The guanine nucleotide exchange factor GBF1 participates in rotavirus replication

Abstract

19Cellular and viral factors participate in the replication cycle of rotavirus. We report that the 20 guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce 21 COPI transport processes, is required for rotavirus replication since knocking down GBF1 22 3 IMPORTANCE 40 Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in 41 children and young animals worldwide. Despite the significant advances in the 42 characterization of the biology of this virus, the mechanisms involved in morphogenesis of 43 the virus particle are still poorly understood. In this work, we show that the guanine 44 nucleotide exchange factor GBF1, relevant for the COPI/Arf1-mediated cellular vesicular 45 transport, participates in the replication cycle of the virus, influencing the correct processing 46 of viral glycoproteins VP7 and NSP4, and the assembly of the virus surface proteins VP7 47 59 Rotaviruses, members of the family Reoviridae, are non-enveloped particles formed by three 60 concentric layers of proteins that surround the eleven genome segments of double-stranded 61 RNA (dsRNA). The innermost layer is composed of the core-shell proteinVP2 that encloses 62 the replication intermediates, composed of the RNA dependent RNA polymerase VP1, and 63 the guanylyl-methyl transferase, VP3. The intermediate layer is formed by VP6 that 64 surrounds the VP2 layer to form double-layered particles (DLPs). Finally, the addition of the 65 glycoprotein VP7 and the spike protein VP4 onto the DLPs forms the infectious triple-layered 66 particles (TLPs) (1, 2). 67 The replication of rotavirus occurs in cytoplasmic non-membranous electron-dense 68 inclusions termed viroplasms composed of NSP2, NSP5, VP1, VP2, VP6 and host 69 components (1, 3). The replication and packaging of the viral genome into newly synthesized 70DLPs take place in these inclusions (4), which then bud into the lumen of the endoplasmic 71 reticulum (ER) through membrane sites modified by the presence of NSP4 (5, 6). NSP4 is a 72 transmembrane ER glycoprotein with two N-linked high mannose glycosylated chains (7) 73 that play a crucial role in the last steps of rotavirus assembly. It has been shown that the 74 cytoplasm oriented-terminus of NSP4 associates with VP4 (8), and binds the VP6 on DLPs 75 acting as a receptor for these particles to mediate their budding into the ER (9, 10). Moreover, 76 NSP4 has been shown to also interact with VP7 through its N-terminus oriented to the ER 77 lumen (11, 12). It has been proposed that these interactions drive the incorporation of the 78 outer layer proteins into the transitory lipid envelope that DLPs acquire during ER membrane 79 budding; this envelope is removed in the lumen of the ER by an unknown process in which 80 NSP4 is eliminated while VP4 and VP7 are assembled to produce the final infectious TLPs 81 All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.110...

A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology

“…The identification of major cellular events regulated by garz/ GBF1 27-29, [47][48][49] has targeted such molecules for health and disease studies 18 . Recently, it has been shown that siRNA knockdown of GBF1 causes intracellular APP accumulation in primary cortical neurons; overexpression of GBF1 contributes to APP trafficking and is dependent on its GEF activity 50 .…”

A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology

“…The identification of major cellular events regulated by garz/GBF1 27-29, [47][48][49] has targeted such molecules for health and disease studies 18 . Recently, it has been shown that siRNA knockdown of GBF1 causes intracellular Amyloid Precursor Protein (APP) accumulation in primary cortical neurons; overexpression of GBF1 contributes to APP trafficking and is dependent on its GEF activity 50 .…”

Role of the Guanine Nucleotide Exchange Factor GBF1 in the Replication of RNA Viruses

“…In recent years, the use of genome-wide RNAi screens has shown that the vesicle transport mediated by the COPI/Arf1 machinery is relevant for virus replication, since knocking down the expression of some of the proteins that integrate such machinery significantly impairs the yield of rotavirus progeny [ 141 , 142 ]; similar results are obtained when the vesicle transport is inhibited by BFA treatment of rotavirus-infected cells [ 76 , 143 ]. This inhibition is related to the activity of GBF1, since interfering specifically with this factor by GCA, or depletion of this nucleotide exchange factor by RNAi, significantly reduced the production of new viral infectious particles [ 76 ]. In addition, the block in the production of infectious virus was associated with the assembly of the virus surface proteins VP7 and VP4, since both BFA and GCA block the assembly of the viral surface proteins onto DLPs, preventing the production of mature infectious TLPs [ 76 ].…”

“…This inhibition is related to the activity of GBF1, since interfering specifically with this factor by GCA, or depletion of this nucleotide exchange factor by RNAi, significantly reduced the production of new viral infectious particles [ 76 ]. In addition, the block in the production of infectious virus was associated with the assembly of the virus surface proteins VP7 and VP4, since both BFA and GCA block the assembly of the viral surface proteins onto DLPs, preventing the production of mature infectious TLPs [ 76 ]. The restriction in the assembly of TLPs was shown to be the result of a defective trimerization of VP7, required for its assembly onto DLPs, rather than a block in the budding of DLPs into the ER lumen.…”

“…It has been shown that the inhibition of GBF1 by either BFA or GCA, or the depletion of this factor by RNAi, alters the electrophoretic mobility of the viral glycoproteins VP7 and NSP4 [ 76 , 143 ]. Although the altered posttranslational modification of VP7 was initially considered to explain its lack of trimerization, increasing evidence has pointed out that the alteration of NSP4 is rather responsible, since depletion of this protein or alteration of its posttranslational processing with tunicamycin, an inhibitor of N-glycosylation, affects the trimerization of VP7 and restricts the maturation of the transient enveloped viral particles to TLPs.…”