scholarly journals Identification of One Critical Amino Acid Residue of the Nucleoprotein as a Determinant for in vitro Viral Replication Fitness of Influenza D Virus

2021 ◽  
Author(s):  
Jieshi Yu ◽  
Chen Huang ◽  
Zizhang Sheng ◽  
Zhao Wang ◽  
Feng Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virus Particle ◽  
Reverse Genetics ◽  
Particle Production ◽  
Infectious Virus ◽  
High Titer ◽  
Production Mechanism ◽  
Replication Fitness ◽  
Virus Particle Production

The newly identified influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range with broad geographical distribution. Despite the first appearance in U.S. pig herds in 2011, subsequent studies demonstrate that IDV is widespread in global cattle populations, supporting a theory that IDV utilizes bovines as a primary reservoir. Our investigation of the two reference influenza D viruses, D/swine/Oklahoma/1334/2011 (OK/11) isolated from swine and D/Bovine/Oklahoma/660/2013 (660/13) from cattle, revealed that 660/13 replicated to titers approximately 100-fold higher than those for OK/11 in multiple cell lines. By using a recently developed IDV reverse genetics system derived from low-titer OK/11, we generated recombinant chimeric OK/11 viruses in that one of the seven genome segments was replaced with its counterpart from high-titer 660/13 virus. Further characterization demonstrated that the replication level of the chimeric OK/11 virus was significantly increased only when harboring the 660/13 nucleoprotein (NP) segment. Finally, through both gain-of-function and loss-of-function experiments, we identified that one amino acid residue at position 381, located in the body domain of NP protein, was a key determinant for the replication difference between the low-titer OK/11 virus and the high-titer 660/13 virus. Taken together, our findings provide important insight into IDV replication fitness mediated by the NP protein, which should facilitate future study of infectious virus particle production mechanism of IDV. IMPORTANCE Little is known about virus infection and production mechanism for newly discovered influenza D virus (IDV) that utilizes bovines as a primary reservoir with frequent spillover to new hosts including swine. In this study, we showed that two well-characterized IDVs, 660/13 replicated more efficiently (approximately 100-fold higher) than OK/11. Using a recently developed IDV reverse genetics system, we identified viral nucleoprotein (NP) as a primary determinant of the different replication capacity observed between these two nearly identical viruses. Mechanistic investigation further revealed that a mutation at NP position 381 evidently modulated virus fitness. Taken together, these observations indicate that IDV NP protein performs a critical role in infectious virus particle production. Our study thus illustrates a NP-based mechanism for efficient IDV infection and production in vitro .

Increased capsid oligomerization is deleterious to dengue virus particle production

2021 ◽  
Vol 102 (8) ◽  
Author(s):  
Sutha Sangiambut ◽  
Natcha Promphet ◽  
Suwipa Chaiyaloom ◽  
Chunya Puttikhunt ◽  
Panisadee Avirutnan ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Virus Particle ◽  
Particle Production ◽  
Infectious Virus ◽  
Hydrophobic Interactions ◽  
Viral Infectivity ◽  
Wild Type ◽  
Mosquito Cells ◽  
Wild Type Counterpart ◽  
Virus Particle Production

The capsid protein (C) of dengue virus is required for viral infectivity as it packages viral RNA genome into infectious particles. C exists as a homodimer that forms via hydrophobic interactions between the α2 and α4 helices of monomers. To identify C region(s) important for virus particle production, a complementation system was employed in which single-round infectious particles are generated by trans-encapsidation of a viral C-deleted genome by recombinant C expressed in mosquito cells. Mutants harbouring a complete α3 deletion, or a dual Ile65-/Trp69-to-Ala substitution in the α3 helix, exhibited reduced production of infectious virus. Unexpectedly, higher proportions of oligomeric C were detected in cells expressing both mutated forms as compared with the wild-type counterpart, indicating that the α3 helix, through its internal hydrophobic residues, may down-modulate oligomerization of C during particle formation. Compared with wild-type C, the double Ile65-/Trp69 to Ala mutations appeared to hamper viral infectivity but not C and genomic RNA incorporation into the pseudo-infectious virus particles, suggesting that increased C oligomerization may impair DENV replication at the cell entry step.

scholarly journals Identification of HNRNPK as Regulator of Hepatitis C Virus Particle Production

2015 ◽  
Vol 11 (1) ◽  
pp. e1004573 ◽  
Author(s):  
Marion Poenisch ◽  
Philippe Metz ◽  
Hagen Blankenburg ◽  
Alessia Ruggieri ◽  
Ji-Young Lee ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Particle ◽  
Particle Production ◽  
Virus Particle Production

scholarly journals Characterization of Sendai virus M protein mutants that can partially interfere with virus particle production

1999 ◽  
Vol 80 (11) ◽  
pp. 2977-2986 ◽  
Author(s):  
Geneviève Mottet ◽  
Virginie Müller ◽  
Laurent Roux
Keyword(s):  
Virus Particle ◽  
Particle Production ◽  
Sendai Virus ◽  
Single Band ◽  
M Protein ◽  
Phenotypic Traits ◽  
Cell Periphery ◽  
Page Migration ◽  
Protein Mutants ◽  
Virus Particle Production

Substitution of Val113 in Sendai virus (SeV) M protein generates non-functional polypeptides, characterized by their exclusion from virus particles and by their ability to interfere with virus particle production. These phenotypic traits correlate with a single-band PAGE migration profile, in contrast to wild-type M (Mwt ), which separates into two species, one of which is a phosphorylated form. The single-band migration is likely to result from a conformational change, as evidenced by the lack of maturation of a native epitope and by a particular tryptic digestion profile, and not from the phosphorylation of all M molecules, an assumption consistent with the PAGE migration feature. One of the M mutants (HA–M30 , an M protein carrying Thr112Met and Val113 Glu substitutions tagged with an influenza virus haemagglutinin epitope) was characterized further in the context of SeV infection, i.e. under conditions of co-expression with Mwt. HA–M 30 is shown (i) to bind mainly to membrane fractions, (ii) not to co-precipitate Mwt, as HA–Mwt does, (iii) to interfere with the binding of nucleocapsids to membranes and (iv) to accumulate in perinuclear regions, in contrast to HA-Mwt , which is also found at the cell periphery. Such mutants constitute potential tools for the identification of critical steps in paramyxovirus assembly and budding.

scholarly journals A Reverse Genetics Approach for Recovery of Recombinant Influenza B Viruses Entirely from cDNA

2002 ◽  
Vol 76 (22) ◽  
pp. 11744-11747 ◽  
Author(s):  
David Jackson ◽  
Andrew Cadman ◽  
Thomas Zurcher ◽  
Wendy S. Barclay
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Gene ◽  
Neuraminidase Inhibitor ◽  
Influenza B Virus ◽  
Influenza B ◽  
B Virus

ABSTRACT The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.

scholarly journals A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Phylicia Dupree ◽  
Inna Lobeck ◽  
Sarah Mowery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biliary Atresia ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Wild Type ◽  
Neonatal Mice ◽  
Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

scholarly journals Determinants of Virulence of Classical Swine Fever Virus Strain Brescia

2004 ◽  
Vol 78 (16) ◽  
pp. 8812-8823 ◽  
Author(s):  
H. G. P. van Gennip ◽  
A. C. Vlot ◽  
M. M. Hulst ◽  
A. J. de Smit ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acid ◽  
Classical Swine Fever Virus ◽  
Reverse Genetics ◽  
Animal Experiments ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Target Cells ◽  
Adaptive Mutations

ABSTRACT Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB 476R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB 476R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB 476S variant into a His residue did not affect virulence. However, the 710His in the C1.1.1/CoBrB virus, together with adaptive mutations 276R, 476R, and 477I in Erns, resulted in reduced virulence in pigs. These results indicated that mutations in Erns and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 Erns may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the 710L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

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