Viperin Restricts Zika Virus and Tick-Borne Encephalitis Virus Replication by Targeting NS3 for Proteasomal Degradation.

Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus, affording the possible availability of new drug targets that can be used for therapeutic intervention.IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses, but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism employed by viperin to inhibit the replication of two flaviviruses: tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV nonstructural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's antiflavivirus activity. Taking the results together, our work shows important similarities and differences among the members of the genus Flavivirus and could lead to the possibility of therapeutic intervention.

Cellular requirements for iron-sulfur cluster insertion into the antiviral radical SAM protein viperin.

Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and 55Fe-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. 55Fe-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for 55Fe/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.

Aetiology of Acute Respiratory Tract Infections in Hospitalised Children in Cyprus.

In order to improve clinical management and prevention of viral infections in hospitalised children improved etiological insight is needed. The aim of the present study was to assess the spectrum of respiratory viral pathogens in children admitted to hospital with acute respiratory tract infections in Cyprus. For this purpose nasopharyngeal swab samples from 424 children less than 12 years of age with acute respiratory tract infections were collected over three epidemic seasons and were analysed for the presence of the most common 15 respiratory viruses. A viral pathogen was identified in 86% of the samples, with multiple infections being observed in almost 20% of the samples. The most frequently detected viruses were RSV (30.4%) and Rhinovirus (27.4%). RSV exhibited a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. While RSV and PIV3 incidence decreased significantly with age, the opposite was observed for influenza A and B as well as adenovirus infections. The data presented expand our understanding of the epidemiology of viral respiratory tract infections in Cypriot children and will be helpful to the clinicians and researchers interested in the treatment and control of viral respiratory tract infections.

The many isoforms of human adenylate kinases.

Adenine nucleotides are involved in a variety of cellular metabolic processes, including nucleic acid synthesis and repair, formation of coenzymes, energy transfer, cell and ciliary motility, hormone secretion, gene expression regulation and ion-channel control. Adenylate kinases are abundant phosphotransferases that catalyze the interconversion of adenine nucleotides and thus regulate the adenine nucleotide ratios in different intracellular compartments. Nine different adenylate kinase isoenzymes have been identified and characterized so far in human tissues, named AK1 to AK9 according to their order of discovery. Adenylate kinases differ in molecular weight, tissue distribution, subcellular localization, substrate and phosphate donor specificity and kinetic properties. The preferred substrate and phosphate donor of all adenylate kinases are AMP and ATP respectively, but some members of the family can phosphorylate other substrates and use other phosphate donors. In addition to their nucleoside monophosphate kinase activity, adenylate kinases were found to possess nucleoside diphosphate kinase activity as they are able to phosphorylate both ribonucleoside and deoxyribonucleoside diphosphates to their corresponding triphosphates. Nucleoside analogues are structural analogues of natural nucleosides, used in the treatment of cancer and viral infections. They are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. Novel data presented in this review confirm the role of adenylate kinases in the activation of deoxyadenosine and deoxycytidine nucleoside analogues.

The human adenylate kinase 9 is a nucleoside mono- and diphosphate kinase.

Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate kinase family of enzymes and accordingly named the protein adenylate kinase 9 (AK9). Initially a full-length cDNA of a hypothetical protein containing a predicted adenylate kinase domain was identified and subsequently cloned and expressed in Escherichia coli. The substrate specificity of the recombinant protein showed that the enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP as phosphate donor, while only AMP and CMP were phosphorylated when GTP was the phosphate donor. The kinetic parameters of AK9 were determined for AMP, dAMP and CMP with ATP as phosphate donor. Interestingly, in addition to the diphosphate products, a nucleoside diphosphate kinase (NDPK) activity was also present with subsequent triphosphates formed. With ATP or GTP as phosphate donor it was possible to detect the production of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and TTP as enzymatic products from the corresponding diphosphate substrates. A number of previously characterized adenylate kinases were also tested and found to possess a broad phosphotransferase activity similar to AK9. These enzymes are accordingly suggested to be regarded as nucleoside mono- and diphosphate kinases with catalytic activities possibly determined by local substrate concentrations.

The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes.

Differences in expression profiles, substrate specificities, kinetic properties and subcellular localization among the AK (adenylate kinase) isoenzymes have been shown to be important for maintaining a proper adenine nucleotide composition for many different cell functions. In the present study, human AK7 was characterized and its substrate specificity, kinetic properties and subcellular localization determined. In addition, a novel member of the human AK family, with two functional domains, was identified and characterized and assigned the name AK8. AK8 is the second known human AK with two complete and active AK domains within its polypeptide chain, a feature that has previously been shown for AK5. The full-length AK8, as well as its two domains AK8p1 and AK8p2, all showed similar AK enzyme activity. AK7, full-length AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP with ATP as the phosphate donor, and also AMP, CMP and dCMP with GTP as the phosphate donor. Both AK7 and full-length AK8 showed highest affinity for AMP with ATP as the phosphate donor, and proved to be more efficient in AMP phosphorylation as compared with the major cytosolic isoform AK1. Expression of the proteins fused with green fluorescent protein demonstrated a cytosolic localization for both AK7 and AK8.

Evidence of an intact N-terminal translocation sequence of human mitochondrial adenylate kinase 4.

Adenylate kinases are abundant nucleoside monophosphate kinases, which catalyze the phosphorylation of AMP by using ATP or GTP as phosphate donors. A previously cloned cDNA was named adenylate kinase 4 (AK4) based on its sequence similarity with known AKs but with no confirmed AK enzyme activity. In the present study the AK4 cDNA was expressed in Escherichia coli and the substrate specificity and kinetic properties of the recombinant protein were characterized. The enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP or GTP as phosphate donors and AK4 also phosphorylated AMP with UTP as phosphate donor. The kinetic parameters of the enzyme were determined for AMP and dAMP with ATP as phosphate donor and for AMP with GTP as phosphate donor. AK4 showed its highest efficiency when phosphorylating AMP with GTP and a slightly lower efficiency for the phosphorylation of AMP with ATP. Among the three reactions for which kinetics were performed, dAMP was the poorest substrate. The AK4 mitochondrial localization was confirmed by expression of AK4 as a fusion protein with GFP in HeLa cells. The mitochondrial import sequence was shown to be located within the first N-terminal 11 amino acid residues, very close to the ATP-binding region of the enzyme. Import analysis suggested that the mitochondrial import sequence was not cleaved and thus the enzyme retained its activity upon entering the mitochondria. Site directed mutagenesis of amino acids Lys 4 and Arg 7 showed that these two residues were essential for mitochondrial import.

Identification of two active functional domains of human adenylate kinase 5.

A full length cDNA that partially corresponded to human adenylate kinase 5 (AK5) was identified and shown to encode for two separate domains. The full length protein could be divided in two distinct functional domains, a previously unidentified domain of 338 amino acids and a second domain of 198 amino acids that corresponded to the protein characterized as AK5, now called AK5p2. The first domain, AK5p1, phosphorylated AMP, CMP, dAMP and dCMP with ATP or GTP as phosphate donors similarly to AK5p2. Our data demonstrate that human AK5 has two separate functional domains and that both have enzymatic activity.