Cauliflower mosaic virus P6 protein interactions: a complex story

Cauliflower mosaic virus P6 protein interactions: a complex story

Bibliographic Details
Main Author: Lutz, Lindy Michelle
Language:English
Published: University of Toledo / OhioLINK 2014
Subjects:
Biology
Plant Biology
Virology
Online Access:http://rave.ohiolink.edu/etdc/view?acc_num=toledo1393863253
id ndltd-OhioLink-oai-etd.ohiolink.edu-toledo1393863253
record_format oai_dc
collection NDLTD
language English
sources NDLTD
topic Biology
Plant Biology
Virology
spellingShingle Biology
Plant Biology
Virology
Lutz, Lindy Michelle
Cauliflower mosaic virus P6 protein interactions: a complex story
author Lutz, Lindy Michelle
author_facet Lutz, Lindy Michelle
author_sort Lutz, Lindy Michelle
title Cauliflower mosaic virus P6 protein interactions: a complex story
title_short Cauliflower mosaic virus P6 protein interactions: a complex story
title_full Cauliflower mosaic virus P6 protein interactions: a complex story
title_fullStr Cauliflower mosaic virus P6 protein interactions: a complex story
title_full_unstemmed Cauliflower mosaic virus P6 protein interactions: a complex story
title_sort cauliflower mosaic virus p6 protein interactions: a complex story
publisher University of Toledo / OhioLINK
publishDate 2014
url http://rave.ohiolink.edu/etdc/view?acc_num=toledo1393863253
work_keys_str_mv AT lutzlindymichelle cauliflowermosaicvirusp6proteininteractionsacomplexstory
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spelling ndltd-OhioLink-oai-etd.ohiolink.edu-toledo13938632532021-08-03T06:22:36Z Cauliflower mosaic virus P6 protein interactions: a complex story Lutz, Lindy Michelle Biology Plant Biology Virology Cauliflower mosaic virus (CaMV), one of the top ten viruses from a molecular plant pathology standpoint, is a plant pararetrovirus whose 8 kb circular double-stranded DNA genome encodes 7 different proteins (P1-P7). CaMV P6, encoded by gene VI has been implicated in a variety of functions such as: translational transactivation, host range control, symptom formation, host hypersensitive responses, RNA silencing suppressor activity, inclusion body (IB) formation and virus infectivity. Because of its multifunctional nature, P6 interacts with many host, and viral proteins including itself. P6 self-association appears to involve four domains (D1-D4). D3 has been implicated in viral infectivity and contains two RNA binding domains, separated by a highly conserved 34 amino acid spacer called D3b. CaMV mutants harboring a deletion of D3b are non-infectious, indicating its importance for viral propagation. To further analyze D3b, full-length P6 constructs were generated that harbored single amino acid substitutions within this region. In general, the mutants bound less efficiently to the individual P6 domains than wild type. Mutations near the amino-terminal end of D3b had a more detrimental effect on self-association domain binding than those near the central portion. Since P6 is an IB protein, we hypothesized that mutations in D3b may influence IB formation. P6 IBs are thought to start out as small aggregations of protein (most likely P6) and ribosomes. They acquire additional materials (viral proteins and nucleic acids) to enlarge to form small bodies. Small bodies are then thought to fuse together to form larger, mature IBs. All mutant P6s formed IBs when expressed as green fluorescent protein (GFP) fusions in transgenic cells. However, the mutant P6s that were most reduced in binding also showed decreased IB size. Hence, the mutations in D3b appear to affect the fusion of small IBs into larger ones. It is possible that IB size is important because it correlated with differences in virus host range. CaMV strain W260 has a much wider host range and more efficiently infects host plants when compared to the CM1841 isolated. Our most recent data show that CM1841 IBs are smaller than those formed by W260 P6. In addition, P6 mutants that showed decreased binding to self-association domains and smaller IB sizes also exhibited much lower total viral DNA levels in inoculated leaves. This was also reflected by systemic symptom formation. Hence, less efficient binding correlates with smaller IB size and reduced local and systemic infection for the mutants. Taken together, these data suggest that fusion of small IBs into larger ones is important for proper viral infections to occur and we have possibly identified mutants in this process. In addition, these data suggest that IB formation is required for viral infection rather than merely being a consequence of itThe CaMV genome encodes seven viral proteins including P6. P6 has been reported to interact with two other viral proteins in addition to itself. Therefore, we also examined P6 for its ability to interact with the other viral gene products. P6 was found to interact with the aphid transmission factor (P2), the virion-associated protein (P3), reverse transcriptase protein (P5), and the protein of unknown function (P7). P2 was previously reported to control the difference in IB stability between CM1841 and W260. Our data indicate that P2 from both viruses bound equally well to P6. The CM1841 P2 is less stable than its W260 counterpart. Taken together, this would suggest that the differences in IB stability for W260 and CM1841 mediated by P2 are due to variation in P2 protein stability rather than P6 binding. Binding of P6 to P3 could help the latter protein form complexes necessary for aphid transmission and virus cell-to-cell movement. P5 has a tri-partite structure with an N-terminal protease domain, a central reverse transcriptase (RT) and a C-terminal RNase H domain. Our pull-down results showed P6 could interact with full-length P5. Based on our preliminary pull-down analyses, P6 could bind inefficiently to the protease but more efficiently to the RT-RNase H (termed P5MC) portion of P5. Perhaps this interaction plays a role in P5 RT regulation. Interestingly, P5MC interactions with P5 showed a similar pattern to the P6 interactions. P5MC was able to self-associate well, but and interacted weakly with full-length P5 and the protease. P6 also interacted with P7, but the significance of this interaction is unknown. Perhaps P7 aids P6 in regulating an aspect of translational transactivation, but this is mere speculation. In addition, P6 can also interact with a variety of host factors. In collaboration with Dr. James Schoelz at the University of Missouri, we found three Arabidopsis proteins: CHUP1, C2CDMT, and FIT that interact with full-length P6. Interestingly, of the four domains involved in P6 self-association, only D2 and D4 bind to CHUP1 and C2CDMT. However, FIT was able to bind to all P6 self-association domains but best to D2. Given that it binds to other host factors, we might speculate that D2 of P6 maybe acts as a host interface domain. In summary P6 interacts with a large number of both viral and host proteins. P6 self-association is needed for proper IB formation and efficient infection. P6 interactions with each of the other viral proteins may be to modulate proper interactions of these proteins with their appropriate partners. Finally, P6 interactions with host factors may play a role in inhibiting host defenses, modulating systemic symptom formation, or mediating inter and intra cellular movement. 2014-08-01 English text University of Toledo / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=toledo1393863253 http://rave.ohiolink.edu/etdc/view?acc_num=toledo1393863253 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

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