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Destruction of cancer cells by genetically modified viral and nonviral vectors has been the aim of many research programs. The ability to target cytotoxic gene therapies to the cells of interest is an essential prerequisite, and the treatment has always had the potential to provide better and more long-lasting therapy than existing chemotherapies. However, the potency of these infectious agents requires effective testing systems, in which hypotheses can be explored both in vitro and in vivo before the establishment of clinical trials in humans. The real prospect of off-target effects should be eliminated in the preclinical stage, if current prejudices against such therapies are to be overcome. In this review we have set out, using adenoviral vectors as a commonly used example, to discuss some of the key parameters required to develop more effective testing, and to critically assess the current cellular models for the development and testing of prostate cancer biotherapy. Only by developing models that more closely mirror human tissues will we be able to translate literature publications into clinical trials and hence into acceptable alternative treatments for the most commonly diagnosed cancer in humans.

The influenza A virus nucleoprotein (NP) is a single-stranded RNA-binding protein that encapsidates the virus genome and has essential functions in viral-RNA synthesis. Here, we report the characterization of a temperature-sensitive (ts) NP mutant (US3) originally generated in fowl plague virus (A/chicken/Rostock/34). Sequence analysis revealed a single mutation, M239L, in NP, consistent with earlier mapping studies assigning the ts lesion to segment 5. Introduction of this mutation into A/PR/8/34 virus by reverse genetics produced a ts phenotype, confirming the identity of the lesion. Despite an approximately 100-fold drop in the viral titer at the nonpermissive temperature, the mutant US3 polypeptide supported wild-type (WT) levels of genome transcription, replication, and protein synthesis, indicating a late-stage defect in function of the NP polypeptide. Nucleocytoplasmic trafficking of the US3 NP was also normal, and the virus actually assembled and released around sixfold more virus particles than the WT virus, with normal viral-RNA content. However, the particle/PFU ratio of these virions was 50-fold higher than that of WT virus, and many particles exhibited an abnormal morphology. Reverse-genetics studies in which A/PR/8/34 segment 7 was swapped with sequences from other strains of virus revealed a profound incompatibility between the M239L mutation and the A/Udorn/72 M1 gene, suggesting that the ts mutation affects M1-NP interactions. Thus, we have identified a late-acting defect in NP that, separate from its function in RNA synthesis, indicates a role for the polypeptide in virion assembly, most likely involving M1 as a partner.

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser(22)). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser(22) is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Herpes simplex virus 1 (HSV-1) replicates in the nucleus of host cells and radically alters nuclear architecture as part of its replication process. Replication compartments (RCs) form, and host chromatin is marginalized. Chromatin is later dispersed, and RCs spread past it to reach the nuclear edge. Using a lamin A-green fluorescent protein fusion, we provide direct evidence that the nuclear lamina is disrupted during HSV-1 infection and that the UL31 and UL34 proteins are required for this. We show nuclear expansion from 8 h to 24 h postinfection and place chromatin rearrangement and disruption of the lamina in the context of this global change in nuclear architecture. We show HSV-1-induced disruption of the localization of Cdc14B, a cellular protein and component of a putative nucleoskeleton. We also show that UL31 and UL34 are required for nuclear expansion. Studies with inhibitors of globular actin (G-actin) indicate that G-actin plays an essential role in nuclear expansion and chromatin dispersal but not in lamina alterations induced by HSV-1 infection. From analyses of HSV infections under various conditions, we conclude that nuclear expansion and chromatin dispersal are dispensable for optimal replication, while lamina rearrangement is associated with efficient replication.

Herpes simplex virus 1 (HSV-1) forms replication compartments (RCs), domains in which viral DNA replication, late-gene transcription, and encapsidation take place, in the host cell nucleus. The formation of these domains leads to compression and marginalization of host cell chromatin, which forms a dense layer surrounding the viral RCs and constitutes a potential barrier to viral nuclear egress or primary envelopment at the inner nuclear membrane. Surrounding the chromatin layer is the nuclear lamina, a further host cell barrier to egress. In this study, we describe an additional phase in RC maturation that involves disruption of the host chromatin and nuclear lamina so that the RC can approach the nuclear envelope. During this phase, the structure of the chromatin layer is altered so that it no longer forms a continuous layer around the RCs but instead is fragmented, forming islands between which RCs extend to reach the nuclear periphery. Coincident with these changes, the nuclear lamina components lamin A/C and lamin-associated protein 2 appear to be redistributed via a mechanism involving the U(L)31 and U(L)34 gene products. Viruses in which the U(L)31 or U(L)34 gene has been deleted are unable to undergo this phase of chromatin reorganization and lamina alterations and instead form RCs which are bounded by an intact host cell chromatin layer and nuclear lamina. We postulate that these defects in chromatin restructuring and lamina reorganization explain the previously documented growth defects of these mutant viruses.

The VP40 protein of Ebola virus can bud from mammalian cells in the form of lipid-bound, virus-like particles (VLPs), and late budding domains (L-domains) are conserved motifs (PTAP, PPxY, or YxxL; where "x" is any amino acid) that facilitate the budding of VP40-containing VLPs. VP40 is unique in that potential overlapping L-domains with the sequences PTAP and PPEY are present at amino acids 7 to 13 of VP40 (PTAPPEY). L-domains are thought to function by interacting with specific cellular proteins, such as the ubiquitin ligase Nedd4, and a component of the vacuolar protein sorting (vps) pathway, tsg101. Mutational analysis of the PTAPPEY sequence of VP40 was performed to understand further the contribution of each individual motif in promoting VP40 budding. In addition, the contribution of tsg101 and a second member of the vps pathway, vps4, in facilitating budding was addressed. Our results indicate that (i) both the PTAP and PPEY motifs contribute to efficient budding of VP40-containing VLPs; (ii) PTAP and PPEY can function as L-domains when separated and moved from the N terminus (amino acid position 7) to the C terminus (amino acid position 316) of full-length VP40; (iii) A VP40-PTAP/tsg101 interaction recruits tsg101 into budding VLPs; (iv) a VP40-PTAP/tsg101 interaction recruits VP40 into lipid raft microdomains; and (v) a dominant-negative mutant of vps4 (E228Q), but not wild-type vps4, significantly inhibited the budding of Ebola virus (Zaire). These results provide important insights into the complex interplay between viral and host proteins during the late stages of Ebola virus budding.

Morphogenesis of influenza virus is a poorly understood process that produces two types of enveloped virion: approximately 100-nm spheres and similar diameter filaments that reach 20 microm in length. Spherical particles assemble at plasma membrane lipid rafts in a process independent of microfilaments. The budding site of filamentous virions is hitherto uncharacterised but their formation involves the actin cytoskeleton. We confirm microfilament involvement in filamentous budding and show that after disruption of cortical actin by jasplakinolide, HA, NP, and M1 redistributed around beta-actin clusters to form novel annular membrane structures. HA in filamentous virions and jasplakinolide-induced annuli was detergent insoluble at 4 degrees C. Furthermore, in both cases HA partitioned into low buoyant density detergent-insoluble glycolipid domains, indicating that filamentous virions and annuli contain reorganised lipid rafts. We propose that the actin cytoskeleton is required to maintain the correct organisation of lipid rafts for incorporation into budding viral filaments.

Influenza virus transcription occurs in the nuclei of infected cells, where the viral genomic RNAs are complexed with a nucleoprotein (NP) to form ribonucleoprotein (RNP) structures. Prior to assembly into progeny virions, these RNPs exit the nucleus and accumulate in the cytoplasm. The mechanisms responsible for RNP export are only partially understood but have been proposed to involve the viral M1 and NS2 polypeptides. We found that the drug leptomycin B (LMB), which specifically inactivates the cellular CRM1 polypeptide, caused nuclear retention of NP in virus-infected cells, indicating a role for the CRM1 nuclear export pathway in RNP egress. However, no alteration was seen in the cellular distribution of M1 or NS2, even in the case of a mutant virus which synthesizes greatly reduced amounts of NS2. Furthermore, NP was distributed throughout the nuclei of infected cells at early times postinfection but, when retained in the nucleus at late times by LMB treatment, was redistributed to the periphery of the nucleoplasm. No such change was seen in the nuclear distribution of M1 or NS2 after drug treatment. Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2. Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution. Consistent with this, NP bound CRM1 in vitro. Overall, these data raise the possibility that RNP export is mediated by a direct interaction between NP and the cellular CRM1 export pathway.