Predicting the host protein interactors of Chandipura virus using a structural similarity-based approach

Arnoldi

Schraner

et al. 2019

Preprint

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...

Section: Discussionmentioning

confidence: 99%

The guanine nucleotide exchange factor GBF1 participates in rotavirus replication

Arnoldi

Schraner

et al. 2019

Preprint

“…Moreover, for VSV as well as for hepatitis C virus (66)(67)(68), mouse hepatitis coronavirus (69), chikungunya virus (70), poliovirus (58,59,71), coxsackievirus (72), and classical swine fever virus (73), COPI/Arf1 has been shown to be necessary for genome replication as well as for viral protein expression. COPI/Arf1 has also been shown to be critical for viral particle assembly of influenza virus and the Chandipura virus (63,74). The specific mechanism through which COPI and Arf1 participate in these processes has not been characterized; however, GBF1, involved in the first step of the COPI/Arf1 transport process, has been shown to play an important role for the replication of some of these viruses.…”

Section: Discussionmentioning

confidence: 99%

The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication

Arnoldi

Schraner

et al. 2019

J Virol

Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4. IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4. KEYWORDS GBF1 nucleotide exchange factor, morphogenesis, rotavirus R otaviruses, members of the family Reoviridae, are nonenveloped particles formed by three concentric layers of proteins that surround the 11 genome segments of double-stranded RNA (dsRNA). The innermost layer is composed of the core shell protein VP2, which encloses the replication intermediates, composed of the RNAdependent RNA polymerase VP1 and the guanylyl-methyl transferase VP3. The intermediate layer is formed by VP6, which surrounds the VP2 layer to form double-layered particles (DLPs). Finally, the addition of the glycoprotein VP7 and the spike protein VP4 onto the DLPs forms the infectious triple-layered particles (TLPs) (1, 2). Downloaded fromFIG 2 BFA and GCA inhibit rotavirus replication at a postentry step. (A) MA104 cells were infected with RRV (MOI ϭ 5) at 37°C for 1 h. Unbound virus was removed, and 0.5 g/ml of BFA or 10 M GCA was added at the indicated times postinf...

“…On the other hand, a structure-based computational analysis of possible protein–protein interactions between human proteins and the proteins of Chandipura virus (CHPV), another member of the Vesiculovirus genus, which has emerged as a pediatric encephalitic virus in India [ 127 , 128 ], revealed that GBF1 and Arf1 exhibit a high potential for interaction with the G and L proteins of this virus [ 129 ]. Although the association of GBF1 and Arf1 with the G protein was rather expected, given the association of the G protein with the secretory pathway, the potential interaction of GBF1 and Arf1 with the polymerase L of CHPV is a surprising finding that might indicate that GBF1 could directly regulate the activity of the L protein during viral RNA replication; the interaction between GBF1 and the L protein was not corroborated by biochemical assays.…”

Section: Negative-sense Rna Virusesmentioning

confidence: 99%

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

Arias

2020

Viruses

The guanine nucleotide exchange factor GBF1 is a well-known factor that can activate different ADP-ribosylation factor (Arf) proteins during the regulation of different cellular vesicular transport processes. In the last decade, it has become increasingly evident that GBF1 can also regulate different steps of the replication cycle of RNA viruses belonging to different virus families. GBF1 has been shown not only to facilitate the intracellular traffic of different viral and cellular elements during infection, but also to modulate the replication of viral RNA, the formation and maturation of viral replication complexes, and the processing of viral proteins through mechanisms that do not depend on its canonical role in intracellular transport. Here, we review the various roles that GBF1 plays during the replication of different RNA viruses.