Inhibition of Endosomal Transport Results in Decreased Synthesis of Nucleocapsid Protein of PHV Virus
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Inhibition of Endosomal Transport Results in Decreased Synthesis of Nucleocapsid Protein of PHV Virus

M. Muyangwa*, E.E. Garanina**

Kazan Federal University, Kazan, 420008 Russia

E-mail: *musalwam@yahoo.co.uk, **kathryn.cherenkova@gmail.com

Received February 1, 2017

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Abstract

This paper is devoted to investigation of molecular mechanisms of transport of nucleocapsid protein of non-pathogenic virus Prospect Hill. Prospect Hill virus (PHV) belongs to the family Bunyaviridae, genus Hantavirus. Hantaviruses utilize the intracellular trafficking pathways for delivery of viral RNA and proteins to the site of viral assembly. While significant progress has been achieved in understanding PHV replication, the role of intracellular transport in virus replication remains largely unknown. Co-localization of PHV nucleocapsid protein and Rab5, Rab7, and Rab11 (early, late, and recycling endosomes, respectively) has been demonstrated using immunofluorescence assay. Western blot analysis has revealed decreased accumulation of nucleocapsid protein when dominant negative (DN) mutant protein forms Rab5 and Rab11were expressed in PHV infected cells. Our data suggest that the functional activity of early, late, and recycling endosomes is essential for replication of HPV proteins. The obtained results are of great importance for comprehension of molecular and cellular mechanisms of pathogenesis of hantaviral infection.

Keywords: recombinant lentivirus, nucleocapsid protein, hantavirus, Prospect Hill Virus, PHV, Rab proteins, endosomes

Acknowledgments. This study was funded by the subsidy allocated to Kazan Federal University as part of the state program for increasing its competitiveness among the world's leading centers of science and education. The work was also supported by the Russian Science Foundation (project no. 15-14-00016).

Figure Captions

Fig. 1. The analysis of the efficiency of transfection of the culture of A549 cells with DNA plasmid encoding for Rab wild-type proteins, 48 h after transfection: A – untransfected control, B – A549 cells culture transfected with DsRed-Rab5-WT plasmid, C – A549 cells culture transfected with DsRed-Rab7-WT plasmid, D – А549 cells culture transfected with DsRed-Rab11-WT plasmid. Scale: 20 ?m.

Fig. 2. Intercellular co-localization of nucleocapsid proteins of PHV hantavirus and proteins of various endosomes. Confocal fluorescence microscopy. Blue fluorescence – DAPI. Green fluorescence – staining to nucleocapsid protein of PHV virus. Red fluorescence – DsRed-Rab5, DsRed-Rab7, or DsRed-Rab11, allows to detect localization of early, late, and recycling endosomes. A – co-localization of nucleocapsid protein of LV-PHV-S recombinant lentivirus with Rab5 protein of early endosomes. B – co-localization of nucleocapsid protein of LV-PHV-S recombinant lentivirus with Rab7 protein of late endosomes. C – co-localization of nucleocapsid protein of LV-PHV-S recombinant lentivirus with Rab11 protein of recycling endosomes. Scale: 20 ?m.

Fig. 3. The analysis of intracellular content of nucleocapsid protein in A549 cell lysates transfected with the plasmid constructions of dominant negative mutant or wild-type Rab5 and Rab11 and transduced with LV-PHV-S recombinant lentivirus. Western blot analysis. A – analysis of the synthesis of nucleocapsid proteins in A549 cell lysates transfected with the plasmid constructions of dominant negative mutant and wild-type Rab5 and Rab11 and transduced after 48 h with LV-PHV-S recombinant lentivirus. NTC – control, non-transduced cells. 1, 3, 5 – cells transfected with DsRed-Rab5-DN. 2, 4, 6 – DsRed-Rab5-WT. 8, 10, 12 – DsRed-Rab11-DN. 9, 11, 13 – DsRed-Rab11-WT. 7, 14 – non-transfected cells. Cells were collected 48 h (1, 2, 7, 8, 9), 72 h (3, 4, 10, 11), and 96 h (5, 6, 12, 13, 14) after transduction. Main band – nucleocapsid protein of hantavirus with the molecular weight of 50 kDa. B – quantitative analysis of intracellular content of nucleocapsid protein of hantavirus of A549 cells transduced with LV-PHV-S recombinant lentivirus 48 h after transfection with the plasmid constructions of dominant negative or wild-type Rab5 and Rab11. Data are presented as mean value ? standard error, p < 0.05. The asterisk marks statistically significant data.

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For citation: Muyangwa M., Garanina E.E. Inhibition of endosomal transport results in decreased synthesis of nucleocapsid protein of PHV virus. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 1, pp. 58–71. (In Russian)


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