Suisheng Zhang

Members of the NF90/NFAR protein group are involved in the life cycle of a positive-strand RNA virus

A major issue of current virology concerns the characterization of cellular proteins that operate as functional components of the viral multiplication process. Here we describe a group of host factors designated as ‘NFAR proteins’ that are recruited by the replication machinery of bovine viral diarrhea virus, a close relative of the human pathogen hepatitis C virus. The NFAR proteins associate specifically with both the termini of the viral RNA genome involving regulatory elements in the 5′ and 3′ non‐translated regions. Modification of the protein interaction sites in the 3′ non‐translated region yielded viral RNAs that were replication deficient. Viral replication was also inhibited by RNAi approaches that reduced the concentration of RNA helicase A, a member of the NFAR group, in the host cells cytoplasm. Further experimental data suggest that NFAR proteins mediate a circular conformation of the viral genome that may be important for the coordination of translation and replication. Because NFAR proteins are presumed components of the antiviral response, we suspect that viral recruitment may also serve to weaken cellular defense mechanisms.

Domain structure of human nuclear DNA helicase II (RNA helicase A).

Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression system. Recombinant NDH II unwound both DNA and RNA. Limited tryptic digestion produced active helicases with molecular masses of 130 and 100 kDa. The 130-kDa helicase missed a glycine-rich domain (RGG-box) at the carboxyl terminus, while the 100-kDa form missed both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and its RGG-box. Hence, the dsRBDs and the RGG-box were dispensable for unwinding. On the other hand, the isolated DEXH core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH2 terminus to the helicase core. Hence, an active helicase required part of the NH2 terminus, the DEXH core, and a C-terminal extension of the core. Both dsRBDs and the RGG-box were bacterially expressed as glutathione S-transferase fusion proteins. The two dsRBDs had a strong affinity to double-stranded RNA and cooperated upon RNA binding, while the RGG-box bound preferentially to single-stranded DNA. A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II.

Centrosomal localization of DNA damage checkpoint proteins

During mitosis, the phosphatidylinositol‐3 (PI‐3) family‐related DNA damage checkpoint kinases ATM and ATR were found on the centrosomes of human cells. ATRIP, an interaction partner of ATR, as well as Chk1 and Chk2, the downstream targets of ATR or ATM, were also localized to the centrosomes. Surprisingly, the DNA‐PK inhibitor vanillin enhanced the level of ATM on centrosomes. Accordingly, DNA‐PKcs, the catalytic subunit of DNA‐PK, was also found on the centrosomes. Vanillin altered the phosphorylation of Chk2 in the centrosomes and in whole cell extracts. Nucleoplasmic ATM co‐immunoprecipitated with Ku70/86, the DNA binding subunits of DNA‐PK, while vanillin diminished this association. Vanillin did not affect microtubule polymerization at the centrosomes but, surprisingly, caused a transient enhancement of α‐tubulin foci in the nucleus. Interestingly, γ‐tubulin was also present in the nucleus and co‐immunoprecipitated with ATR or BRCA1. DNA damage led to a reduction of the mentioned checkpoint proteins on the centrosomes but increased the level of γ‐tubulin at this organelle. Taken together, these results indicate that DNA damage checkpoint proteins may control the formation of γ‐tubulin and/or the kinetics of microtubule formation at the centrosomes, and thereby couple them to the DNA damage response. J. Cell. Biochem. 101: 451–465, 2007.

Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2)

The telomeric repeat binding factor 2 (TRF2) specifically recognizes TTAGGG tandem repeats at chromosomal ends. Unexpectedly immunofluorescence studies revealed a prominent nucleolar localization of TRF2 in human cells, which appeared as discrete dots with sizes similar to those present in the nucleoplasm. The TRF2 dots did not overlap with dots stemming from the upstream binding factor (UBF) or the B23 protein. After treatment with a low concentration of actinomycin D (0.05 μg/ml), TRF2 remained in the nucleolus, although this condition selectively inhibited RNA polymerase I and led to a relocalization of UBF and B23. TRF2 was prominent in the nucleolus at G0 and S but seemed to diffuse out of the nucleolus in G2 phase. During mitosis TRF2 dispersed from the condensed chromosomes and returned to the nucleolus at cytokinesis. Treatment with low doses of actinomycin D delayed the release of TRF2 from the nucleolus as cells progressed from G2 phase into mitosis. With actinomycin D present TRF2 was detected in discrete foci adjacent to UBF in prophase, while in metaphase a complete overlap between TRF2 and UBF was observed. TRF2 was present in DNase-insensitive complexes of nucleolar extracts, whereas DNA degradation disrupted the protein-DNA complexes consisting of Ku antigen and B23. Following treatment with actinomycin D some of the mitotic cells displayed chromosome end-to-end fusions. This could be correlated to the actinomycin D-suppressed relocalization of TRF2 from the nucleolus to the telomeres during mitosis. These results support the view that the nucleolus may sequester TRF2 and thereby influences its telomeric functions.

Werner syndrome helicase (WRN), nuclear DNA helicase II (NDH II) and histone γH2AX are localized to the centrosome

Werner syndrome helicase (WRN) was found in the centrosome of human cells, both in interphase and in mitosis. Nuclear DNA helicase II (NDH II), also called RNA helicase A (RHA), an interaction partner of WRN, was also present in the centrosome. NDH II localized to the centrosome in interphase but left the centrosome with the ongoing progression of mitosis. The localization of NDH II to the centrosome was hardly affected by cytochalasin D that depolymerizes actin filaments. In contrast, treatment by the microtubules disrupting agent nocodazole strikingly detached NDH II from the centrosome, which was in contrast to WRN that remained there under this condition. Treatment of cells with the DNA damaging agent 4‐nitroquinoline‐1‐oxide (4NQO) released NDH II, but not WRN from the centrosome. Surprisingly, the double‐stranded DNA break repair‐induced histone variant γH2AX was also found in centrosomes of interphase and mitotic cells. Following DNA damage by 4NQO, γH2AX left the centrosome with similar kinetics as NDH II. In vitro pull‐down assays confirmed a direct physical interaction between these two proteins. Since NDH II associated with γH2AX after DNA damage, we suggest that complex formation between NDH II and γH2AX may occur in pre‐assembled complexes at the centrosome, which are subsequently recruited to sites of damaged DNA for inducing the repair process.