Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts

CREB Regulates AChE-R-Induced Proliferation of Human Glioblastoma Cells

The cyclic adenosine monophosphate (AMP) response element-binding protein, CREB, often modulates stress responses. Here, we report that CREB suppresses the glioblastoma proliferative effect of the stress-induced acetylcholinesterase variant, AChE-R. In human U87MG glioblastoma cells, AChE-R formed a triple complex with protein kinase C (PKC) ε and the scaffold protein RACK1, enhanced PKCε phosphorylation, and facilitated BrdU incorporation. Either overexpressed CREB, or antisense destruction of AChE-R mRNA, PKC, or protein kinase A (PKA) inhibitors—but not CREB combined with PKC inhibition suppressed—this proliferation, suggesting that CREB's repression of this process involves a PKC-mediated pathway, whereas impaired CREB regulation allows AChE-R-induced, PKA-mediated proliferation of glioblastoma tumors

The complex regulation of HIC (Human I-mfa domain containing protein) expression.

Human I-mfa domain containing protein (HIC) differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb). Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5' and 3' untranslated regions (UTRs) compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3'-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5'-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions

Amino Terminal Region of Dengue Virus NS4A Cytosolic Domain Binds to Highly Curved Liposomes

Dengue virus (DENV) is an important human pathogen causing millions of disease cases and thousands of deaths worldwide. Non-structural protein 4A (NS4A) is a vital component of the viral replication complex (RC) and plays a major role in the formation of host cell membrane-derived structures that provide a scaffold for replication. The N-terminal cytoplasmic region of NS4A(1–48) is known to preferentially interact with highly curved membranes. Here, we provide experimental evidence for the stable binding of NS4A(1–48) to small liposomes using a liposome floatation assay and identify the lipid binding sequence by NMR spectroscopy. Mutations L6E;M10E were previously shown to inhibit DENV replication and to interfere with the binding of NS4A(1–48) to small liposomes. Our results provide new details on the interaction of the N-terminal region of NS4A with membranes and will prompt studies of the functional relevance of the curvature sensitive membrane anchor at the N-terminus of NS4A

The Role of NS1 Protein in the Diagnosis of Flavivirus Infections

Nonstructural protein 1 (NS1) is a glycoprotein among the flavivirus genus. It is found in both membrane-associated and soluble secreted forms, has an essential role in viral replication, and modulates the host immune response. NS1 is secreted from infected cells within hours after viral infection, and thus immunodetection of NS1 can be used for early serum diagnosis of dengue fever infections instead of real-time (RT)-PCR. This method is fast, simple, and affordable, and its availability could provide an easy point-of-care testing solution for developing countries. Early studies show that detecting NS1 in cerebrospinal fluid (CSF) samples is possible and can improve the surveillance of patients with dengue-associated neurological diseases. NS1 can be detected postmortem in tissue specimens. It can also be identified using noninvasive methods in urine, saliva, and dried blood spots, extending the availability and effective detection period. Recently, an enzyme-linked immunosorbent assay (ELISA) assay for detecting antibodies directed against Zika virus NS1 has been developed and used for diagnosing Zika infection. This NS1-based assay was significantly more specific than envelope protein-based assays, suggesting that similar assays might be more specific for other flaviviruses as well. This review summarizes the knowledge on flaviviruses’ NS1′s potential role in antigen and antibody diagnosis

Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway

Abstract Background The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here. Findings Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs). Conclusions These findings add TBC1D20 to the network of host factors regulating HIV replication cycle.</p

Structure and membrane interaction of the N-terminal region of Dengue virus NS4A protein

Dengue virus (DENV) infection presents a serious public health threat with more than one third of the world population at risk. DENV is a mosquito-transmitted virus that causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. There is no vaccine available against DENV and no specific treatment for dengue fever. DENV is believed to replicate its RNA genome in association with modified intracellular membranes. However, the details of the assembly of this replication complex are incompletely understood. We focused on the DENV non-structural protein 4A (NS4A) which has been implicated in the formation of the viral RNA replication complex. Sequence analysis identified conserved regions in the N-terminal 48 amino acids of NS4A that might form amphipathic helices (AH). Mutations (L6E; M10E) designed to reduce the amphipathic character of the predicted AH, abolished viral replication and reduced NS4A oligomerization [1]. However, little is known about the three dimensional structure of NS4A(1-48). We used solution state nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to study the structure of wild type NS4A(1-48) and of the double mutant NS4A(1-48, L6E;M10E) in the presence and absence of model membranes. The hydrodynamic radius of liposomes and detergent micelles was determined by dynamic light scattering (DLS). Both peptides were recombinantly produced in E.coli and are basically unstructured in aqueous buffer [2]. Addition of liposomes made of POPC or POPC/DOPS mixtures induced formation of α-helical secondary structure in case of the wild type NS4A(1-48) but not for the mutant peptide. The degree of helicity of wt NS4A(1-48) is sensitive to the lipid composition and to the size of the liposomes. Formation of α-helical secondary structure was observed for both wt and mutant NS4A(1-48) upon addition of various membrane mimicking detergent micelles (SDS, DPC, DHPC, DM). The degree of helix formation depends on the type and concentration of the detergent and reaches a maximum at about 100 mM SDS or DPC. Solution state NMR spectroscopy provided a detailed picture of the structure and micelle interaction for both peptides in presence of 100 mM SDS. Backbone resonance assignment followed by analysis of secondary chemical shifts allowed us to identify two α-helical segments in each peptide which cover amino acid residues 5-10 and 15-29 in NS4A(1-48) and residues 4-9 and 15-29 in NS4A(1-48, L6E;M10E). Analysis of paramagnetic relaxation enhancement after addition of paramagnetic Mn2+ to the SDS micelle-containing buffer allowed us to distinguish buffer exposed from buried amino acid residues.[1] O. Stern et al. (2013) J. Virol. 87:4080-85[2] Y.F. Hung et al. (2014) PLoS One. 9: e8648