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2015 Vol.30(6)

Phage display is a method for the study of protein- or peptide-biological molecule interactions that utilize bacteriophages to connect peptides with the genetic information that encodes them. The filamentous bacteriophage M13, which can be functionalized by genetic engineering and chemical conjugation of its exterior surface coat proteins, has been applied as a biological scaffold in biomaterials, imaging, biocatalysis, and drug delivery. In this issue, a research group lead by Dr. Xiaosheng Liang, reports an engineered tyrosinedisplaying M13 phage that can aggregate under a wide range of concentration of ferric and ferrous ions. This engineered phage could be used as a probe for detection of ferric or ferrous ions without complicated instruments or reagents. The cover image shows the filamentous tyrosine-displaying phages aggregated side by side after incubation with Fe3+ and Fe2+. See page 410- 416 for details.

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Review

RNA chaperones encoded by RNA viruses

Jie Yang, Hongjie Xia, Qi Qian, Xi Zhou

2015, 30(6): 401 doi: 10.1007/s12250-015-3676-2

Received: 05 November 2015 Accepted: 11 December 2015 Published: 22 December 2015
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RNAs are functionally diverse macromolecules whose proper functions rely strictly upon their correct tertiary structures. However, because of their high structural flexibility, correct folding of RNAs is challenging and slow. Therefore, cells and viruses encode a variety of RNA remodeling proteins, including helicases and RNA chaperones. In RNA viruses, these proteins are believed to play pivotal roles in all the processes involving viral RNAs during the life cycle. RNA helicases have been studied extensively for decades, whereas RNA chaperones, particularly virus-encoded RNA chaperones, are often overlooked. This review describes the activities of RNA chaperones encoded by RNA viruses, particularly the ones identified and characterized in recent years, and the functions of these proteins in different steps of viral life cycles, and presents an overview of this unique group of proteins.
Research Article

Application of an M13 bacteriophage displaying tyrosine on the surface for detection of Fe3+ and Fe2+ ions

Xiaohua Guo, Chuncheng Niu, Yunhua Wu, Xiaosheng Liang

2015, 30(6): 410 doi: 10.1007/s12250-015-3651-y

Received: 16 September 2015 Accepted: 03 December 2015 Published: 14 December 2015
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Ferric and ferrous ion plays critical roles in bioprocesses, their influences in many fields have not been fully explored due to the lack of methods for quantification of ferric and ferrous ions in biological system or complex matrix. In this study, an M13 bacteriophage (phage) was engineered for use as a sensor for ferric and ferrous ions via the display of a tyrosine residue on the P8 coat protein. The interaction between the specific phenol group of tyrosine and Fe3+ / Fe2+ was used as the sensor. Transmission electron microscopy showed aggregation of the tyrosine-displaying phages after incubation with Fe3+ and Fe2+. The aggregated phages infected the host bacterium inefficiently. This phenomenon could be utilized for detection of ferric and ferrous ions. For ferric ions, a calibration curve ranging from 200 nmol/L to 8 μmol/L with a detection limit of 58 nmol/L was acquired. For ferrous ions, a calibration curve ranging from 800 nmol/L to 8 μmol/L with a detection limit of 641.7 nmol/L was acquired. The assay was specific for Fe3+ and Fe2+ when tested against Ni2+, Pb2+, Zn2+, Mn2+, Co2+, Ca2+, Cu2+, Cr3+, Ba2+, and K+. The tyrosine displaying phage to Fe3+ and Fe2+ interaction would have plenty of room in application to biomaterials and bionanotechnology.

Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller, Cnaphalocrocis medinalis

Shan Zhang, Zheng Zhu, Shifeng Sun, Qijin Chen, Fei Deng, Kai Yang

2015, 30(6): 417 doi: 10.1007/s12250-015-3658-4

Received: 29 September 2015 Accepted: 24 November 2015 Published: 18 December 2015
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The complete genome of Cnaphalocrocis medinalis granulovirus (CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system (GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames (ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17kb gene block inversions, and does not contain any typical homologous region (hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis, based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV, and would benefit to the utilization of CnmeGV as pest control reagent for rice production.

Cloning, expression, and antiviral activity of interferon β from the Chinese microbat, Myotis davidii

Ying-Zi Liang, Li-Jun Wu, Qian Zhang, Peng Zhou, Mei-Niang Wang, Xing-Lou Yang, Xing-Yi Ge, Lin-Fa Wang, Zheng-Li Shi

2015, 30(6): 425 doi: 10.1007/s12250-015-3668-2

Received: 16 October 2015 Accepted: 27 November 2015 Published: 07 December 2015
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Bats are natural reservoir hosts for many viruses that produce no clinical symptoms in bats. Therefore, bats may have evolved effective mechanisms to control viral replication. However, little information is available on bat immune responses to viral infection. Type I interferon (IFN) plays a key role in controlling viral infections. In this study, we report the cloning, expression, and biological activity of interferon β (IFNβ) from the Chinese microbat species, Myotis davidii. We demonstrated the upregulation of IFNB and IFN-stimulated genes in a kidney cell line derived from M. davidii after treatment with polyI:C or infection with Sendai virus. Furthermore, the recombinant IFNβ inhibited vesicular stomatitis virus and bat adenovirus replication in cell lines from two bat species, M. davidii and Rhinolophus sinicus. We provide the first in vitro evidence of IFNβ antiviral activity in microbats, which has important implications for virus interactions with these hosts.

Conserved termini and adjacent variable region of Twortlikevirus Staphylococcus phages

Xianglilan Zhang, Huaixing Kang, Yuyuan Li, Xiaodong Liu, Yu Yang, Shasha Li, Guangqian Pei, Qiang Sun, Peng Shu, Zhiqiang Mi, Yong Huang, Zhiyi Zhang, Yannan Liu, Xiaoping An, Xiaolu Xu, Yigang Tong

2015, 30(6): 433 doi: 10.1007/s12250-015-3643-y

Received: 06 September 2015 Accepted: 13 November 2015 Published: 14 December 2015
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Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing cause of serious infection, both in the community and hospital settings. Despite sophisticated strategies and efforts, the antibiotic options for treating MRSA infection are narrowing because of the limited number of newly developed antimicrobials. Here, four newly-isolated MRSA-virulent phages, IME-SA1, IMESA2, IME-SA118 and IME-SA119, were sequenced and analyzed. Their genome termini were identified using our previously proposed "termini analysis theory". We provide evidence that remarkable conserved terminus sequences are found in IME-SA1/2/118/119, and, moreover, are widespread throughout Twortlikevirus Staphylococcus phage G1 and K species. Results also suggested that each phage of the two species has conserved 5′ terminus while the 3′ terminus is variable. More importantly, a variable region with a specific pattern was found to be present near the conserved terminus of Twortlikevirus S. phage G1 species. The clone with the longest variable region had variable terminus lengths in successive generations, while the clones with the shortest variable region and with the average length variable region maintained the same terminal length as themselves during successive generations. IME-SA1 bacterial infection experiments showed that the variation is not derived from adaptation of the phage to different host strains. This is the first study of the conserved terminus and variable region of Twortlikevirus S. phages.

HIV-1 Vpr protein activates the NF-κB pathway to promote G2/M cell cycle arrest

Zhibin Liang, Ruikang Liu, Yongquan Lin, Chen Liang, Juan Tan, Wentao Qiao

2015, 30(6): 441 doi: 10.1007/s12250-015-3654-8

Received: 24 September 2015 Accepted: 24 November 2015 Published: 14 December 2015
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Viral protein R (Vpr) plays an important role in the replication and pathogenesis of Human immunodeficiency virus type 1 (HIV-1). Some of the various functions attributed to Vpr, including the induction of G2/M cell cycle arrest, activating the NF-κB pathway, and promoting viral reverse transcription, might be interrelated. To test this hypothesis, a panel of Vpr mutants were investigated for their ability to induce G2/M arrest and to activate the NF-κB pathway. The results showed that the Vpr mutants that failed to activate NF-κB also lost the activity to induce G2/M arrest, which suggests that inducing G2/M arrest via Vpr depends at least partially on the activation of NF-κB. This latter possibility is supported by data showing that knocking down the key factors in the NF-κB pathway – p65, RelB, IKKα, or IKKβ – partially rescued the G2/M arrest induced by Vpr. Our results suggest that the NF-κB pathway is probably involved in Vpr-induced G2/M cell cycle arrest.

N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies

Dezhi Li, Jie Liu, Li Zhang, Tianshu Xu, Junheng Chen, Liping Wang, Qi Zhao

2015, 30(6): 449 doi: 10.1007/s12250-015-3664-6

Received: 09 October 2015 Accepted: 14 December 2015 Published: 21 December 2015
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The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662–667 in the MPER) but also several other residues (aa 652–655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.
Letter

Morphological diversity of cultured cold-active lytic bacteriophages isolated from the Napahai plateau wetland in China

Xiuling Ji, Chunjing Zhang, Anxiu Kuang, Jiankai Li, Yinshan Cui, Kunhao Qin, Lianbing Lin, Benxu Cheng, Qi Zhang, Yunlin Wei

2015, 30(6): 457 doi: 10.1007/s12250-015-3674-4

Published: 16 December 2015
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In summary, rod-shaped bacterial cells and Pseudomonas spp. dominated the bacterial community in the Napahai plateau wetland. Based on our TEM results, bacteriophages with larger capsids (60–140 nm) and Siphoviridae members were dominant. We were able to isolate a batch of cultured cold-active bacteriophages and their host bacterial strains, which should provide opportunities for further qualitative or quantitative analysis of bacteriophage function in wetland ecology. Specifically, these new isolates can help clarify bacteriophage coldadaptation mechanisms, structure, and assembly, as well as improve our understanding of phage-host co-evolution and interactions.

Epidemiological study and variation analysis of the porcine kobuvirus 3D gene in Sichuan province, China

Pengjuan Liu, Ping Li, Wenting Lyu, Xinqiong Li, Song Li, Fan Yang, Jianbo Huang, Zhiwen Xu, Ling Zhu

2015, 30(6): 460 doi: 10.1007/s12250-015-3632-1

Published: 30 November 2015
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As a viral RNA-dependent polymerase, porcine kobuvirus 3D protein is involved in regulating viral RNA replication and plays an important role in virus proliferation (Verma et al., 2013). Therefore, we first conducted molecular and phylogenic analysis of the porcine kobuvirus 3D region. According to the phylogenic tree, the porcine kobuvirus 3D gene was relatively conserved, and the virus has not evolved dramatically since it was first isolated in 2007. A report by Chen showed that the nucleotide and amino acid identities of the porcine kobuvirus VP1 region range from 97.5%–98.8% and 98.2%–98.7%, respectively (Chen et al., 2013); this variability was less than that observed in our current study. Moreover, some point mutations were observed in the analyzed sequences; these mutations may have changed the conformation and function of the 3D protein. These current findings provide insights into the epidemiology and genetic characteristics of this newly emerging virus.

Molecular characterization of DENV-3 circulating during the post-monsoon period of 2013-14 in Delhi, India

Sharma Pankaj, Mittal Veena, Chhabra Mala, Roop Kumari, Priyanka Singh, Dipesh Bhattacharya, Srinivas Venkatesh, Arvind Rai

2015, 30(6): 464 doi: 10.1007/s12250-015-3649-5

Published: 14 December 2015
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The present study characterizes the recently circulating DENV-3 strain in the metropolitan city of Delhi during post monsoon period of 2013-14. Partial molecular characterization of 12 DENV3 isolates was carried out on the basis of envelope (E) and non-structural 1 (NS1) gene regions. Phylogenetic analysis showed all these 12 isolates grouped under lineage III of genotype III with recent isolates from China and Pakistan. The point mutation L430I in the Env region appears to be the unique molecular signature for the 2013 strains. Another unique substitution, I167V in NS1 protein was observed in a single isolate among those 12 samples. The study describing the molecular characterization could be significant if these unique substitutions cause the poor B cell responses.

Molecular characterization of chikungunya virus from three regions of Cameroon

Demanou Maurice, Sadeuh-Mba Serge Alain, Vanhecke Christophe, Ndikweti Rene, Kouna Tsala Irene, Inais Nsizoa Marthe, Njouom Richard

2015, 30(6): 470 doi: 10.1007/s12250-015-3663-7

Published: 21 December 2015
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Our results indicate that CHIKV strains circulating in these three regions of Cameroon have a common origin and do not differ from strains circulating in Central Africa as they all clustered in the central African lineage. The presence of the E1-A226V mutation indicates the possible virulence of the Cameroonian strains and might explain the emergence or re-emergence of chikungunya in Central Africa during the last decade after the introduction of A. albopictus.