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 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 .

scholarly journals Construction and Characterization of a Temperature-Sensitive Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutant

1998 ◽  
Vol 72 (3) ◽  
pp. 2047-2054 ◽  
Author(s):  
Mingjun Huang ◽  
Ralf Zensen ◽  
Michael Cho ◽  
Malcolm A. Martin
Keyword(s):  
Virus Particle ◽  
Particle Production ◽  
Wild Type Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Virus Particle Production

ABSTRACT A temperature-sensitive (ts) human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutant was generated by charged-cluster-to-alanine mutagenesis. The mutant virus, containing three charged residues within the RT finger domain changed to alanine (K64A, K66A, and D67A), replicated normally at 34.5 but not 39.5°C. Quantitating virus particle production by p24 antigen capture or virion-associated RT activity and virus infectivity by the MAGI cell assay, we found that (i) mutant virions produced at the permissive temperature were indistinguishable from wild-type virus in assays performed at the nonpermissive temperature, suggesting that thets mutation did not impair early steps in the virus replication cycle and that the mutant RT enzyme was not ts; and (ii) virus particle production in cells transfected with thets mutant at the nonpermissive temperature was comparable to that of wild-type virus. However, the particle-associated RT activity and infectivity of mutant virions produced at the nonpermissive temperature were greatly reduced when assays were conducted at the permissive temperature. These results are consistent with an irreversible ts event affecting RT that occurs during virus particle production. Radioimmunoprecipitation analyses revealed that both p66 and p51 RT subunits were absent from mutant virions generated at 39.5°C. The presence of normal levels of HIV-1 integrase in mutant particles produced at the nonpermissive temperature was inconsistent with defective Gag-Pol synthesis or Gag-Pol incorporation into progeny virions. Furthermore, wild-type levels of the mutant Pr160 gag-pol were detected in virions produced at the nonpermissive temperature when the HIV-1 protease was inactivated by site-specific mutagenesis. Taken together, these results are most consistent with a ts defect affecting the degradation or aberrant processing of the mutated RT during its processing/maturation within nascent particles.

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 Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production

2018 ◽  
Vol 132 (1) ◽  
pp. jcs217042 ◽  
Author(s):  
Susan Lassen ◽  
Cordula Grüttner ◽  
Van Nguyen-Dinh ◽  
Eva Herker
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Particle ◽  
Particle Production ◽  
Perilipin 2 ◽  
Virus Particle Production

scholarly journals Gag-Processing Defect of Human Immunodeficiency Virus Type 1 Integrase E246 and G247 Mutants Is Caused by Activation of an Overlapping 5′ Splice Site

2007 ◽  
Vol 82 (3) ◽  
pp. 1600-1604 ◽  
Author(s):  
Dibyakanti Mandal ◽  
Zehua Feng ◽  
C. Martin Stoltzfus
Keyword(s):  
Virus Particle ◽  
Splice Site ◽  
Rna Splicing ◽  
Particle Production ◽  
Gag Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Virus Particle Production ◽  
Integrase Protein

ABSTRACT We have previously described several human immunodeficiency virus type 1 (HIV-1) mutants that are characterized by an excessive-RNA-splicing phenotype and reduced virus particle production. In one of these mutants (NLD2up), the sequence of 5′ splice site D2 was changed to a consensus splice donor site. This splice site overlaps the HIV-1 integrase reading frame, and thus, the NLD2up mutant also bears a G-to-W change at amino acid 247 of the integrase. A previously described E-to-K mutant at position 246 of the C-terminal domain of the integrase, which resulted in a G-to-A mutation at the +3 position of overlapping splice donor D2 (NLD2A3), was also shown to affect virus particle production and Gag protein processing. By using second-site mutations to revert the excessive-splicing phenotype, we show that the effects on Gag protein processing and virus particle production of both the NLD2up and NLD2A3 mutants are caused by excessive viral RNA splicing due to the activation of the overlapping 5′ splice site and not to the changes in the integrase protein. Both integrase protein mutations, however, are lethal for virus infectivity. These studies suggest that changes in the usage of overlapping splice sites may be a possible alternative explanation for a defective virus phenotype resulting from changes in protein-coding sequences or in the nucleotide sequence during codon optimization.

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