- Volume 84, Issue 3, 2003

Adenoviruses are promising vectors for human cancer gene therapy. However, the extensively used adenoviruses serotypes 2 and 5 (Ad2 and Ad5) from species C have a major disadvantage in being highly prevalent; thus, most adults have an immunity against the two viruses. Furthermore, the expression of coxsackievirus and adenovirus receptors for Ad2 and Ad5 varies in different cells. This study aims to identify adenovirus serotypes with specific tropism for endothelial cells and epithelial tumour cells. Comparison of the binding affinities of Ad31, Ad11, Ad5, Ad37, Ad4 and Ad41, belonging to species A–F, respectively, to established cell lines of hepatoma (HepG2), breast cancer (CAMA and MG7), prostatic cancer (DU145 and LNCaP) and laryngeal cancer (Hep2), as well as to endothelial cells (HMEC), was carried out by flow cytometric analysis. Ad11 from species B showed markedly higher binding affinity than Ad5 for the endothelial cell line and all carcinoma cell lines studied. Ad4 showed a specific binding affinity for hepatoma cells and laryneal carcinoma cells. The ability of Ad11, Ad4 and Ad5 to be expressed in hepatoma, breast cancer and endothelial cell lines was studied by immunostaining and 35S-labelling of viral proteins in infected cells. Ad11 and Ad4 manifested a higher proportion of infected cells and a higher degree of hexon expression than Ad5.

Efficient gene delivery of a baculovirus-derived vector (BV-p53-lacZ) to a human osteogenic sarcoma cell line, Saos-2, was serendipitously found while evaluating the vector for gene delivery to human p53-null tumour cells in a previous study. Therefore, we investigated other human, rat and mouse osteogenic sarcoma and other types of tumour cell lines for transduction efficiency via baculovirus vectors containing a lacZ reporter gene under the control of either a cytomegalovirus or Rous sarcoma virus promoter. The expression of β-galactosidase protein, assessed by X-Gal staining and β-galactosidase ELISA, demonstrated an extremely high level of transduction efficiency in some osteogenic sarcoma cell lines, such as U-2OS, Saos-2 and Saos-LM2. These human osteogenic sarcoma cell lines showed levels of β-galactosidase expression 5–40 times greater than HepG2 cells, which were previously thought to be the mammalian cells most susceptible to baculovirus-mediated gene delivery. The level of acetylated histone proteins in these tumour lines did not correlate well with the high level of reporter gene expression. These results strongly suggest that some osteogenic sarcoma cells are highly susceptible to baculovirus-mediated gene delivery and that a baculovirus-derived vector is an efficient gene delivery vehicle into human osteogenic sarcoma cells.

Ld652Y cells derived from the gypsy moth, Lymantria dispar, were infected with seven different nucleopolyhedroviruses (NPVs) including those from Autographa californica, Bombyx mori (BmNPV), Hyphantria cunea (HycuNPV), Spodoptera exigua (SeMNPV), L. dispar, Orgyia pseudotsugata (OpMNPV) and Spodoptera litura (SpltMNPV). The results showed that Ld652Y cells infected with BmNPV, HycuNPV, SeMNPV, OpMNPV and SpltMNPV underwent apoptosis, displaying apoptotic bodies, characteristic DNA fragmentation and increased caspase-3-like protease activity; HycuNPV induced the most severe apoptosis. In HycuNPV-infected Ld652Y cells, a considerable amount of viral DNA was synthesized although there was no detectable yield of budded virions and polyhedrin. Northern blot and immunoblot analyses revealed that HycuNPV inhibitor of apoptosis 3 (IAP3), which has been shown to function in Sf9 cells, was expressed in HycuNPV-infected Ld652Y cells at a level higher than or comparable with that in HycuNPV-infected SpIm cells, which produced a high titre of progeny virions without any apoptotic response. These results imply that the relative ease of apoptosis induction in NPV-infected Ld652Y cells is largely dependent on inherent cellular properties rather than functions of the respective NPVs, and indicate that the defect in progeny virion production is not merely due to the virus-induced apoptosis in HycuNPV-infected Ld652Y cells.

Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-associated membranes at early stages of infection. This study reports that TMV movement protein (MP)-specific protein kinases (PKs) associated with the ER of tobacco were capable of phosphorylating Thr104 in TMV MP. The MP-specific PKs with apparent molecular masses of about 45–50 kDa and 38 kDa were revealed by gel PK assays. Two types of mutations were introduced in TMV MP gene of wild-type TMV U1 genome to substitute Thr104 by neutral Ala or by negatively charged Asp. Mutation of Thr104 to Ala did not affect the size of necrotic lesions induced by the mutant virus in Nicotiana tabacum Xanthi nc. plants. Conversely, mutation of Thr to Asp mimicking Thr104 phosphorylation strongly inhibited cell-to-cell movement. The possible role of Thr104 phosphorylation in TMV MP function is discussed.

The sequences of the coat protein gene of a representative sample of 40 isolates of Rice yellow mottle virus (RYMV) from 11 African countries were analysed. The overall level of nucleotide diversity was high ( ∼14 %). Great geographical distances between the sites where isolates were collected were consistently associated with high genetic distances. In contrast, a wide range of genetic distances occurred among isolates spread over short geographical distances. There was no evidence of long-range dispersal. RYMV diversity in relation to land area was eight times greater in East Africa than in West/Central Africa. West/Central African isolates with up to 9 % divergence belonged to a monophyletic group, whereas the East African isolates with up to 13 % divergence fell into distantly related groups. In East Africa, each Tanzanian strain had a specific and restricted geographical range, whereas West/Central African strains had large and partially overlapping geographical distributions. Overall, our results suggest an earlier RYMV diversification in East Africa and a later radiation in West/Central Africa. The West African situation was consistent with virus adaptation to savanna, forest and other ecological conditions. In contrast East Africa, as exemplified by the Tanzanian situation, with numerous physical barriers (mountain chains, sea channel, lakes), suggested that RYMV strains resulted from divergence under isolated conditions. For RYMV and for two other viruses, phylogenetic relationships were established between isolates from Madagascar and isolates from the Lake Victoria region.

Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus.

Single point mutations in the pathogenicity domain of Potato spindle tuber viroid (PSTVd) can have a dramatic effect on disease expression, and only three substitutions are required for the spontaneous conversion of the type strain PSTVd-Intermediate to the rapidly replicating, highly pathogenic variant RG1 (Gruner et al., Virology 209, 60–69, 1995) . To identify available evolutionary pathways linking these two variants, we mutagenized five positions in an infectious cDNA copy of PSTVd-Intermediate and screened the resulting mixture of 768 sequences for neutral or near-neutral mutants. Numerical simulations based on the bioassay data indicate that the 23 variants recovered represent >80 % of all such sequences. RG1 was the only naturally occurring variant recovered, and the overall pattern of sequence changes observed indicates that PSTVd-Int occupies a comparatively steep peak within the fitness landscape.