Viral determinants in the NS3 helicase and 2K peptide that promote West Nile virus resistance to antiviral action of 2′,5′-oligoadenylate synthetase 1b

The interferon-inducible 2′,5′-oligoadenylate synthetase 1b (Oas1b) protein inhibits West Nile virus (WNV) infection by preventing viral RNA (vRNA) accumulation in infected cells. Serial passage of WNV in Oas1b-expressing mouse cells selected a virus variant with improved growth capacity. Two major amino acid substitutions were identified in this Oas1b-resistant WNV variant: NS3-S365G in the ATPase/helicase domain of NS3 and 2K-V9M in the C-terminal segment of NS4A. To assess their effect on antiviral activity of Oas1b, the NS3 and 2K mutations were engineered into an infectious WNV cDNA clone. The NS3 mutation alters requirement of ATP for ATPase activity and attenuates Oas1b-mediated suppression of vRNA accumulation. However, growth of NS3-mutant virus remains impaired in Oas1b-expressing cells. Only the 2K-V9M mutation efficiently rescued viral growth by promoting vRNA replication. Thus, WNV resistance to Oas1b antiviral action could be attributed to the 2K-V9M substitution with a potential role of NS3-S365G through rescue of vRNA accumulation.     

The 5′CL-PCBP RNP complex, 3′ poly(A) tail and 2A are required for optimal translation of poliovirus RNA

In this study, we showed that the 5′CL-PCBP complex, 3′ poly(A) tail and viral protein 2A^pro are all required for optimal translation of PV RNA. The 2A^pro-mediated stimulation of translation was observed in the presence or absence of both the 5′CL and the 3′ poly(A) tail. Using protein-RNA tethering, we established that the 5′CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5′CL-PCBP complex and the 3′ poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5′CL and the 3′ poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5′CL-PCBP complex interacts with the 3′ poly(A)-PABP complex to form a 5′-3′ circular complex that facilitates ribosome reloading and stimulates PV RNA translation.     

Synergistic antiviral activity of Sofosbuvir and type‐I interferons (α and β) against Zika virus

Zika virus (ZIKV) is transmitted by mosquitoes and causes Dengue‐like illness, neurological symptoms such as Guillain‐Barré Syndrome and microcephaly in children born to infected pregnant mothers. Recently, the World Health Organization (WHO) declared ZIKV infection as a Global Health Emergency. However, there are no known prophylactic or therapeutic measures against this virus. As a proof of concept toward combination therapeutic strategy against ZIKV, combinations of host‐targeted (Interferon‐α and Interferon‐β) and direct acting (Sofosbuvir) antivirals were evaluated in a hepatic cell line (Huh7) using a Cytoprotection (CP) assay. The combination of these antivirals resulted in synergistic inhibition of ZIKV infection in the in vitro CP assay. Additional testing in a ZIKV yield assay demonstrated that combination treatment of these antivirals conferred >2‐log reduction in the release of viral RNA. Measurement of ZIKV proteins in the cells infected with multiple ZIKV strains isolated from different geographical regions (Americas, Asia, and Africa) using an immunofluorescence assay confirmed the effective antiviral activity of this combination against ZIKV. These results demonstrate the in vitro proof of concept (POC) for using a combination approach utilizing the strengths of both virus and host‐targeted antivirals. These results suggest the effectiveness of the combination strategy in combating ZIKV, in the in vitro systems. Further evaluation of such combination therapies in vivo might provide an impetus for the development of effective ZIKV therapeutic strategies.     

Host-dependent roles of the viral 5′ untranslated region (UTR) in RNA stabilization and cap-independent translational enhancement mediated by the 3′ UTR of Red clover necrotic mosaic virus RNA1

The genome of Red clover necrotic mosaic virus (RCNMV) consists of RNA1 and RNA2, both lacking a cap structure and a poly(A)tail. RNA1 has a translational enhancer element (3′TE-DR1) in the 3′ untranslated region (UTR). In this study, we analyzed the roles of 5′ and 3′ UTRs of RNA1 in 3′TE-DR1-mediated cap-independent translation in cowpea and tobacco BY-2 protoplasts using a dual-luciferase (Luc) reporter assay system. Most mutations introduced into RNA1 5′ UTR in reporter Luc mRNA abolished or greatly reduced cap-independent translation in BY-2 protoplasts, whereas those mutations had no or much milder effects if any on translational activity in cowpea protoplasts. Our results suggest that a stem-loop structure predicted in the 5′ proximal region of RNA1 plays important roles in both translation and RNA stability. We also show that 3′TE-DR1-mediated cap-independent translation relies on a ribosome-scanning mechanism in both protoplasts.     

Blockade of central β-adrenoceptors attenuates tremor induced by 5-hydroxytryptamine (5-HT)-receptor activation in rats

The effect of β-adrenoceptor antagonists, varying in lipophilicity and receptor selectivity, were studied on tremor elicited by l -5-hydroxytryptophan (L-5-HTP) in rats pretreated with a peripherally acting decarboxylase inhibitor and a monoamine oxidase inhibitor, or by the directly acting 5-HT agonist s-methoxy-N,N-dimethyltryptamine (5-MeODMT). Plasma levels of the β-adrenoceptor antagonists were determined simultaneously. The non-selective lipophilic adrenoceptor antagonist propranolol was found to dose-dependently reduce tremor intensity, whereas the non-selective hydrophilic adrenoceptor antagonist sotalol had no effect, indicating a central site of action. Furthermore, β₁-selective blockade with the adrenoceptor antagonist metoprolol had no effect on tremor intensity, whereas the β₂-selective antagonist ICI 118,551 dose-dependently suppressed tremor intensity, suggesting that the β-adrenoceptor subtype involved is of the β₂-type. These results suggest that blockade of centrally located β₂-adrenoceptors are able to attenuate the tremor response following 5-hydroxytryptamine receptor activation.     

鹅流感病毒2/96-H5N1亚型毒株RNA1-3及RNA5节段核苷酸全序测定

目的　明确广东鹅流感病毒２９６Ｈ５ Ｎ１ 亚型毒株ＲＮＡ１３ 和ＲＮＡ５ 节段核苷酸全序列及其所编码蛋白的氨基酸序列,以及这些基因节段与香港禽流感病毒１５６９７Ｈ５ Ｎ１ 亚型毒株相应节段间的关系。方法　病毒粒ＲＮＡ经逆转录合成ｃＤＮＡ,经聚合酶链反应（ＰＣＲ） 扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定。结果　广东鹅流感病毒２９６Ｈ５ Ｎ１ 亚型毒株ＲＮＡ１３ 和ＲＮＡ５ 节段长度分别为２３４１,２ ３４１ ,２ ２３３ 和１５６５ 个核苷酸。它们分别编码ＰＢ２（ 含７５９ 个氨基酸）,ＰＢ１（ 含７５７个氨基酸） ,ＰＡ（ 含７１６ 个氨基酸） 和ＮＰ蛋白（ 含４９８ 个核苷酸） 。这些蛋白与香港禽流感病毒１５６９７Ｈ５ Ｎ１ 亚型毒株相应蛋白氨基酸序列的同源性分别为９６－４％ ,９７－２％ ,９７－３ ％ 和９７－０％ 。结论　本毒株ＲＮＡ１３ 和ＲＮＡ５ 节段长度分别为２ ３４１,２ ３４１,２ ２３３ 和１ ５６５ 个核苷酸,它们与香港１５６９７Ｈ５ Ｎ１ 亚型毒株间存在着差异     

5-HT2C (HTR2C) serotonin receptor gene polymorphism associated with the human personality trait of reward dependence: Interaction with dopamine D4 receptor (D4DR) and dopamine D3 receptor (D3DR) polymorphisms

We recently reported an association between the long repeat allele of the dopamine D4 exon III receptor polymorphism and a human personality dimension, novelty seeking, as measured by the tridimensional personality questionnaire (TPQ), a personality instrument designed by Cloninger to reflect heritable facets of human temperament. The D4 receptor polymorphism (D4DR) accounts for only a small percent of the variance for this trait, suggesting that additional genes influence both novelty seeking as well as the other temperaments that are inventoried by the Cloninger TPQ. In the current investigation, we examined, in the original cohort of 120 normal volunteers, two additional coding region polymorphisms, a glycine to serine substitution in the dopamine D3 receptor (D3DR) and a cysteine to serine substitution in the 5-HT_2C serotonin receptor (HTR2C). Three-way analysis of variance (TPQ score grouped by D4DR, D3DR and 5-HT_2C) demonstrated that reward dependence and persistence scores were significantly reduced by the presence of the less common 5-HT_2Cser polymorphism. The effect of the serine substitution in this X-linked serotonin receptor polymorphism on reward dependence was also observed when male and female subject groups were separately analyzed. There was also a significant interaction between the two dopamine receptor polymorphisms and the serotonin polymorphism on reward dependence. In particular, the effect of the 5-HT_2C polymorphism on reward dependence was markedly accentuated in individuals who had the long version of the D4DR exon III repeat polymorphism. When present in the same individual, the 5-HT_2C and dopamine receptor polymorphisms account for 30% of the observed variance for persistence (RD2) and 13% of the variance for reward dependence scores (RD134). However, the number of subjects with both less common D4DR and 5-HT_2C polymorphisms is small, underscoring the importance of verifying this interaction in a larger cohort. Am. J. Med. Genet. 74:65–72, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of β-thalassemia mutations in the United Arab Emirates provides evidence for recurrent origin of the IVSINT 5 (G-C) mutation

β-thalassemia mutations were characterized in a sample of 70 patients from United Arab Emirates (U.A.E.), resulting in an enlargement of the spectrum of types found in the country. The complete association between the most common IVS I nt 5 (G-C) mutation and a specific haplotype reveals an independent origin of this mutation in U.A.E. © 1995 Wiley-Liss, Inc.     

The interaction of beta 2-microglobulin (β2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse β2m

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8⁺ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and β₂-microglobulin (β₂m) have been used to examine the assembly of the trimolecular MHC class I/β₂m/peptide complex. Recombinant human β₂m and mouse β₂m² have been generated to compare the binding of the two β₂m to mouse class I. It is frequently assumed that human β₂m binds to mouse class I heavy chain with a much higher affinity than mouse β₂m itself. We find that human β₂m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse β₂m. In addition, we compared the effect of the two β₂m upon peptide binding to mouse class I. The ability of human β₂m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse β₂m only facilitated peptide binding when mouse β₂m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse β₂m to support peptide binding could not be attributed to a reduced affinity of mouse β₂m/MHC class I complexes for peptides or to a reduction in the fraction of mouse β₂m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in β₂m binding. We propose that mouse β₂m interacts with the minor peptide binding (i.e. the “empty”) fraction with a lower affinity than human β₂m does, whereas mouse and human β₂m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse β₂m is less efficient than human β₂m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human β₂m preferentially.