Putative N-linked Glycosylation Sites Research Articles

Establishment of a CPER reverse genetics system for Powassan virus defines attenuating NS1 glycosylation sites and an infectious NS1-GFP11 reporter virus.

Powassan virus (POWV) is an emerging tick-borne Flavivirus that causes lethal encephalitis and long-term neurologic damage. Currently, there are no POWV therapeutics, licensed vaccines, or reverse genetics systems for producing infectious POWVs from recombinant DNA. Using a circular polymerase extension reaction (CPER), we generated recombinant LI9 (recLI9) POWVs with attenuating NS1 protein mutations and a recLI9-split-eGFP reporter virus. NS1 proteins are highly conserved glycoproteins that regulate replication, spread, and neurovirulence. POWV NS1 contains three putative N-linked glycosylation sites that we modified individually in infectious recLI9 mutants (N85Q, N208Q, and N224Q). NS1 glycosylation site mutations reduced replication kinetics and were attenuated, with 1-2 log decreases in titer. Severely attenuated recLI9-N224Q exhibited a 2- to 3-day delay in focal cell-to-cell spread and reduced NS1 secretion but was lethal when intracranially inoculated into suckling mice. However, footpad inoculation of recLI9-N224Q resulted in the survival of 80% of mice and demonstrated that NS1-N224Q mutations reduce POWV neuroinvasion in vivo. To monitor NS1 trafficking, we CPER fused a split GFP11-tag to the NS1 C-terminus and generated an infectious reporter virus, recLI9-NS1-GFP11. Cells infected with recLI9-NS1-GFP11 revealed NS1 trafficking in live cells and the novel formation of large NS1-lined intracellular vesicles. An infectious recLI9-NS1-GFP11 reporter virus permits real-time analysis of NS1 functions in POWV replication, assembly, and secretion and provides a platform for evaluating antiviral compounds. Collectively, our robust POWV reverse genetics system permits analysis of viral spread and neurovirulence determinants in vitro and in vivo and enables the rational genetic design of live attenuated POWV vaccines. IMPORTANCE Our findings newly establish a mechanism for genetically modifying Powassan viruses (POWVs), systematically defining pathogenic determinants and rationally designing live attenuated POWV vaccines. This initial study demonstrates that mutating POWV NS1 glycosylation sites attenuates POWV spread and neurovirulence in vitro and in vivo. Our findings validate a robust circular polymerase extension reaction approach as a mechanism for developing, and evaluating, attenuated genetically modified POWVs. We further designed an infectious GFP-tagged reporter POWV that permits us to monitor secretory trafficking of POWV in live cells, which can be applied to screen potential POWV replication inhibitors. This robust system for modifying POWVs provides the ability to define attenuating POWV mutations and create genetically attenuated recPOWV vaccines.

Insights into the role of N‐linked glycosylation in directing plasma membrane localization of GPCRs expressed in Saccharomyces cerevisiae

Glycosylation is the biochemical process of the attachment of sugar moieties or carbohydrates to proteins during co‐translational or post‐translational modification (PTM). It is one of many types of protein modifications that naturally occurs during the process of protein expression in organisms like Saccharomyces cerevisiae (Baker’s yeast). For natively expressed proteins, glycosylation can be critical for proper protein processing and can also be important for the production of proteins in a stable and biologically active state. For proteins that are non‐natively, or heterologously, expressed in these organisms, glycosylation can lead to improper processing and ultimately produce proteins with little to no biological relevance. This proves to be an undesirable effect for the elucidation of protein structure. However, large quantities of protein are required for structural analysis, presenting a significant obstacle for structural biologists and those who use heterologous expression as a means to acquire large quantities of protein. This problem becomes significantly more challenging for membrane proteins.Membrane proteins are an elusive class of biomolecules that reside or closely associate with the lipid bilayers of cells and encompass transporters, receptors and channel pores, to name a few. They play an intrinsic role in maintaining proper cell function including signaling and selective uptake of extracellular ions and small molecules. G‐protein coupled receptors (GPCRs) constitute one of the largest groups of membrane proteins across the plant and animal kingdoms. Despite being of scientific and medical interest, GPCRs and many integral membrane proteins are notoriously difficult to isolate and work with because of their hydrophobicity, structural flexibility and instability in the absence of the cell membrane. Optimized experimental conditions often vary from protein to protein and necessitate a systematic approach to achieve. This typically requires labor and resource intensive efforts. The goal of this work is to uncover new insights into the role of post‐translational processing and protein localization in the cell. Using a combination of fluorescence microscopy and bioinformatics we formulated an approach to investigate the effects of N‐linked glycosylation on plasma membrane localization of GPCRs expressed in S. cerevisiae. We postulate a critical link between plasma membrane localization and expression of “ligand active” or structurally relevant GPCRs. Previous studies of the human adenosine A2A receptor (hA2AR) show it readily localizes to the plasma membrane during expression in yeast, while the human dopamine D2 receptor (hD2R) does not. A sequence comparison of the two reveals three putative N‐linked glycosylation sites at the N‐terminal extracellular region of hD2R which is lacking in hA2AR. Using a chimera of these two receptors prepared with a C‐terminal GFP tag, we show the chimera does not traffic to the plasma membrane similarly to full‐length hA2AR, suggesting N‐linked glycosylation strongly influences functional high‐level expression of GPCRs and possibly other membrane proteins in yeast.

The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity.

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.

Functional disruption of transferrin expression alters reproductive physiology in Anopheles culicifacies.

BackgroundIron metabolism is crucial to maintain optimal physiological homeostasis of every organism and any alteration of the iron concentration (i.e. deficit or excess) can have adverse consequences. Transferrins are glycoproteins that play important role in iron transportation and have been widely characterized in vertebrates and insects, but poorly studied in blood-feeding mosquitoes.ResultsWe characterized a 2102 bp long transcript AcTrf1a with complete CDS of 1872bp, and 226bp UTR region, encoding putative transferrin homolog protein from mosquito An. culicifacies. A detailed in silico analysis predicts AcTrf1a encodes 624 amino acid (aa) long polypeptide that carries transferrin domain. AcTrf1a also showed a putative N-linked glycosylation site, a characteristic feature of most of the mammalian transferrins and certain non-blood feeding insects. Structure modelling prediction confirms the presence of an iron-binding site at the N-terminal lobe of the transferrin. Our spatial and temporal expression analysis under altered pathophysiological conditions showed that AcTrf1a is abundantly expressed in the fat-body, ovary, and its response is significantly altered (enhanced) after blood meal uptake, and exogenous bacterial challenge. Additionally, non-heme iron supplementation of FeCl3 at 1 mM concentration not only augmented the AcTrf1a transcript expression in fat-body but also enhanced the reproductive fecundity of gravid adult female mosquitoes. RNAi-mediated knockdown of AcTrf1a causes a significant reduction in fecundity, confirming the important role of transferrin in oocyte maturation.ConclusionAll together our results advocate that detailed characterization of newly identified AcTrf1a transcript may help to select it as a unique target to impair the mosquito reproductive outcome.

Effects of N224 Glycosylation in Saccharomycopsis fibuligera R64 α-Amylase on Enzyme Activity and Stability

Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. Objective: To test whether N224 plays a key role in enzyme activity and stability. Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activities of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

Dual ELISA using SARS-CoV-2 nucleocapsid protein produced in E. coli and CHO cells reveals epitope masking by N-glycosylation

Spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-SP, SARS-CoV-2-NP) are the main immunogenic targets for antibodies. We herein demonstrate that the glycosylation of SARS-CoV-2-NP masks some of its antibody epitopes. In many cases, this can lead to false-negative serological tests. Deglycosylation of SARS-CoV-2-NP significantly increased the number of positive tests. The glycosylation pattern analysis of this protein revealed that the putative N-linked glycosylation sites, at the amino acid positions 48 and 270, co-located with two of the main immunodominant B cell epitopes.

Fasciola hepatica Extracellular Vesicles isolated from excretory-secretory products using a gravity flow method modulate dendritic cell phenotype and activity.

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.

Genetic characterization of bovine ephemeral fever virus in southern China, 2013-2017.

Bovine ephemeral fever virus (BEFV) can cause bovine ephemeral fever and is an economically important arbovirus of cattle. To expand the knowledge of the molecular epidemiology of BEFV in southern China, the complete surface glycoprotein G gene of BEFV was sequenced from samples collected in five restricted outbreaks from 2013 to 2017, namely 2013ZH, 2014HM, 2015GX, 11082-2016, and qy2017. It was noted that both 2014HM and 11082-2016 were detected in cattle regularly vaccinated with inactivated vaccine. Phylogenetic analysis demonstrated that all five strains grouped into cluster I. However, qy2017 was closer to the BEFV strains identified in Thailand, Japan, and Taiwan after 2000, while 2013ZH, 2014HM, 2015GX, and 11082-2016 were closer to the Chinese strains in 2011 and the Turkey strains in 2012. The analysis of antigenic sites indicated that several amino acid changes occurred between the five strains and the vaccine strain. Importantly, one novel amino acid mutation site was observed in the putative N-linked glycosylation sites of 2013ZH, 2014HM, 2015GX, and 11082-2016. Our study indicated novel genetic characteristics of the newly emerging BEFV strains in southern China and the necessity of updating the component of commercially available inactivated BEFV vaccines in China.

Identification, characterization, and expression analysis of a serotonin receptor involved in the reproductive process of the Pacific abalone, Haliotis discus hannai.

Serotonin receptor (5-HT) is a biogenic amine acting as a neurotransmitter and neuromodulator that mediates various aspects of reproduction and gametogenesis. The full-length nucleotide sequence of Haliotis discus hannai encodes a protein of 417 amino acids with a predicted molecular mass of 46.54kDa and isoelectric point of 8.94. The structural profile of 5-HTHdh displayed key features of G protein-coupled receptors, including seven hydrophobic transmembrane domains, putative N-linked glycosylation sites, and several phosphorylation consensus motifs. It shares the highest homology of its amino acid sequence with the 5-HT receptor from Haliotis asinina, and to lesser extent of human 5-HT receptor. The cloned sequence possesses two cysteine residues (Cys-115 and Cys-193), which are likely to form a disulfide bond. Phylogenetic comparison with other known 5-HT receptor genes revealed that the 5-HTHdh is most closely related to the 5-HTHa receptor. The three-dimensional structure of the 5-HTHdh showed multiple alpha helices which is separated by a helix-loop-helix (HLH) structure. Quantitative PCR demonstrated that the receptor mRNA was predominantly expressed in the pleuropedal ganglion. Significant differences in the transcriptional activity of the 5-HTHdh gene were observed in the ovary at the ripening stage. An exclusive expression was detected in pleuropedal ganglion, testis, and ovary at higher effective accumulative temperature (1000°C). In situ hybridization showed that the 5-HTHdh expressing neurosecretory cells were distributed in the cortex of the pleuropedal ganglion. Our results suggest that 5-HTHdh synthesized in the neural ganglia may be involved in oocyte maturation and spawning of H. discus hannai.

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~ 50,000 high-quality full-length HIV envelope sequences (> 2200bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

Cloning, identification, and functional analysis of bone marrow stromal cell antigen-2 from sika deer (Cervus nippon)

BST-2(tetherin/CD317/HM1.24) has been identified as a cellular antiviral factor that inhibits the release of a wide range of enveloped viruses from infected cells. Orthologs of BST-2 have been identified in several species including humans, monkeys, cows, sheep, pigs, and mice. In this study, we cloned the gene and characterized the protein of the BST-2 homolog from sika deer (Cervus nippon). cnBST-2 shares 37.8% and 74.2% identity with the BST-2 homologs from Homo sapiens and Ovis aries, respectively. The extracellular domain of cnBST-2 has two putative N-linked glycosylation sites and three potential dimerization sites. cnBST-2 was shown to be expressed on the cell surface, like human BST-2. Exogenous expression of cnBST-2 resulted in potent inhibition of HIV-1 particle release in 293T cells; however, this activity resisted antagonism by HIV-1 Vpu. Moreover, cnBST-2 was not able to activate nuclear factor-κB, in contrast to human BST-2. This study is the first report of the isolation and characterization of BST-2 from C. nippon.

Genomic analysis of a recombinant NADC30-like porcine reproductive and respiratory syndrome virus in china.

Recently, NADC30-like porcine reproductive and respiratory syndrome viruses (PRRSVs), which are genetically similar to the NADC30 strain isolated in the United States of America in 2008, have become prevalent in China. Here, a novel variant PRRSV strain named HNhx was successfully isolated on porcine alveolar macrophages from Henan province and the full-length genome sequence was determined. Phylogenetic analysis indicated that HNhx strain was classified into the NADC30-like PRRSV subgroup, in which all the strains had the unique discontinuous 131-amino acid deletion relative to that of the nonstructural protein 2 (Nsp2) of the VR2332 strain. Genetically, HNhx shared 92.9% nucleotide similarity to NADC30. Furthermore, HNhx strain contained extensive amino acid mutations in GP5. In particular, the S32H, N33D, D34N, and S36G variations resulted in that HNhx lost all the putative N-linked glycosylation sites at amino acid positions 30, 32, 33, 34, and 35. Recombination analysis revealed that HNhx was the result of recombination between the NADC30 strain and the highly pathogenic PRRSV vaccine strain circulating in China in Nsp4 (nt 5261) to Nsp9 (nt 7911). The novel genome data of HNhx will be helpful for understanding the evolution and epidemiology of PRRSV in China.

Targeted Cell-to-Cell Delivery of Protein Payloads via the Granzyme-Perforin Pathway

There is great potential for engineering cellular therapeutics by repurposing biological systems. Here, we report utilization of the granzyme-perforin pathway of cytotoxic lymphocytes as a cell-to-cell protein delivery module. We designed and constructed granzyme B-derived chaperone molecules fused to a fluorescent protein payload and expressed these constructs in natural killer (NK) cells. Using confocal microscopy and flow cytometry, we investigated the co-localization of the chaperones with lytic granules and the chaperone-mediated transfer of the fluorescent protein payload from NK to target cells in co-culture experiments. A synthetic chaperone consisting of the granzyme B ER signal peptide and a domain encompassing putative N-linked glycosylation sites in granzyme B is insufficient for payload transfer to target cells, whereas full-length granzyme B is sufficient for payload delivery. Combining our functional data with an analysis of the crystal structure of granzyme B suggests that the necessary motifs for granzyme B loading into lytic granules are dispersed throughout the primary amino acid sequence and are only functional when contiguous in the tertiary structure. These results illustrate that by using granzyme B as a molecular chaperone the granzyme-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

Heterologous Protection against Asian Zika Virus Challenge in Rhesus Macaques.

BackgroundZika virus (ZIKV; Flaviviridae, Flavivirus) was declared a public health emergency of international concern by the World Health Organization (WHO) in February 2016, because of the evidence linking infection with ZIKV to neurological complications, such as Guillain-Barre Syndrome in adults and congenital birth defects including microcephaly in the developing fetus. Because development of a ZIKV vaccine is a top research priority and because the genetic and antigenic variability of many RNA viruses limits the effectiveness of vaccines, assessing whether immunity elicited against one ZIKV strain is sufficient to confer broad protection against all ZIKV strains is critical. Recently, in vitro studies demonstrated that ZIKV likely circulates as a single serotype. Here, we demonstrate that immunity elicited by African lineage ZIKV protects rhesus macaques against subsequent infection with Asian lineage ZIKV.Methodology/Principal FindingsUsing our recently developed rhesus macaque model of ZIKV infection, we report that the prototypical ZIKV strain MR766 productively infects macaques, and that immunity elicited by MR766 protects macaques against heterologous Asian ZIKV. Furthermore, using next generation deep sequencing, we found in vivo restoration of a putative N-linked glycosylation site upon replication in macaques that is absent in numerous MR766 strains that are widely being used by the research community. This reversion highlights the importance of carefully examining the sequence composition of all viral stocks as well as understanding how passage history may alter a virus from its original form.Conclusions/SignificanceAn effective ZIKV vaccine is needed to prevent infection-associated fetal abnormalities. Macaques whose immune responses were primed by infection with East African ZIKV were completely protected from detectable viremia when subsequently rechallenged with heterologous Asian ZIKV. Therefore, these data suggest that immunogen selection is unlikely to adversely affect the breadth of vaccine protection, i.e., any Asian ZIKV immunogen that protects against homologous challenge will likely confer protection against all other Asian ZIKV strains.

Effects of N-glycosylation of the human cation channel TRPA1 on agonist-sensitivity

Determining the functional significance of post-translational modifications advances our understanding of many broadly-expressed proteins, and particularly ion channels. The enzymes that catalyze these modifications are often expressed in a cell-type specific manner, resulting in considerable structural diversity among post-translationally modified proteins that are expressed across a variety of cell types. TRP channels exhibit notably variable behavior between cell types in vitro and in vivo , and they are frequently modified with N-glycans that contribute to protein function. TRPA1 possesses two putative N-linked glycosylation sites at N747 and N753 that have not yet been studied in detail. Here, we show that both of these sites can be modified with an N-glycan and that the glycan at position N747 modulates agonist-sensitivity of TRPA1 in vitro Additionally, we found that N-glycosylation also modulates cooperative effects of temperature and the agonist cinnamaldehyde on TRPA1 channel activation. Collectively, these findings suggest a dynamic role played by the N-glycosylation of human TRPA1. They also provide further evidence of the versatility of N-glycans and will assist in efforts to fully understand the complex regulation of TRPA1 activity.

Temporal dynamics of ‘HoBi’-like pestivirus quasispecies in persistently infected calves generated under experimental conditions

‘HoBi’-like virus is an atypical group within the Pestivirus genus that is implicated in economic losses for cattle producers due to both acute and persistent infections. Pestivirus strains exist as quasispecies (swarms of individual viruses) in infected animals and the viral populations making up the quasispecies differ widely in size and diversity in each animal. In the present study the viral quasispecies circulating in persistently infected (PI) calves, generated and maintained under experimental conditions using two different ‘HoBi’-like strains, was observed over time. An increase in genetic variability and the development of certain mutations was observed over time. Mutations observed included the loss of a putative N-linked glycosylation site in the E2 region and the change of specific residues in E1/E2. It is hypothesized that these changes may be the results on continued adaption of the pestivirus to individual hosts. This is the first study characterizing variation in the viral swarms of animals persistently infected with HoBi-like viruses over time. Studies of the shifts in PI viral swarms will contribute to our understanding of the host and viral mechanisms that function in the maintenance of pestivirus persistent infections.

Emerging of two new subgenotypes of porcine reproductive and respiratory syndrome viruses in Southeast China

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the leading swine pathogens and causes major economic loss to the global swine industry. In this study, a total of 49 PRRSV isolates were collected from different swine herds in seven provinces in Southeast China from 2014 to 2015. All the ORF5 genes and some Nsp2 genes were sequenced. Phylogenetic analysis showed that all the isolates belonged to the North America genotype. Among them, five isolates formed a new subgenotype IV derived from highly pathogenic PRRSV (HP-PRRSV). Six isolates formed subgenotype III, which were closely related to the NADC30 strain in the US. These isolates formed 13 putative N-linked glycosylation site (NGS) patterns based on N30, 33, 34, 35, 44 and 51. There were fewer NGSs of isolates in subgenotype IV than in subgenotype III. This indicates that the two new subgenotypes of PRRSV strains with different NGS patterns were spreading in those regions of China. The genetic diversity should be considered for the control and prevention of this disease.

In silico analysis of Mn transporters (NRAMP1) in various plant species.

Manganese (Mn) is an essential micronutrient in plant life cycle. It may be involved in photosynthesis, carbohydrate and lipid biosynthesis, and oxidative stress protection. Mn deficiency inhibits the plant growth and development, and causes the various plant symptoms such as interveinal chlorosis and tissue necrosis. Despite its importance in plant life cycle, we still have limited knowledge about Mn transporters in many plant species. Therefore, this study aimed to identify and characterize high affinity Arabidopsis Mn root transporter NRAMP1 orthologs in 17 different plant species. Various in silico methods and digital gene expression data were used in identification and characterization of NRAMP1 homologs; physico-chemical properties of sequences were calculated, putative transmembrane domains (TMDs) and conserved motif signatures were determined, phylogenetic tree was constructed, 3D models and interactome map were generated, and gene expression data was analyzed. 49 NRAMP1 homologs were identified from proteome datasets of 17 plant species using AtNRAMP1 as query. Identified sequences were characterized with a NRAMP domain structure, 10-12 putative TMDs with cytosolic N- and C-terminuses, and 10-14 exons encoding a protein of 500-588 amino acids and 53.8-64.3 kDa molecular weight with basic characteristics. Consensus transport residues, GQSSTITGTYAGQY(/F)V(/I)MQGFLD(/E/N) between TMD-8 and 9 were identified in all sequences but putative N-linked glycosylation sites were not highly conserved. In phylogeny, NRAMP1 sequences demonstrated divergence in lower and higher plants as well as in monocots and dicots. Despite divergence of lower plant Physcomitrella patens in phylogeny, it showed similarity in superposed 3D models. Phylogenetic distribution of AtNRAMP1 and 6 homologs inferred a functional relationship to NRAMP6 sequences in Mn transport, while distribution of OsNRAMP1 and 5 homologs implicated an involvement of NRAMP1 sequences in Mn transport or a cross-talk between in Fe-Mn homeostasis. Interactome analysis further confirmed this cross-talk between Mn and Fe pathways. Gene expression profile of AtNRAMP1 under Fe-, K-, P- and S-deficiencies, and cold, drought, heat and salt stresses revealed various proteins involving in transcription regulation, cofactor biosynthesis, diverse developmental roles, carbohydrate metabolism, oxidation-reduction reactions, cellular signaling and protein degradation pathways. Mn deficiency or toxicity could cause serious adverse effects in plants as well as in humans. To reduce these adversities mainly rely on understanding the molecular mechanisms underlying Mn uptake from the soil. However, we still have limited knowledge regarding the structural and functional roles of Mn transporters in many plant species. Therefore, identification and characterization of Mn root uptake transporter, NRAMP1 orthologs in various plant species will provide valuable theoretical knowledge to better understand Mn transporters as well as it may become an insight for future studies aiming to develop genetically engineered and biofortified plants.

Analysis of Dominant HIV Quasispecies Suggests Independent Viral Evolution Within Spinal Granulomas Coinfected with Mycobacterium tuberculosis and HIV-1 Subtype C.

Extrapulmonary tuberculosis (TB) is a significant public health challenge in South Africa and worldwide, largely fuelled by the HIV epidemic. In spinal TB, Mycobacteria infect the spinal column without dissemination to the spinal cord. The immune microenvironment, target cell characteristics, and other evolutionary forces within granulomas during HIV/TB coinfection are poorly characterized. We investigated whether spinal TB granulomas represent a sequestered anatomical site where independent HIV evolution occurs, and assessed the role of macrophages as a target cell for both HIV and mycobacteria. RNA was extracted from plasma and granulomatous tissue from six antiretroviral-naive HIV-1/spinal TB-coinfected patients, RT-PCR amplified, and the C2-V5 env segment was cloned and sequenced. Analysis of genetic diversity, phylogeny and coalescence patterns was performed on clonal sequences. To investigate their role in HIV sequestration, macrophages and the HIV-1 p24 protein were immune localized and ultrastructural features were studied. Intercompartment diversity measurements and phylogenetic reconstruction revealed anatomically distinct monophyletic HIV-1 clusters in four of six patients. Genotypic CCR5-tropic variants were predominant (98.9%) with conservation of putative N-linked glycosylation sites in both compartments. CD68(+) reactivity was associated with higher tissue viral load (r = 1.0; p < 0.01) but not greater intrapatient diversity (r = 0.60; p > 0.05). Ultrastructural imaging revealed the presence of bacterial and virus-like particles within membrane-bound intracellular compartments of macrophages. Spinal tuberculosis granulomas may form anatomically discreet sites of divergent viral evolution. Macrophages in these granulomas harbored both pathogens, suggesting that they may facilitate the process of viral sequestration within this compartment.

Biological Property of Recombinant Hemagglutinin-Neuraminidase Protein of Avian Paramyxovirus Type 6 Expressed by Recombinant Baculovirus

Hemagglutination inhibition (HI) test employing whole virus antigen is a prescribed serological test for serotyping, diagnosis and surveillance for avian paramyxoviruses (APMVs). For use as alternative to the virus antigen, hemagglutininneuraminidase (HN) protein gene of the wild duck isolate APMV-6/WB12-163FS of APMV serotype 6 (APMV-6) was amplified, cloned and expressed in Spodoptera frugiperda insect cells. The HN gene of 1,842 bps in length showed nucleotide and amino acid homology of 93.4% and 97.1%, respectively with that of APMV-6 prototype strain. Putative sialic acid binding motif and potential N-linked glycosylation sites were conserved. In Western blot analysis, the expressed protein had a molecular mass of 66 kDa and reacted specifically with antiserum to APMV-6. In addition, the recombinant HN protein showed biological properties such as hemagglutination (HA) and elution. The recombinant HN protein produced from infected cells showed high HA titers (approximately 2 13 HA unit/ml). The HA activity of the recombinant HN protein was inhibited by antisera to APMV-6. In cross HA inhibition test, the recombinant HN protein had the highest titers with antisera to homologous APMV serotype, although there was weak cross reaction with some of antisera to other APMV serotypes. Our results indicated that recombinant APMV-6 HN protein would have the potential as alternative to the APMV-6 antigen in HI assays.