set of MLTU mRNAs (16,33,44). At the same time, the MLP is further activated by IVa2 protein (30, 45) working with L4-22K and/or L4-33K (2, 33, 38).The essential role of L4-22K and L4-33K in producing full late-phase expression from the Ad5 MLTU creates a paradox since, according to the current model of Ad5 gene expression, their expression is achieved only as a consequence of this activation process. Here, we show that a...

Human adenovirus serotype 5 encodes three proteins, E1b 55K, E4 Orf3 and E4 Orf6, which interact with each other and with components of the nucleus to regulate mRNA processing and export, viral DNA replication and p53-dependent apoptosis. Previous studies have shown that, during wild-type infection, 55K associates initially with structures termed ND10, which are sites of localization of the promyelocytic leukaemia protein, and then moves, dependent upon its interaction with Orf6, to the establishing virus replication centres. Absence of either Orf3 or Orf6 affects the localization of 55K and so may affect its function. In this study, the influence of Orf3 and Orf6 expression on the association of 55K with the insoluble matrix fraction of the nucleus and with ND10 particularly was examined. Overexpression of Orf6 was sufficient to block the association of 55K with this fraction, irrespective of the presence of Orf3. This effect depended upon the two proteins being able to interact. However, the association of 55K with ND10, which persists throughout infection in the absence of Orf6, required Orf3 to be present, thus distinguishing two subsets of matrix-associated 55K. A modified form of 55K, formation of which was blocked by mutating the known site of SUMO-1 attachment, was more abundant in the absence of Orf6 but unaffected by the absence of Orf3. Thus, this modification is favoured when 55K remains associated with the matrix but does not correlate with its stable association with ND10, many components of which are modified by SUMO-1.

A genome rearrangement involving RNA segment 11 of a bovine rotavirus has been analysed by molecular cloning and sequencing. This revealed that the rearranged genome segment was generated by a head to tail concatemerisation of two almost full length copies of segment 11. The upstream copy of the gene has lost its 3' end and the downstream copy its 5' end. The truncation of the upstream copy of the gene occurs within the termination codon for VP11 converting it from a UAG to a UGA, the rearranged gene is therefore still able to encode a normal VP11. The possible mechanisms by which this rearrangement may have been generated are discussed.