Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity.

UNLABELLED: PA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. W. Jagger, H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger, and P. Digard, 2012, Science 337:199-204, http://dx.doi.org/10.1126/science.1222213, and E. A. Desmet, K. A. Bussey, R. Stone, and T. Takimoto, 2013, J Virol 87:3108-3118, http://dx.doi.org/10.1128/JVI.02826-12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus. IMPORTANCE: Influenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes, to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.

Kaposi's sarcoma-associated herpesvirus-encoded LANA recruits topoisomerase IIß for latent DNA replication of the terminal repeats.

The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) plays a major role in maintaining latency and is critical for the perpetual segregation of viral episomes to the progeny nuclei of newly divided cells. LANA binds to KSHV terminal repeat (TR) DNA and tethers the viral episomes to host chromosomes through the association of chromatin-bound cellular proteins. TR elements serve as potential origin sites of KSHV replication and have been shown to play important roles in latent DNA replication and transcription of adjacent genes. Affinity chromatography and proteomics analysis using KSHV TR DNA and the LANA binding site as the affinity column identified topoisomerase IIß (TopoIIß) as a LANA-interacting protein. Here, we show that TopoIIß forms complexes with LANA that colocalize as punctuate bodies in the nucleus of KSHV-infected cells. The specific TopoIIß binding region of LANA has been identified to its N terminus and the first 32 amino acid residues containing the nucleosome-binding region crucial for binding. Moreover, this region could also act as a dominant negative to disrupt association of TopoIIß with LANA. TopoIIß plays an important role in LANA-dependent latent DNA replication, as addition of ellipticine, a selective inhibitor of TopoII, negatively regulated replication mediated by the TR. DNA break labeling and chromatin immunoprecipitation assay using biotin-16-dUTP and terminal deoxynucleotide transferase showed that TopoIIß mediates a transient DNA break on viral DNA. These studies confirm that LANA recruits TopoIIß at the origins of latent replication to unwind the DNA for replication.

A Case of Lenvatinib-Induced Focal Segmental Glomerulosclerosis (FSGS) in Metastatic Medullary Thyroid Cancer.

We describe a case of lenvatinib (E7080) associated focal segmental glomerulosclerosis (FSGS) encountered during the treatment of metastatic medullary thyroid cancer. Proteinuria is a relatively common side effect of VEGF-targeted treatments and can occasionally result in treatment discontinuation. Here, we describe a patient who developed secondary FSGS necessitating discontinuation of treatment at first but who was subsequently rechallenged with anti-VEGF-targeted treatment without recurrence of proteinuria. Lenvatinib was a novel experimental agent at the time the treatment took place; however, its recent licensing for the treatment of thyroid malignancies in the UK makes reporting of these adverse effects all the more important now.

Inhibition of Tip60 Reduces Lytic and Latent Gene Expression of Kaposi's Sarcoma-Associated Herpes Virus (KSHV) and Proliferation of KSHV-Infected Tumor Cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus responsible for the development of Kaposi's sarcoma, primary effusion lymphoma (PEL), and Multicentric Castleman's disease in immunocompromised individuals. Despite the burden of these diseases there are few treatment options for afflicted individuals, due in part to our limited understanding of virus-host interactions. Tip60, a histone aceytltransferase (HAT) has been previously shown to interact with both the KSHV latency associated nuclear antigen protein (LANA), which is the main factor in maintaining the viral latent state, and ORF36, a viral kinase expressed in the lytic phase. We further investigated Tip60-virus interaction to ascertain Tip60's role in the viral life cycle and its potential as a target for future therapeutics. Through modulation of Tip60 expression in HEK293T cells harboring a plasmid containing the KSHV viral episome, Bac36, we found that Tip60 is vital for both lytic replication as well as efficient expression of latent genes. Interestingly, Tip60 small molecule inhibitors, MG149 and NU9056, similarly inhibited latent and lytic genes, and reduced virion production in wild-type KSHV+/EBV- PEL, BCBL-1 cells. Long-term treatment with these Tip60 inhibitors selectively decreased the viability of KSHV-infected B lymphoma cells compared to uninfected cells. From this study, we conclude that Tip60 is important for KSHV infection and its associated cancer development, and Tip60 is therefore a potential target for future antiviral and anticancer therapeutics.

Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture.

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.

Antidepressants and unresponsive episodes in the elderly.

We present a case where initiation of antidepressant therapy was associated with an episode of unresponsiveness. Providing the opportunity to examine the possible pitfalls in geriatric prescribing.