bTranscription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes. Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) belongs to the gammaherpesvirus subfamily, which also includes Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV-68). KSHV is etiologically linked to the AIDS-associated malignancy Kaposi's sarcoma, as well as two rare lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD) (1). During the latent phase of infection, the KSHV genome is maintained as a circular episome in the nucleus (2), where only a few genes are transcribed as part of the latency program (3, 4). The lytic phase begins with the induction of immediate early (IE) genes, which in turn promotes the expression of early (E) genes (5). The transcription of late genes depends on lytic replication of viral DNA, a process carried out by the core DNA replication machinery composed of KSHV-encoded proteins (6). Chemical inhibitors of the herpesvirus-encoded DNA polymerase (e.g., phosphonoacetic acid [PAA]) efficiently prevent expression of late genes but do not affect genes of the other kinetic classes (5).Several studies have begun to shed light on the regulation of late gene expression in beta-and gammaherpesviruses. Genetic studies of MHV-68 and EBV identified six gene products (open reading frame 18 [ORF18], -24, -30, -31, -34, and -66 in the case of KSHV), each of which is required for the transcription of late genes but dispensable for viral DNA replication and the expression of IE and E genes (7-11). Many of these evolutionarily conserved genes of the beta-and gammaherpesviruses (␤-␥ genes) have since been shown to have similar effects on the gene expression of murine cytomegalovirus (MCMV) (12, 13), human CMV (HCMV) (14-16), and KSHV (17, 18). Of note, none of these six ␤-␥ genes are found in the alphaherpesvirus subfamily, suggesting that a mechanistically distinct process controls late gene expression in the alpha subfamily. Recent studies suggest that the ␤-␥ gene products assemble into a viral preinitiation complex (vPIC) that also includes RNA polymerase II (RNAPII) (11,18). One of the vPIC components is ORF24, which was identified as a viral protein distantly related to the cellular TATA ...

At the tip of flagella, an array of LC8 dimers binds to the spoke protein RSP3 in radial spoke precursors, triggering phosphorylation, stalk base formation, and axoneme targeting.

Amphipathic helices in the A-kinase anchoring protein RSP3 bind to spoke proteins involved in the assembly and modulation of the flagellar radial spoke complex, expanding the repertoire of these versatile helical protein motifs.

BackgroundFlagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical ‘9 + 2’ cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain.ResultsWe found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus.ConclusionIt appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0103-y) contains supplementary material, which is available to authorized users.

Axonemal complexes in flagella are largely pre-packaged in the cell body. As such, one mutation often results in the absence of the co-assembled components and permanent motility deficiencies. For example, a Chlamydomonas mutant defective in RSP4 in the radial spoke (RS), which is critical for bend propagation, has paralyzed flagella that also lack the paralogue RSP6 and three additional RS proteins. Intriguingly, recent studies showed that several mutant strains contain a mixed population of swimmers and paralyzed cells despite their identical genetic background. Here we report a cause underlying these variations. Two new mutants lacking RSP6 swim processively and other components appear normally assembled in early log phase indicating that, unlike RSP4, this paralogue is dispensable. However, swimmers cannot maintain the typical helical trajectory and reactivated cell models tend to spin. Interestingly the motile fraction and the spokehead content dwindle during stationary phase. These results suggest that 1) intact RS is critical for maintaining the rhythm of oscillatory beating and thus the helical trajectory; 2) assembly of the axonemal complex with subtle defects is less efficient and the inefficiency is accentuated in compromised conditions, leading to reversible dyskinesia. Consistently, several organisms only possess one RSP4/6 gene. Gene duplication in Chlamydomonas enhances RS assembly to maintain optimal motility in various environments.