Drosophila male courtship behavior is modulated by ecdysteroids.

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd(1) adult Drosophila melanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed toward females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.

trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells.

A BHK cell line persistently expressing a Kunjin (KUN) virus replicon RNA (repBHK, similar to our recently described ME/76Neo BHK cell line [A. A. Khromykh and E. G. Westaway, J. Virol. 71:1497-1505, 1997]) was used for rescue and propagation of KUN viruses defective in the RNA polymerase gene (NS5). A new infectious full-length KUN virus cDNA clone, FLSDX, prepared from our previously described cDNA clone pAKUN (A. A. Khromykh and E. G. Westaway, J. Virol. 68:4580-4588, 1994) and possessing approximately 10(5)-fold higher specific infectivity than that of pAKUN, was used for preparation of defective mutants. Deletions of the predicted RNA polymerase motif GDD (producing FLdGDD) and of one of the predicted methyltransferase motifs (S-adenosylmethionine [SAM] binding site, producing FLdSAM) were introduced separately into FLSDX. Transcription and transfection of FLdGDD and FLdSAM RNAs into repBHK cells but not into normal BHK cells resulted in their replication and the recovery of defective viruses able to replicate only in repBHK cells. Reverse transcription-PCR and sequencing analyses showed retention of the introduced deletions in the genomes of the recovered viruses. Retention of these deletions, as well as our inability to recover viruses able to replicate in normal BHK cells after prolonged incubation (for 7 days) of FLdGDD- or FLdSAM-transfected repBHK cells, excluded the possibility that recombination had occurred between the deleted defective NS5 genes present in transfected RNAs and the functional NS5 gene present in the repBHK cells. An RNA with a point mutation in the GDD motif (FLGVD) was also complemented in transfected repBHK cells, and defective virus was recovered by day 3 after transfection. However, in contrast to the results with FLdGDD and FLdSAM RNAs, prolonged (4 days or more) incubation of FLGVD RNA in normal BHK cells allowed recovery of a virus in which the GVD mutation had reverted via a single base change to the wild-type GDD sequence. Overall, these results represent the first demonstration of trans-complementation of defective flavivirus RNAs with deleterious deletions in the flavivirus RNA polymerase gene NS5. The complementation system described here may prove to be useful for the in vivo complementation of deletions and mutations affecting functional domains or the essential secondary structure in any of the other flavivirus nonstructural proteins.

Ultrastructure of Kunjin virus-infected cells: colocalization of NS1 and NS3 with double-stranded RNA, and of NS2B with NS3, in virus-induced membrane structures.

The subcellular location of the nonstructural proteins NS1, NS2B, and NS3 in Vero cells infected with the flavivirus Kunjin was investigated using indirect immunofluorescence and cryoimmunoelectron microscopy with monospecific antibodies. Comparisons were also made by dual immunolabelling using antibodies to double-stranded RNA (dsRNA), the putative template in the flavivirus replication complex. At 8 h postinfection, the immunofluorescent patterns showed NS1, NS2B, NS3, and dsRNA located in a perinuclear rim with extensions into the peripheral cytoplasm. By 16 h, at the end of the latent period, all patterns had changed to some discrete perinuclear foci associated with a thick cytoplasmic reticulum. By 24 h, this localization in perinuclear foci was more apparent and some foci were dual labelled with antibodies to dsRNA. In immuno-gold-labelled cryosections of infected cells at 24 h, all antibodies were associated with clusters of induced membrane structures in the perinuclear region. Two important and novel observations were made. First, one set of induced membranes comprised vesicle packets of smooth membranes dual labelled with anti-dsRNA and anti-NS1 or anti-NS3 antibodies. Second, adjacent masses of paracrystalline arrays or of convoluted smooth membranes, which appeared to be structurally related, were strongly labelled only with anti-NS2B and anti-NS3 antibodies. Paired membranes similar in appearance to the rough endoplasmic reticulum were also labelled, but less strongly, with antibodies to the three nonstructural proteins. Other paired membranes adjacent to the structures discussed above enclosed accumulated virus particles but were not labelled with any of the four antibodies. The collection of induced membranes may represent virus factories in which translation, RNA synthesis, and virus assembly occur.

Proteins C and NS4B of the flavivirus Kunjin translocate independently into the nucleus.

The subcellular locations in infected Vero cells of Kunjin (KUN) virus core protein C and NS4B were analyzed by immunofluorescence (IF) and by immunoelectron microscopy using monospecific antibodies. Selection of appropriate fixation methods for IF showed that both proteins were associated at all times with perinuclear membranes spreading outward in a reticular pattern and they entered the nucleus late during the latent period. Subsequently NS4B was also dispersed through the nucleoplasm, while C appeared in the nucleolus and the nucleoplasm. These nuclear locations were confirmed by immunogold labeling of cryosections of infected cells at 24 hr postinfection. Labeling of NS4B in cryosections was especially enriched in the perinuclear membranes of the endoplasmic reticulum. When C and NS4B were each expressed separately in stably transformed cell lines, both cytoplasmic and nuclear localization was observed by IF and confirmed by immunoelectron microscopy. Thus the two proteins translocated to the nucleus independently of each other and of other viral proteins. Dual IF with antibodies to double-stranded RNA showed that cytoplasmic locations of C and NS4B were apparently associated in part with the sites of viral RNA synthesis which were resistant to solubilization by Triton X-100.

Homozygous intragenic deletion in the WT1 gene in a sporadic Wilms' tumour associated with high levels of expression of a truncated transcript.

We have examined a panel of 21 sporadic Wilms' tumours for rearrangements in the Wilms' tumour suppressor gene, WT1. In one tumour with specific allele loss in chromosome 11p13, a homozygous deletion in the 3' end of the gene, encompassing exon 10 and the 3' untranslated region, was identified. High levels of a truncated WT1 transcript, predicted to encode a polypeptide missing the fourth zinc finger were expressed in this tumour. All other samples showed normal patterns of digestion on Southern blots. This observation confirms previous findings that large deletions in the gene occur infrequently in sporadic Wilms' tumours and that the zinc-finger region of the encoded polypeptide is critical for correct functioning of the gene.