scholarly journals Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ricardo S. Cardoso ◽  
Lucas Alves Tavares ◽  
Bruna Lais S. Jesus ◽  
Miria F. Criado ◽  
Andreia Nogueira de Carvalho ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Secretory Pathway ◽  
Human Respiratory Syncytial Virus ◽  
Structural Proteins ◽  
Sorting Nexin ◽  
Syncytial Virus ◽  
N Proteins

ABSTRACT Human respiratory syncytial virus (HRSV) envelope glycoproteins traffic to assembly sites through the secretory pathway, while nonglycosylated proteins M and N are present in HRSV inclusion bodies but must reach the plasma membrane, where HRSV assembly happens. Little is known about how nonglycosylated HRSV proteins reach assembly sites. Here, we show that HRSV M and N proteins partially colocalize with the Golgi marker giantin, and the glycosylated F and nonglycosylated N proteins are closely located in the trans-Golgi, suggesting their interaction in that compartment. Brefeldin A compromised the trafficking of HRSV F and N proteins and inclusion body sizes, indicating that the Golgi is important for both glycosylated and nonglycosylated HRSV protein traffic. HRSV N and M proteins colocalized and interacted with sorting nexin 2 (SNX2), a retromer component that shapes endosomes in tubular structures. Glycosylated F and nonglycosylated N HRSV proteins are detected in SNX2-laden aggregates with intracellular filaments projecting from their outer surfaces, and VPS26, another retromer component, was also found in inclusion bodies and filament-shaped structures. Similar to SNX2, TGN46 also colocalized with HRSV M and N proteins in filamentous structures at the plasma membrane. Cell fractionation showed enrichment of SNX2 in fractions containing HRSV M and N proteins. Silencing of SNX1 and 2 was associated with reduction in viral proteins, HRSV inclusion body size, syncytium formation, and progeny production. The results indicate that HRSV structural proteins M and N are in the secretory pathway, and SNX2 plays an important role in the traffic of HRSV structural proteins toward assembly sites. IMPORTANCE The present study contributes new knowledge to understand HRSV assembly by providing evidence that nonglycosylated structural proteins M and N interact with elements of the secretory pathway, shedding light on their intracellular traffic. To the best of our knowledge, the present contribution is important given the scarcity of studies about the traffic of HRSV nonglycosylated proteins, especially by pointing to the involvement of SNX2, a retromer component, in the HRSV assembly process.

scholarly journals The replication of the human respiratory syncytial virus in a T cell line has multiple ineffective steps

2020 ◽  
Author(s):  
Ricardo de Souza Cardoso ◽  
Ana Carolina Lunardello Coelho ◽  
Bruna Laís Santos de Jesus ◽  
Brenda Cristina Vitti ◽  
Juliano de Paula Souza ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
T Cell ◽  
Cell Line ◽  
Inclusion Bodies ◽  
Secretory Pathway ◽  
Human Respiratory Syncytial Virus ◽  
Lymphoid Cells ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a CD4+ T cell line. We found by flow cytometry and fluorescent focus assay that A3.01 cells are susceptible but virtually not permissive to HRSV infection. De-quenching experiments revealed that the fusion process of HRSV in A3.01 cells is reduced in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by qPCR determined that the replication of HRSV in A3.01 cells was modest. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, we found by fluorescence in situ hybridization that the inclusion body-associated granules (IBAG’s) are almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins co-localized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments are present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, with virion production hampered by low fusion, hypofunctional inclusion bodies, altered trafficking of viral proteins to the plasma membrane.

Immunovirological studies on human respiratory syncytial virus structural proteins

1986 ◽  
Vol 32 (1) ◽  
pp. 15-21 ◽  
Author(s):  
Michel Trudel ◽  
Francine Nadon ◽  
Cécile Séguin ◽  
Simone Ghoubril ◽  
Pierre Payment ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Neutralizing Antibodies ◽  
Sodium Dodecyl ◽  
Human Respiratory Syncytial Virus ◽  
Structural Proteins ◽  
Membrane Glycoproteins ◽  
Protein Dimer ◽  
Syncytial Virus ◽  
Nucleoprotein N

Immunovirological studies suggest that human respiratory syncytial virus may well be composed of five structural proteins as are other members of the Paramyxoviridae family: the two external membrane glycoproteins H (90 000) and Fo (F1, 49 000; F2, 20 000; disulfide linked), the internal membrane protein M (34 000), the nucleoprotein N (42 000), and a protein (78 000) designated P that could be the equivalent of the polymerase of the morbillivirus and paramyxovirus genus. Neutralizing monoclonal antibodies showed, by immunoprecipitation and immunoblotting, that the fusion protein carries neutralizing epitopes. One monoclonal antibody, which shows a high neutralizing titer, immunoblotted directly with the F1 fragment (49 000) of the fusion protein. Analysis in mice of the immunogenicity of the structural proteins separated on sodium dodecyl sulphate gels indicated that, under our conditions, only the fusion protein dimer Fo and its F1 fragment were capable of inducing neutralizing antibodies.

scholarly journals Requirements for Human Respiratory Syncytial Virus Glycoproteins in Assembly and Egress from Infected Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Melissa Batonick ◽  
Gail W. Wertz
Keyword(s):  
Plasma Membrane ◽  
Respiratory Syncytial Virus ◽  
G Proteins ◽  
Matrix Protein ◽  
Protein Localization ◽  
Viral Proteins ◽  
Human Respiratory Syncytial Virus ◽  
The Other ◽  
Infected Cells ◽  
Syncytial Virus

Human respiratory syncytial virus (HRSV) is an enveloped RNA virus that assembles and buds from the plasma membrane of infected cells. The ribonucleoprotein complex (RNP) must associate with the viral matrix protein and glycoproteins to form newly infectious particles prior to budding. The viral proteins involved in HRSV assembly and egress are mostly unexplored. We investigated whether the glycoproteins of HRSV were involved in the late stages of viral replication by utilizing recombinant viruses where each individual glycoprotein gene was deleted and replaced with a reporter gene to maintain wild-type levels of gene expression. These engineered viruses allowed us to study the roles of the glycoproteins in assembly and budding in the context of infectious virus. Microscopy data showed that the F glycoprotein was involved in the localization of the glycoproteins with the other viral proteins at the plasma membrane. Biochemical analyses showed that deletion of the F and G proteins affected incorporation of the other viral proteins into budded virions. However, efficient viral release was unaffected by the deletion of any of the glycoproteins individually or in concert. These studies attribute a novel role to the F and G proteins in viral protein localization and assembly.

scholarly journals Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 231
Author(s):  
Ricardo de Souza Cardoso ◽  
Rosa Maria Mendes Viana ◽  
Brenda Cristina Vitti ◽  
Ana Carolina Lunardello Coelho ◽  
Bruna Laís Santos de Jesus ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Respiratory Syncytial Virus ◽  
T Cell ◽  
Virus Infection ◽  
Cell Line ◽  
Inclusion Bodies ◽  
Human Respiratory Syncytial Virus ◽  
Lymphoid Cells ◽  
Human T Cell ◽  
Syncytial Virus

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.

scholarly journals N6 -Methyladenosine Negatively Regulates Human Respiratory Syncytial Virus Replication

2021 ◽  
Vol 9 ◽  
Author(s):  
Fabian Figueroa ◽  
Alonso Vega-Gibson ◽  
Joseline Catrileo ◽  
Aracelly Gaete-Argel ◽  
Sebastian Riquelme-Barrios ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Viral Replication ◽  
Inclusion Bodies ◽  
Opposite Effect ◽  
Human Respiratory Syncytial Virus ◽  
Genomic Rna ◽  
Syncytial Virus ◽  
Rna Accumulation ◽  
The Impact ◽  
Viral Genomic Rna

N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and several viral RNA including human respiratory syncytial virus (HRSV). Here, we evaluated the impact of m6A writers, erasers and readers on HRSV genomic RNA accumulation and inclusion bodies assembly during viral replication. We observed that the METTL3/METTL14 m6A writer complex plays a negative role in HRSV protein synthesis and viral titers, while m6A erasers FTO and ALKBH5 had the opposite effect. We also observed that m6A readers YTHDF1-3 bind to the viral genomic RNA inducing a decrease in its intracellular levels and thus, inhibiting viral replication. Finally, we observed that overexpression of YTHDFs proteins caused a decrease in the size of inclusion bodies (IBs), accompanied by an increase in their number. METTL3 knockdown cells showed an opposite effect indicating that the dynamics of IBs assembly and coalescence are strongly affected by m6A readers in a mechanism dependent on m6A writers. Taken together, our results demonstrated that the m6A modification negatively affects HRSV replication, possibly through a mechanism involving the assembly of inclusion bodies, the main factories of viral genomic RNA synthesis.

Sign in / Sign up

Export Citation Format

Share Document