scholarly journals Viral Entry Properties Required for Fitness in Humans Are Lost through Rapid Genomic Change during Viral Isolation

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Sho Iketani ◽  
Ryan C. Shean ◽  
Marion Ferren ◽  
Negar Makhsous ◽  
Dolly B. Aquino ◽  
...  
Keyword(s):  
Primary Culture ◽  
Parainfluenza Virus ◽  
Clinical Samples ◽  
Genomic Change ◽  
Viral Isolation ◽  
Dimer Interface ◽  
Genome Wide ◽  
A Genome ◽  
Immortalized Cells ◽  
Human Parainfluenza Virus

ABSTRACTHuman parainfluenza viruses cause a large burden of human respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, human parainfluenza virus 3 (HPIV-3) evolves in culture. Cultured viruses differ in their properties compared to clinical strains. We present a genome-wide survey of HPIV-3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates (zero passage virus). Nonsynonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins—the receptor binding protein hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV-3 primary culture isolates and 23 HPIV-3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN’s dimerization/F-interaction site, a site critical for activation of viral fusion. Alterations in HN dimer interface residues known to favor infection in culture occurred within 4 days (H552 and N556). A novel cluster of residues at a different face of the HN dimer interface emerged (P241 and R242) and imply a role in HPIV-3-mediated fusion. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV-3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity. These results utilize a method for identifying genome-wide changes associated with brief adaptation to culture to highlight the notion that even brief exposure to immortalized cells may affect key viral properties and underscore the balance of features of the HN-F complex required for fitness by circulating viruses.IMPORTANCEHuman parainfluenza virus 3 is an important cause of morbidity and mortality among infants, the immunocompromised, and the elderly. Using deep genomic sequencing of HPIV-3-positive clinical material and its subsequent viral isolate, we discover a number of known and novel coding mutations in the main HPIV-3 attachment protein HN during brief exposure to immortalized cells. These mutations significantly alter function of the fusion complex, increasing fusion promotion by HN as well as generally decreasing neuraminidase activity and increasing HN-receptor engagement. These results show that viruses may evolve rapidly in culture even during primary isolation of the virus and before the first passage and reveal features of fitness for humans that are obscured by rapid adaptation to laboratory conditions.

scholarly journals SATB2 Overexpression Promotes the Proliferation, Migration and Invasion of Oral Squamous Cell Carcinoma by Up-Regulating NOX4

2020 ◽  
Author(s):  
Jie Gao ◽  
Ying Meng ◽  
Xin Ge ◽  
Chen-xing Hou ◽  
Qing-hai Zhu ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Ectopic Expression ◽  
Migration And Invasion ◽  
Clinical Samples ◽  
Genome Wide ◽  
A Genome ◽  
Sirna Knockdown

Abstract Background: While atypical expression of special AT-rich sequence-binding protein 2 (SATB2) has been approved associated with tumor progression, metastasis and unfavourable prognosis in various carcinomas. However, in oral squamous cell carcinoma (OSCC), both the expressive state and associated functions of SATB2’s are still undefined.Methods: Real-time PCR, western blotting, and immunohistochemistry were used to examine SATB2 expression. In vitro experiments including Flow Cytometry, CCK8 assay, migration assay, wound-healing assay were used to investigate the effects of SATB2 on HN4 cell proliferation, migration and invasion ability. Additionally, an orthotopic implantation assay was performed in nude mice to confirm the effects of SATB2 in vivo. Furthermore, a genome wide siRNA knockdown experiment was performed to explore the potential downstream regulatory mechanism of SATB2 in OSCC.Results: We found that , in clinical samples from a retrospective cohort of 58 OSCC patients, high expression of SATB2 is associated with poor prognosis of OSCC patients. In this study, we investigated SATB2 is highly expressed in OSCC tissues and cell lines ,which can promotes OSCC cells’ proliferation, migration, invasion and tumor growth. Following a genome wide siRNA knockdown experiment, we identified NOX4, a bona fide downstream target of SATB2, which can partially suppress OSCC proliferation. Furthermore, NOX4 knockdown inhibits tumorigenicity, which can be rescued partially by ectopic expression of SATB2.Conclusion: Our findings not only indicate overexpression of SATB2 triggers the proliferative, migratory and invasive mechanisms which are important in the malignant phenotype of OSCC, but also identify NOX4 as the downstream gene for SATB2. These findings indicate that SATB2 may play a key role in OSCC tumorigenicity and may be a future target for the development of new therapeutic regimens.

scholarly journals SARS-CoV-2 genetic variations associated with COVID-19 severity

2020 ◽  
Author(s):  
Pakorn Aiewsakun ◽  
Patompon Wongtrakoongate ◽  
Yuttapong Thawornwattana ◽  
Suradej Hongeng ◽  
Arunee Thitithanyanont
Keyword(s):  
Genome Wide Association Study ◽  
Genetic Variations ◽  
Genome Wide Association ◽  
Clinical Samples ◽  
Structural Protein ◽  
Coding Region ◽  
Patient Status ◽  
Genome Wide ◽  
A Genome ◽  
Asymptomatic Infections

AbstractHerein, we performed a genome-wide association study on SARS-CoV-2 genomes to identify genetic variations that might be associated with the COVID-19 severity. 152 full-length genomes of SARS-CoV-2 that were generated from original clinical samples and whose patient status could be determined conclusively as either “asymptomatic” or “symptomatic” were retrieved from the GISAID database. We found that nucleotide variations at the genomic position 11,083, locating in the coding region of non-structural protein 6, were associated with the COVID-19 severity. While the 11083G variant (i.e. having G at the position 11,083) was more commonly found in symptomatic patients, the 11083T variant appeared to associate more often with asymptomatic infections. We also identified three microRNAs that differentially target the two variants, namely miR-485-3p, miR-539-3p, and miR-3149. This may in part contribute to the differential association of the two SARS-CoV-2 variants with the disease severity.

scholarly journals Adaptation of Human Parainfluenza Virus to Airway Epithelium Reveals Fusion Properties Required for Growth in Host Tissue

mBio ◽  
2012 ◽  
Vol 3 (3) ◽  
Author(s):  
Samantha G. Palmer ◽  
Matteo Porotto ◽  
Laura M. Palermo ◽  
Luis F. Cunha ◽  
Olga Greengard ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Membrane ◽  
Fusion Protein ◽  
Airway Epithelium ◽  
Parainfluenza Virus ◽  
Enveloped Viruses ◽  
Human Airway Epithelium ◽  
Sialic Acid Binding ◽  
Immortalized Cells ◽  
Human Parainfluenza Virus

ABSTRACTParamyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN’s secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN’s secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism.IMPORTANCEHuman parainfluenza viruses (HPIVs) are paramyxoviruses that cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide, but there are currently no vaccines or antivirals available for treatment. Enveloped viruses must fuse their membrane with the target cell membrane in order to initiate infection. Parainfluenza virus fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. The receptor-binding hemagglutinin-neuraminidase (HN), upon receptor engagement, activates the fusion protein (F) to penetrate the target cell and mediate viral entry. In this study, we show that the precise balance of fusion activation properties of these two glycoproteins during entry is key for infection. In clinically relevant tissues, viruses evolve to acquire a set of fusion features that provide key clues about requirements for infection in human beings.

scholarly journals The Genome Length of Human Parainfluenza Virus Type 2 Follows the Rule of Six, and Recombinant Viruses Recovered from Non-Polyhexameric-Length Antigenomic cDNAs Contain a Biased Distribution of Correcting Mutations

2003 ◽  
Vol 77 (1) ◽  
pp. 270-279 ◽  
Author(s):  
Mario H. Skiadopoulos ◽  
Leatrice Vogel ◽  
Jeffrey M. Riggs ◽  
Sonja R. Surman ◽  
Peter L. Collins ◽  
...  
Keyword(s):  
Virus Type ◽  
Genomic Sequence ◽  
Full Length ◽  
Parainfluenza Virus ◽  
Genome Length ◽  
A Genome ◽  
Nucleotide Insertions ◽  
Human Parainfluenza Virus ◽  
Rule Of Six

ABSTRACT Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the “rule of six.” To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.

scholarly journals Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Rui Xu ◽  
Samantha G. Palmer ◽  
Matteo Porotto ◽  
Laura M. Palermo ◽  
Stefan Niewiesk ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Virus Type ◽  
Receptor Binding ◽  
Target Cell ◽  
Structural Features ◽  
Parainfluenza Virus ◽  
Structural Explanation ◽  
Dimer Interface ◽  
Human Parainfluenza Virus

ABSTRACTParamyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infectionin vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growthin vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infectionin vivo.IMPORTANCEHuman parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine.In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN’s altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virusin vivo.

scholarly journals Features of Circulating Parainfluenza Virus Required for Growth in Human Airway

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Laura M. Palermo ◽  
Manik Uppal ◽  
Lucy Skrabanek ◽  
Paul Zumbo ◽  
Soren Germer ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Receptor Binding ◽  
Parainfluenza Virus ◽  
Receptor Interaction ◽  
Whole Genome ◽  
Clinical Strains ◽  
Immortalized Cells ◽  
Human Parainfluenza Virus

ABSTRACT Respiratory paramyxoviruses, including the highly prevalent human parainfluenza viruses, cause the majority of childhood croup, bronchiolitis, and pneumonia, yet there are currently no vaccines or effective treatments. Paramyxovirus research has relied on the study of laboratory-adapted strains of virus in immortalized cultured cell lines. We show that findings made in such systems about the receptor interaction and viral fusion requirements for entry and fitness—mediated by the receptor binding protein and the fusion protein—can be drastically different from the requirements for infection in vivo . Here we carried out whole-genome sequencing and genomic analysis of circulating human parainfluenza virus field strains to define functional and structural properties of proteins of circulating strains and to identify the genetic basis for properties that confer fitness in the field. The analysis of clinical strains suggests that the receptor binding-fusion molecule pairs of circulating viruses maintain a balance of properties that result in an inverse correlation between fusion in cultured cells and growth in vivo . Future analysis of entry mechanisms and inhibitory strategies for paramyxoviruses will benefit from considering the properties of viruses that are fit to infect humans, since a focus on viruses that have adapted to laboratory work provides a distinctly different picture of the requirements for the entry step of infection. IMPORTANCE Mechanistic information about viral infection—information that impacts antiviral and vaccine development—is generally derived from viral strains grown under laboratory conditions in immortalized cells. This study uses whole-genome sequencing of clinical strains of human parainfluenza virus 3—a globally important respiratory paramyxovirus—in cell systems that mimic the natural human host and in animal models. By examining the differences between clinical isolates and laboratory-adapted strains, the sequence differences are correlated to mechanistic differences in viral entry. For this ubiquitous and pathogenic respiratory virus to infect the human lung, modulation of the processes of receptor engagement and fusion activation occur in a manner quite different from that carried out by the entry glycoprotein-expressing pair of laboratory strains. These marked contrasts in the viral properties necessary for infection in cultured immortalized cells and in natural host tissues and animals will influence future basic and clinical studies.

Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
F Ponthan ◽  
D Pal ◽  
J Vormoor ◽  
O Heidenreich
Keyword(s):  
Rnai Screen ◽  
Novel Genes ◽  
Genome Wide ◽  
A Genome

A genome-wide linkage scan for familial partial lipodystrophy susceptibility genes in a German kindreds

2007 ◽  
Vol 30 (4) ◽  
pp. 86
Author(s):  
M. Lanktree ◽  
J. Robinson ◽  
J. Creider ◽  
H. Cao ◽  
D. Carter ◽  
...  
Keyword(s):  
Subcutaneous Fat ◽  
Visceral Obesity ◽  
Fat Distribution ◽  
Dominant Negative ◽  
Molecular Forms ◽  
Partial Lipodystrophy ◽  
Genome Wide ◽  
A Genome ◽  
Familial Partial Lipodystrophy ◽  
Promoter Mutations

Background: In Dunnigan-type familial partial lipodystrophy (FPLD) patients are born with normal fat distribution, but subcutaneous fat from extremities and gluteal regions are lost during puberty. The abnormal fat distribution leads to the development of metabolic syndrome (MetS), a cluster of phenotypes including hyperglycemia, dyslipidemia, hypertension, and visceral obesity. The study of FPLD as a monogenic model of MetS may uncover genetic risk factors of the common MetS which affects ~30% of adult North Americans. Two molecular forms of FPLD have been identified including FPLD2, resulting from heterozygous mutations in the LMNA gene, and FPLD3, resulting from both heterozygous dominant negative and haploinsufficiency mutations in the PPARG gene. However, many patients with clinically diagnosed FPLD have no mutation in either LMNA or PPARG, suggesting the involvement of additional genes in FPLD etiology. Methods: Here, we report the results of an Affymetrix 10K GeneChip microarray genome-wide linkage analysis study of a German kindred displaying the FPLD phenotype and no known lipodystrophy-causing mutations. Results: The investigation identified three chromosomal loci, namely 1q, 3p, and 9q, with non-parametric logarithm of odds (NPL) scores >2.7. While not meeting the criteria for genome-wide significance, it is interesting to note that the 1q and 3p peaks contain the LMNA and PPARG genes respectively. Conclusions: Three possible conclusions can be drawn from these results: 1) the peaks identified are spurious findings, 2) additional genes physically close to LMNA, PPARG, or within 9q, are involved in FPLD etiology, or 3) alternative disease causing mechanisms not identified by standard exon sequencing approaches, such as promoter mutations, alternative splicing, or epigenetics, are also responsible for FPLD.

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