Drosophila twin spot clones reveal cell division dynamics in regenerating imaginal discs.

Cell proliferation is required for tissue regeneration, yet the dynamics of proliferation during regeneration are not well understood. Here we investigated the proliferation of eye and leg regeneration in fragments of Drosophila imaginal discs. Using twin spot clones, we followed the proliferation and fates of sister cells arising from the same mother cell in the regeneration blastema. We show that the mother cell gives rise to two sisters that participate equally in regeneration. However, when cells switch disc identity and transdetermine to another fate, they fail to turn off the cell cycle and continue dividing long after regeneration is complete. We further demonstrate that the regeneration blastema moves as a sweep of proliferation, in which cells are displaced. Our results suggest that regenerating cells stop dividing once the missing parts are formed, but if they undergo a switch in cell fate, the proliferation clock is reset.

The twin spot generator for differential Drosophila lineage analysis.

In Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.

Hepatitis B: modern concepts in pathogenesis--APOBEC3 cytidine deaminases as effectors in innate immunity against the hepatitis B virus.

PURPOSE OF REVIEW: APOBEC3 editing enzymes inhibit retroviruses by cytidine deamination in minus-strand cDNA, leading to G to A hypermutated proviruses, and by less well characterized inhibition of retroviral replication independently of catalysis. This review focuses on the effects of APOBEC3 enzymes on the pararetrovirus hepatitis B virus. RECENT FINDINGS: The cytidine deaminases APOBEC3B, APOBEC3C, APOBEC3F and APOBEC3G deaminate cytidine residues in hepatitis-B-virus minus-strand cDNA, resulting in G to A hypermutated genomes in the serum of hepatitis-B-virus-infected patients. APOBEC3B, APOBEC3F and APOBEC3G directly inhibit hepatitis-B-virus reverse transcription independently of deaminase activity. In human liver, APOBEC3B, APOBEC3F and APOBEC3G are expressed to low levels, but in human primary hepatocytes stimulated with interferon-alpha, APOBEC3G is induced to levels sufficient for hepatitis-B-virus inhibition. APOBEC3B inhibits hepatitis-B-virus gene transcription, and APOBEC3B and APOBEC3G preferentially mutate the hepatitis-B-virus x gene leading to the truncated hepatitis-B-virus x variants in hepatitis-B-virus-associated liver cancer. SUMMARY: The interferon-inducible APOBEC3G and the other APOBEC3s restrict hepatitis B virus by cytidine deamination in hepatitis-B-virus minus-strand cDNA and by direct inhibition of hepatitis-B-virus reverse transcriptase. The nuclear localized APOBEC3B is implicated in liver cancer development. To what extent these enzymes contribute to noncytolytic clearance of hepatitis B virus in vivo remains to be defined, yet the APOBEC3 cytidine deaminases are likely to play a role in these processes.

Effects of point mutations in the cytidine deaminase domains of APOBEC3B on replication and hypermutation of hepatitis B virus in vitro.

APOBEC3 cytidine deaminases hypermutate hepatitis B virus (HBV) and inhibit its replication in vitro. Whether this inhibition is due to the generation of hypermutations or to an alternative mechanism is controversial. A series of APOBEC3B (A3B) point mutants was analysed in vitro for hypermutational activity on HBV DNA and for inhibitory effects on HBV replication. Point mutations inactivating the carboxy-terminal deaminase domain abolished the hypermutational activity and reduced the inhibitory activity on HBV replication to approximately 40 %. In contrast, the point mutation H66R, inactivating the amino-terminal deaminase domain, did not affect hypermutations, but reduced the inhibition activity to 63 %, whilst the mutant C97S had no effect in either assay. Thus, only the carboxy-terminal deaminase domain of A3B catalyses cytidine deaminations leading to HBV hypermutations, but induction of hypermutations is not sufficient for full inhibition of HBV replication, for which both domains of A3B must be intact.

Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication.

Hypermutations in hepatitis B virus (HBV) DNA by APOBEC3 cytidine deaminases have been detected in vitro and in vivo, and APOBEC3G (A3G) and APOBEC3F (A3F) have been shown to inhibit the replication of HBV in vitro, but the presumably low or even absent hepatic expression of these enzymes has raised the question as to their physiological impact on HBV replication. We show that normal human liver expresses the mRNAs of APOBEC3B (A3B), APOBEC3C (A3C), A3F, and A3G. In primary human hepatocytes, interferon alpha (IFN-alpha) stimulated the expression of these cytidine deaminases up to 14-fold, and the mRNAs of A3G, A3F, and A3B reached expression levels of 10%, 3%, and 3%, respectively, relative to GAPDH mRNA abundance. On transfection, the full-length protein A3B(L) inhibited HBV replication in vitro as efficiently as A3G or A3F, whereas the truncated splice variant A3B(S) and A3C had no effect. A3B(L) and A3B(S) were detected predominantly in the nucleus of uninfected cells; however, in HBV-expressing cells both proteins were found also in the cytoplasm and were associated with HBV viral particles, similarly to A3G and A3F. Moreover, A3G, A3F, and A3B(L), but not A3B(S), induced extensive G-to-A hypermutations in a fraction of the replicated HBV genomes. In conclusion, the editing enzymes A3B(L), A3F, and most markedly A3G, which are expressed in liver and up-regulated by IFN-alpha in hepatocytes, are candidates to contribute to the noncytolytic clearance of HBV.

Cloning, characterization and analysis by RNA interference of various genes of the Chelonus inanitus polydnavirus.

Successful parasitism of some endoparasitic wasps depends on an obligately symbiotic association with polydnaviruses. These unique viruses have a segmented genome consisting of circles of double-stranded (ds) DNA and do not replicate in the parasitized host. They are produced in the wasp's ovary and injected into the host along with the egg. Chelonus inanitus is an egg-larval parasitoid; its polydnavirus (CiV) has been shown to protect the parasitoid larva from the host's immune system and to induce developmental arrest in the prepupal stage. The genome of CiV consists of at least 10-12 segments and five have been sequenced up to now. Here, the complete (CiV12g2) or partial (CiV12g1, CiV16.8g1) cloning of three new CiV genes is reported. All three occur only on one viral segment and have no similarity to other known polydnavirus genes, with the exception of a high similarity of CiV12g1 to CiV14g1 and CiV12g2 to CiV14g2. Furthermore, the first attempt of in vivo application of RNA interference to study the function of polydnavirus genes is shown. Injection of dsRNA of two late- and one early- and late-expressed CiV genes into CiV/venom-containing host eggs partially rescued last-instar larvae from developmental arrest. Injection of the same dsRNAs into parasitized eggs partially reduced parasitoid survival, mainly by preventing the successful emergence of the parasitoid from the host. These viral genes thus seem to be involved in inducing developmental arrest and in keeping the cuticle soft, which appears to be necessary for parasitoid emergence and host feeding.

Stage-dependent expression of Chelonus inanitus polydnavirus genes in the host and the parasitoid.

Chelonus inanitus (Braconidae) is a solitary egg-larval parasitoid of Spodoptera littoralis (Noctuidae). Along with the egg it also injects polydnaviruses (CiV) and venom, which are prerequisites for successful parasitoid development. CiV protects the parasitoid from encapsulation by the host's immune system and induces a developmental arrest in the prepupal stage. The polydnavirus genome consists of several double-stranded circular DNA segments. Proviral DNA is integrated in the wasp's genome and virus replication is restricted to the wasp's ovary. Here, the analysis of eight CiV genes located on five different segments revealed four patterns of expression in the course of parasitization: early, late, persistent but variable, and early and late. The comparison between parasitized and CiV/venom only containing hosts indicated that the presence of the parasitoid larva modulates transcript levels. Haemocytes, fat body and nervous tissue contained viral transcripts, values being highest in haemocytes. Small amounts of CiV transcripts were also observed in parasitoid larvae and pupae, suggesting transcription from the proviral integrated form of viral DNA. This is the first comparative analysis of the expression patterns of several viral genes in both parasitized and CiV/venom only containing hosts over the entire period of parasitization, and it reveals intricate interactions between the parasitoid, the polydnavirus and the host.