Adenoviruses target their double-stranded DNA genome and its associated core proteins to the interphase nucleus; this core structure then enters through the nuclear pore complex. We have used digitonin permeabilized cell import assays to study the cellular import factors involved in nuclear entry of virus DNA and the core proteins, protein V and protein VII. We show that inhibition of transportin results in aberrant localization of protein V and that transportin is necessary for protein V to accumulate in the nucleolus. Furthermore, inhibition of transportin results in inhibition of protein VII and DNA import, whereas disruption of the classical importin aimportin b import pathway has little effect. We show that mature protein VII has different import preferences from the precursor protein, preVII from which it is derived by proteolytic processing. While bacterially expressed glutathione S-transferase (GST)-preVII primarily utilizes the pathway mediated by importin a-importin b, bacterially expressed GST-VII favours the transportin pathway. This is significant because while preVII is important during viral replication and assembly only mature VII is available during viral DNA import to a newly infected cell. Our results implicate transportin as a key import receptor for the nuclear localization of adenovirus core.

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Adenovirus infection subverts nucleolar structure and function. B23 is a nucleolar protein present in two isoforms (B23.1 and B23.2) and both isoforms have been identified as stimulatory factors for adenovirus DNA replication. Here, it is demonstrated that the two isoforms of B23, B23.1 and B23.2, interact and co-localize differently with viral DNA replication proteins pTP and DBP in adenovirus-infected cells. Thus, the mechanism by which the two proteins stimulate viral DNA replication is likely to differ. These data also demonstrate the importance of testing both isoforms of B23 for interactions with viral proteins and nucleic acids.

We have identified a novel internal ribosome entry site (IRES) within a latently expressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene (vCyclin) that controls the expression of a downstream open reading frame encoding an inhibitor of apoptosis (vFLIP). This IRES is the first such element to be identified in a DNA virus and may represent a novel mechanism through which this virus controls gene expression. We have used a dual luciferase reporter assay to identify important sequence elements essential for the activity of the IRES. A sequence of 32 nucleotides incorporating a polypyrimidine tract (PPT) was found to be required for the proper functioning of the IRES. We also show, using an electrophoretic mobility shift assay (EMSA), that proteins specific to a KSHV-infected cell line (BCP-1) but not a KSHV-negative cell line (HEK293) were able to form complexes with the IRES. By using an in vitro RNA binding assay, the cellular polypyrimidine tract binding protein (PTB, hnRNP-I) was found to bind to the IRES RNA. These results suggest that the interaction of PTB with the PPT may contribute to the correct functioning of the KSHV IRES in infected cells. INTRODUCTIONKaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV-8), is the most recently identified member of the herpesvirus family to infect humans (Chang et al., 1994). KSHV, a gamma-2-herpesvirus, has been proposed as the aetiological agent for Kaposi's sarcoma as well as other malignancies such as primary effusion lymphoma (PEL) (Cesarman et al., 1995) and multicentric Castleman's disease (MCD) (Soulier et al., 1995).KSHV is closely related to three other herpesviruses with oncogenic potential; herpesvirus saimiri (HVS), murine gammaherpesvirus (MHV-68) and, more distantly, to Epstein-Barr virus (EBV). The complete nucleotide sequence of KSHV DNA has revealed several genes which have probably been captured from the host cell during virus evolution, and whose products could also play a role in cellular transformation and tumour induction (Neipel et al., 1997;Russo et al., 1996) The observation that a bicistronic transcript (Talbot et al., 1999) encodes vCyclin and vFLIP led to the investigation of the mechanism of translation of the vFLIP ORF. We (Bieleski & Talbot, 2001) and others (Grundhoff & Ganem, 2001;Low et al., 2001) were able to identify a novel internal ribosome entry site (IRES) within the latently expressed vCyclin gene that controls the expression of the downstream vFLIP ORF. This IRES is the first such element to be identified in a DNA virus. Recently, an IRES element has been described in the untranslated region of the Epstein-Barr nuclear antigen-1 (EBNA1) gene, which may contribute to the regulation of latent gene expression (Isaksson et al., 2003).IRES elements were first identified in the 59 untranslated regions (UTR) of picornaviruses and are essential for the cap-independent translation of the viral polyprotein (Jang et al., 1988;Pelletier & Sonenberg, 1988). More recently IRES elements have been chara...