Project summary
Title:
Cell-to-cell movement of Potyviruses
Background:
Potyviridae is the largest family of plant viruses with the majority of the members in the
Potyvirus genus. Various aspects of these viruses have been well studied, but the cell-to-cell
movement is still unclear. Currently, it is a mystery as to the form (virion or ribonucleoprotein
complex) and mechanism in which the virus moves. Many of the proteins have been
characterized and their function(s) determined, but no dedicated movement protein has been
found. Studies have alluded to the formation of a complex to faciliatate movement because
mutational analysis of several proteins impact the ability of the virus to move to neighboring
cells. Therefore, studies on the movement of Potyviruses will supplement the known
characteristics of this vast genus.
Hypothesis:
Potyviruses move cell-to-cell through the plasmodesmata on the cellular cytoskeleton network as
a virion faciliatated by a complex of proteins.
The goal is to characterize Potyvirus cell-to-cell movement with the following specific aims:
1. Visualize Potyvirus movement during an infection using TuMV.
a. Use electron microscopy to observe TuMV movement in infected tissue.
b. Use fluorescently labeled protein(s) to observe TuMV movement in infected tissue.
2. Determine mechanism of transport to neighboring cells.
a. Identify viral movement protein and specific cytoskeleton component interactions
and if disruption influences viral movement.
b. Identify secretion pathway interactions and investigate if inhibition of pathway
inhibits viral movement.
Rationale/Significance:
Many Potyviruses such as Potato virus Y, Plum pox virus, Turnip mosaic virus, Soybean mosaic
virus, and Papaya ringspot virus cause major yield losses in food crops. Movement is required
for systemic viral infection, therefore, study of virus movement may lead to new antiviral
strategies. Studies of viral movement will provide insight into the spread of infection which is an
important stage of the viral life cycle.
Project Description
Title:
Cell-to-cell movement of Potyviruses
Background:
Potyviridae is the largest family of plant
viruses, encompassing approximately a third of all
known plant viruses. This family consists of six genera
including Potyvirus, which contains nearly 200
members (38). Insects such as aphids and mites
facilitate transmission of these plant viruses. As nonenveloped flexuous rods, they range from 650 to 900
nm long and 10 to 15 nm wide (Fig. 1) (37).
Potyviruses are positive sense RNA viruses with an
approximately 10 Kb genome that contains one large
Fig 1) Electron micrograph of purified
open reading frame (ORF) and a small overlapping
Potyvirus virions from Walsh and
ORF (Fig. 2) (28). Ten of the viral proteins are
Jenner 2002. These viruses are flexuous
translated as a polyprotein, which is then processed
filaments that range from 650 to 900
into 10 mature proteins by several viral proteases (28).
nm long and 10 to 15 nm in diameter.
Although a majority of the mature proteins has
multiple defined functions, none has movement as their primary function (Table 1) (36).
Fig 2) Genome organization of Potyviruses. This is a depiction of the infectious clone of Turnip
mosaic virus (TuMV) with green fluorescent protein (GFP) inserted between two viral proteins. GFP
will be a free mature protein after cleavage by viral proteases.
A varying number of proteins are need for movement of either ribonucleoprotein (RNP)
complexes or virions, the two forms of virus cell-to-cell movement. Tobamoviruses require the
movement protein (MP) for cell-to-cell movement as either an RNP complex or movement
vesicle complex (22). Potexviruses use a group of three proteins known as the triple gene block
to facilitate RNP complex movement through the plasmodesmata (PD) (18). The MP of Cowpea
mosaic virus forms tubular structures to potentiate virion spread to neighboring cells (26).
Multiple proteins have secondary functions involved in movement with the main hypothesized
contributors as coat, viral protein that is genome linked (VPg), and cytoplasmic inclusion (CI)
Page 1 of 8
Project Description
proteins (36). Coat
Proteins
Properties
protein forms the
P1 (32–64 K)
Trypsin-like serine proteinase,
virion with
C-terminal autocleavage
approximately 2,000
Symptomatology
individual coat
HC-Pro (56–58 K) Aphid transmission
Self-interaction
proteins to form the
Systemic movement
helical virion with a
Suppression of gene silencing
3.4 nm pitch (36).
Synergism and symptom development
Mutations within the
Papain-like cysteine proteinase,
coat protein have been
C-terminal autocleavage
found to retain virion
P3 (37 K)
Plant pathogenicity
formation while
6K1
N/A
abolishing cell-to-cell
CI (70 K)
ATPase:RNA helicase
movement of the virus
Cell-to-cell movement
(7, 8). The VPg is
6K2
Anchoring the viral replication complex to membranes
attached to the 5’end
NIa (49 K)
Cellular localization
of the viral genome to
VPg involved in genome replication
mimic a cap structure
Trypsin-like serine proteinase (acts in cis and in trans)
and stabilize the RNA
Protein-protein interaction
molecule. The VPg is
NIb (58 K)
RNA-dependent RNA polymerase (RdRp)
involved in replication
Involved in genome replication
and encapsidation
CP (28–40 K)
Aphid transmission
Cell-to-cell and systemic movement
(35). Within the
Virus assembly
virion, VPg can be
P3NPIPO (26 K)
N/A
detected at a specific
Plasmodesmata localization
end with an epitope
Table 1) Adapted from Urcuqui-Inchima et al. 2001 listing the mature viral
probe to determine
proteins along with weight and function.
polarity and may be
involved in directional
movement (27). Cytoplasmic inclusion (CI) protein generates the characteristic cytopathic effect
in the form of pinwheel structures for Potyviruses. Studies indicate the CI in cell-to-cell
movement observing multimers transversing the PD that connect neighboring cells (29). In a
recent paper, P3NPIPO was found to localize to the PD and hypothesized to be a part of the
movement complex, but actual function is still not known (40, 41). HC-Pro participates in long
distance movement, rather than cell-to-cell movement and facilitates viral systemic infection
through the vascular tissue (5). For Potyviruses, it seems a complex of proteins is involved in
virus movement.
Whether the virus moves as an RNP complex or as a virion, movement generally utilizes
cellular machinery to transport the virus to neighboring cells through the PD (13, 23). Multiple
components of the cellular cytoskeleton, microfilaments, intermediate filaments and
Page 2 of 8
Project Description
microtubules, associate with viral proteins during cell-to-cell movement (2, 17). Localization to
the cytoskeleton occurs during various stages of infection as in viral replication and cell-to-cell
movement, both through a vesicle complex. Tobacco mosaic virus movement protein associates
with microtubules and microfilaments and may potentiate cell-to-cell spread via movement
vesicles complexes (3, 20).
Currently, the exact mode and mechanism of Potyvirus cell-to-cell mediated movement is
unknown. It is unclear whether the virus moves as a virion or as a ribonucleoprotein complex.
What is clear is that the virus requires multiple proteins in order for movement to occur.
Hypothesis:
Potyviruses move cell-to-cell through the PD on the cellular cytoskeleton network as a virion
facilitated by a complex of proteins. This proposal will investigate the mechanism of potyviral
cell-to-cell mediated movement using Turnip mosaic virus (TuMV).
The goal is to characterize Potyvirus cell-to-cell movement with the following specific aims:
1. Visualize Potyvirus movement during an infection using TuMV.
a. Use electron microscopy to observe TuMV movement in infected tissue.
b. Use fluorescently labeled protein(s) to observe TuMV movement in infected tissue.
2. Determine mechanism of transport to neighboring cells.
a. Identify viral movement protein and specific cytoskeleton component interactions
and if disruption influences viral movement.
b. Identify secretion pathway interactions and investigate if inhibition of pathway
inhibits viral movement.
Rationale and significance:
This proposal will use multiple microscopy techniques to observe TuMV viral movement
in infected Arabidopsis tissue. Microscopy has made great advances with resolution and
increased number of available markers used to characterize a variety of virus infections. Electron
microscopy can be used to visualize viral virions and protein complexes within infected tissue. A
large variety of fluorescent protein labels are commercially available, standardized, and
commonly used for scientific research in localization studies. TuMV infection in a model plant
organism such as Arabidopsis has many benefits including availability of a large library of
mutants and characterized genome to facilitate identification of possible host-viral protein
interactors.
Mechanism of viral movement is a crucial characteristic that needs to be uncovered for
the largest family of plant viruses. Potyviruses infect a wide range of economically important
crops as in soybeans, potatoes, and stone fruits (38). If the mechanism of viral spread can be
determined, then an antiviral strategy may be developed from the knowledge gained with this
proposal. Crops would be protected to increase yield, which would be beneficial to farmers and
augment the available food supply.
Page 3 of 8
Project Description
Experimental approach:
Aim 1: Visualize Potyvirus movement during an infection using TuMV.
Viruses have to spread to neighboring cells to proliferate, thereby continuing viral
propagation to establish an infection. In plant viruses, both cell-to-cell and long distance
movement are required to achieve systemic infection, often facilitated via a movement protein.
PD are cytoplasmic connections to neighboring cells that viruses exploit for cell-to-cell
movement (1, 14, 25). This allows the virus to spread without encountering the major obstacle of
plant cell walls. Numerous studies have observed this phenomenon using various microscopy
techniques. Electron and fluorescent microscopy will be employed in this proposal to observe
TuMV cell-to-cell movement.
Transmission electron
microscopy (TEM) can
resolve objects in the
nanometer range including
nucleic acids and proteins.
This method was used to
visualize Cowpea mosaic
virus and Grapevine fanleaf
a)
b)
virus MP tubules formed
during virus movement (Fig.
Fig 3) a) An excerpt from Silva et al. 2002 depicting CPMV MP
3) (21, 32). RNP complexes
tubules traversing PD to allow virions to move between bundle
can also be observed with
sheath cell (BSC) and phloem parenchyma cell (PPC). b) From
TEM as in the case of ORF3
Laporte et al. 2003 showing the tubular formation of GFLV MP
expression of Groundnut
across PD.
rosette virus (Fig. 4) (34). An
ultrathin layer of sample is stained and presented to an
electron beam within a pressure-controlled chamber. A
digital camera collects electron waves passing through the
specimen to generate an image. The thickness and staining of
the sample influences the quality of the image. Often, tissues
require a microtome to produce ultrathin samples that can be
used for TEM. Heavy atom, negative, stains such as uranyl
acetate are used to absorb electrons to enhance contrast
around the specimen. Optimization of both the tissue
sectioning and staining technique will need to be done to
Fig. 4) TEM of RNP complexes
obtain the highest quality image. A consultant with the Iowa
formed within cytoplasmic
State Microscopy and Nanoimaging Facility will assist in
inclusion. Inset is a crossoptimizing the TEM process for observing TuMV movement
section of an RNP complex.
in infected Arabidopsis leaf tissue.
To obtain infected tissue for TEM, wild type Col-0
Page 4 of 8
(Taliansky and Robinson 2003)
Project Description
Arabidopsis thaliana plants will be grown and infected with TuMV. Short day length, 12 hours,
will be used to grow the Arabidopsis to maximize leaf surface area during the rosette stage.
Then, TuMV infectious clone DNA will be bombarded into the leaf tissue to inoculate and allow
for establishment of infection. Progress of the infection front will be tracked by green
fluorescence due to the GFP insertion into the TuMV genome (Fig. 2). Tissue at the infection
front will be used for TEM to observe virus movement to adjacent cells.
Electron microscopy is a costly and time intensive process, but in planta observations of
the movement process with relation to the rest of the cell will provide data about cytopathic
effects along with movement. A recent paper by Otulak and Garbaczewska noted Potato virus Y
(PVY) movement during early infection with TEM (24). They concluded that PVY has two
forms, encapsidated and non-encapsidated, and both can be transported to neighboring cells. It is
difficult to discern in
the figures due to the
low magnification
and darkness of the
specimen (Fig. 5)
(25). Potyviruses may
move as virions due
a)
b)
to the requirement of
the coat protein for
Fig. 5) a) TEM of PVY infection with presence of particles within the PD. b)
cell-to-cell
TEM from Overall and Blackman 1996 showing PD with potentially enough
movement. With
optimization to visualize virus movement through the PD.
TEM, it may be
difficult to distinguish between virions and RNP complexes. Optimization can provide a clearer
picture of Potyvirus cell-to-cell movement. If these obstacles cannot be overcome, then
fluorescent microscopy is an alternative approach to examine cell-to-cell movement. Both
electron and fluorescent microscopy will be used to maximize the probability of identifying the
form in which the virus moves.
Fluorescent microscopy is more commonly used than TEM. Fluorescently labeled
antibodies allow visualization of the spatial orientation of particular targets, often to confirm
interactions and/or co-localizations, when used to probe fixed tissues. Known proteins located
within the PD will be probed along with viral proteins to observe cell-to-cell movement (40).
TuMV infectious clone will have to be modified to remove GFP from in between P1 and HC-Pro
to avoid background fluorescent and to allow potential use of GFP labeled antibodies (9). The
untagged infectious clone will be bombarded into Arabidopsis leaf tissue for fluorescence
microscopy as in preparation for TEM. An advantage of antibodies rather than GFP fusion
proteins is the ability to probe multiple viral proteins simultaneously with antibodies containing
varying flourochromes (11). This method has been used to visualize the co-localization of MPs
and PD of several viruses including Tobacco mosaic virus (Fig 6) (15, 20, 30). A disadvantage of
fluorescence microscopy is only visualizing labeled targets, so unknown interactors are unseen
Page 5 of 8
Project Description
and nonspecific labeling
is a major issue. The
specificity of the
antibody label can be
easily determined with an
immunoblot to test for
cross-reaction with
nonspecific cellular
proteins. Both or either
microscopy techniques
should allow detection of
Potyviral cell-to-cell
mediated movement.
Aim 2: Determine
mechanism of transport
to neighboring cells.
Fig. 6) Kawakami et al. 2004 showing TMV MP move cell-to-cell as part
of the viral replication complex (VRC) vesicle and co-localization to the
PD. These vesicles travel upon microfilaments. Green is TMV MP and
blue is VRC with the arrows indicating co-localization.
All viruses usurp
and manipulate cellular
function for proliferation
including egress from the
already infected cell. Plant
viruses often utilize the
a)
b)
cytoskeleton to facilitate
transport to the PD or increase
Fig. 7) a) Disruption of F-actin by treatment with CMV MP in J
the size exclusion limit of the
and without in K (Su et al. 2010) b) PVX TGBp2 containing
PD by disrupting cytoskeletal
vesicles budding, indicated by arrows, from the plasma
structures for cell-to-cell
membrane (red) by Ju et al. 2007.
movement(1). TMV interacts
with myosin to move RNP complex vesicles to the PD (Fig. 6) (16, 20). Cucumber mosaic virus
(CMV) MP severs actin filaments to alter the PD to allow larger weight molecules to pass (Fig.
7a) (33). Some viruses such as Potato virus X require the endoplasmic reticulum and secretory
pathway for viral spread (Fig. 7b) (18, 19).
First, a yeast two-hybrid screen will identify any interactions between potential
movement proteins from TuMV and Arabidopsis cytoskeleton components. In a yeast twohybrid screen, a transcription factor that activates an essential gene such as Gal4 is broken into
two domains called the binding domain (BD) and an activation domain (AD) (10). A bait protein
is fused to the BD and a prey protein is fused to the AD. If there is an interaction between the
bait and prey proteins, then activation of the essential gene occurs to allow the yeast to grow on
selective media. The bait protein is usually known, viral proteins for this proposal, and used to
identify new interacting partners (12). An Arabidopsis cDNA library can be purchased for the
Page 6 of 8
Project Description
construction of all the prey proteins. Positive interactions
between several viral proteins and cytoskeleton components
are expected because replication vesicles of TuMV move on
microfilaments (Fig. 8) (4). This previous observation
suggests that TuMV cell-to-cell movement will perpetuate
this interaction with cytoskeleton components. Yeast twohybrid screens are prone to false positives, therefore proper
controls and multiple layers of selection are required to
identify true interactions (10). In addition, the interactions
are identified under in vitro conditions and may not hold true
in vivo. To confirm these interactions in planta, fluorescent
microscopy and co-immunoprecipitation will be used.
Fig. 8) TuMV viral replication
complex vesicles are colocalized to and trafficked
upon microfilaments. Each
vesicle contains a single viral
genome. (Cotton et al. 2009)
Fluorescent microscopy will be used to confirm
interactions discovered by the yeast two-hybrid screen of
TuMV viral proteins involved in movement and host
proteins in planta (11). A variety of antibodies is
commercially available with guaranteed specificity.
Labeling of cytoplasmic structures and interacting viral proteins for fluorescent visualization can
verify co-localization within the cell. A more sensitive approach is bimolecular fluorescent
complementation (31). This technique, similar to the yeast two-hybrid, splits a fluorescent
protein into two halves and fuses them to potential interacting partners. If the partner proteins
interact in planta, then the fluorescent protein is reconstituted and functional. Fluorescence of the
split proteins indicates subcellular localization and specific interaction because the two halves
have to be spatially close to function.
A second approach to confirm protein interaction is with co-immunoprecipitation (12).
The interacting partners are each tagged with a different polypeptide tag. Expression of these
proteins in planta allows natural interactions to occur. Then, total proteins are extracted from the
plant tissue and separated via affinity column chromatography. The one of the tagged protein
pairs is bound to the column under conditions that preserve protein complexes. The bound
proteins are eluted and separated on a SDS-PAGE gel. Then, the proteins are transferred to
membrane and immunoblotted for the other partner with an antibody. If present, then the
interaction is confirmed.
To understand the precise mechanism TuMV usurps to facilitate movement, disruption of
select cytoskeleton components will be done to observe impact upon viral cell-to-cell spread.
Previous research localizes P3NPIPO to the PD, which may suggest a similar function to that of
CMV MP (6, 40). Mayhap, P3NPIPO will disrupt actin filaments at PD similar to chemical
destabilizers such as Latrunculin A. Because disruption of microfilaments may influence virus
transport to the PD, viral interaction with other components such as microtubules and
intermediate filaments may facilitate virus movement to and across the PD.
Page 7 of 8
Project Description
All of these various techniques starting with the yeast two-hybrid screen will also be used
to identify potential viral protein interactions with secretory pathway proteins. TuMV replication
complex vesicles are generated by budding of the ER in a secretory pathway dependent process
(39). Therefore, TuMV movement may require both the secretory pathway in conjunction with
trafficking on cytoskeletal structures for cell-to-cell movement.
Future directions:
More time, money and manpower will further progress the research and understanding of
Potyviruses. With the movement mechanism determined, focus can be directed towards
identifying the proteins, host or viral, involved in the formation of the movement complex that
facilitate virus spread. Knowing what form the virus moves as will narrow the search for protein
interaction to target particular viral proteins required for movement. Research on the potential
differential requirement of proteins and conditions to promote cell-to-cell and/or systemic
movement of the virus will expand knowledge of plant viruses. Practical applications can be
discovered by identifying inhibitors, mutants of host proteins and/or chemicals, of virus spread
that has minimal impact of the growth of the plant. Identification of dominant inhibition of
particular viral proteins involved in movement could be another avenue of exploration for
practical application.
Timeline:
Aim 1
Aim 2a
Aim 2b
Year 1
Electron micrograph
Cytoskeleton
Secretion pathway and
visualization of TuMV component and MP(s)
MP(s) interaction
movement
interaction
Year 2
Disruption of
Disruption of
Fluorescent label
cytoskeleton
secretory pathway
Year 3 visualization of TuMV
components involved
involved in viral
movement
in viral movement
movement
This proposal will be accomplished in three years with people working simultaneously on
various aspects. A post-doctoral scientist will visualize TuMV movement. Aim 2 will be the
majority of the doctoral work of a graduate student. Both will become proficient at TEM and
fluorescent microscopy by participating in a course offered by the Iowa State Microscopy and
Nanoimaging Facility. Each aim has multiple parts, independent of each other, and new
knowledge gained by completion of each part, without losing the relevance of each component.
Page 8 of 8
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37.
38.
39.
40.
41.
Torrance, L., I. A. Andreev, R. Gabrenaite-Verhovskaya, G. Cowan, K. Makinen, and M. E.
Taliansky. 2006. An unusual structure at one end of potato potyvirus particles. J Mol Biol 357:18.
Urcuqui-Inchima, S., A. L. Haenni, and F. Bernardi. 2001. Potyvirus proteins: a wealth of
functions. Virus Res 74:157-75.
Walsh, J. A., and C. E. Jenner. 2002. Turnip mosaic virus and the quest for durable resistance.
Mol Plant Pathol 3:289-300.
Ward, C. W., and D. D. Shukla. 1991. Taxonomy of potyviruses: current problems and some
solutions. Intervirology 32:269-96.
Wei, T., and A. Wang. 2008. Biogenesis of cytoplasmic membranous vesicles for plant potyvirus
replication occurs at endoplasmic reticulum exit sites in a COPI- and COPII-dependent manner. J
Virol 82:12252-64.
Wei, T., C. Zhang, J. Hong, R. Xiong, K. D. Kasschau, X. Zhou, J. C. Carrington, and A. Wang.
Formation of complexes at plasmodesmata for potyvirus intercellular movement is mediated by
the viral protein P3N-PIPO. PLoS Pathog 6:e1000962.
Wen, R. H., and M. R. Hajimorad. Mutational analysis of the putative pipo of soybean mosaic
virus suggests disruption of PIPO protein impedes movement. Virology 400:1-7.
Page 3 of 3
Budget Justification
Personnel ($233,028)
This will provide for a graduate student and postdoctoral fellow. University rates are used where
graduate students and postdoctoral fellows receive a 13.3% and 20.2% fringe benefits rate
respectively. The graduate student will identify the potential interaction(s) of various viral
proteins and cellular components involved in spread of the virus. Once the cellular components
have been identified, determine if disruption of normal function inhibits viral movement. The
postdoctoral fellow will discover the form of viral cell-to-cell movement using fluorescent and
electron microscopy.
Equipment ($30,000)
A fluorescent microscope will facilitate the progression of the project. All aims of the proposal
involve fluorescent microscopy and access to a shared microscope may be limited. Therefore, a
microscope solely dedicated to this project will be obtained.
Travel ($9,000)
Domestic and foreign travel is required to share results and attend conferences related to the field
of research like American Society for Virology, RNA Society, International Congress of
Virology and All-Iowa Virology Symposium. At these meetings, discussions and potential
collaborations may form to assist in productive advancement of Potyvirus research and broaden
knowledge base of researchers.
Other Direct Costs ($114,203)
A 3% inflation rate has been implemented.
Materials and supplies ($61,818)
General materials and supplies include general molecular biology laboratory expenses such as
nuclease free tubes and tips, chemicals for solutions, cloning materials to make constructs,
transcription kits to generate RNA, and microscope supplies as in slides and coverslips. Iowa
State University DNA Synthesis and Sequencing Facility services are required to confirm
sequence of constructs ($6/sequencing run and $0.30/base for 50 nmol DNA oligo synthesis).
Funding is required to maintain upkeep of laboratory equipment and growth chambers for plants.
Publication costs ($4,636)
Funds are required to offset cost of publishing results to share with the scientific community.
Color prints are particularly expensive, but will be required due to the nature of the data
presented.
Consultant services ($15,000)
The Iowa State Microscopy and Nanoimaging Facility will be consulted for electron microscopy
to observe viral cell-to-cell movement. One hundred hours are budgeted to included microscope
time, supplies, and consultation.
Computer services ($3,091)
Up to date computers and software will be required to analyze data. High resolution data
collected from microscopy need to be analyzed without loss of resolution to produce quality
figures for presentations.
Graduate Student Tuition ($29,658)
Tuition support is needed for the graduate student. The rates are set for a one-quarter time nonengineering graduate student at Iowa State University for the next three years.
Indirect Costs ($96,558)
These are the agreed upon percentage cost for Iowa State University.
Worksheet for Project Budget
You should see the following tabs at the
bottom of the page. You may have to
scroll across to see all of them.
effective 07/01/2010
revision 07/07/2010
Principal Investigator:
- Start Here (yellow)
- Salaries & Wages (purple)
- Equip-Travel-Participants (green)
- Other Direct (blue)
- Subcontracts (orange)
- Cumulative Budget (red)
- Links (pink)
Sponsor: Iowa State Univeersity
Project Title: Cell-to-cell movement of Potyviruses
Start
Project Period: Date
8/15/11
mm/dd/yyyy
Indirect Cost Rate:
USDA-20%
End
Date
8/15/14
Total
Years
3
mm/dd/yyyy
25.00%
Please refer to the Indirect Cost Rate and Policy links on the
Links Tab
NOTE: Classification must be selected for each person in each
year that funding is requested.
Scroll to the Right to enter
Cost Sharing Amounts > > >
Running Total for Cumulative Budget Amount =
$
482,789.17
Worksheet for Project Budget - Salaries & Wages
Year 1
Please select an
escalation rate for all
salaries below
0.00%
This Fringe Benefit
rate is based on the
Classification
selected at the left.
Senior Personnel
Prefix
First Name
Middle
Init.
Last Name
Classification
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
0.00
0.00
$0
0.0%
$0
$0
$0
Fringe Benefit $
1
Select one
2
Select one
$0
0.0%
$0
$0
$0
3
Select one
$0
0.0%
$0
$0
$0
4
Select one
$0
0.0%
$0
$0
$0
5
Select one
$0
0.0%
$0
$0
$0
6
Select one
$0
0.0%
$0
$0
$0
7
Select one
$0
0.0%
$0
$0
$0
8
Select one
$0
0.0%
$0
$0
$0
Total Senior Personnel
$0
Total Senior Personnel
Other Personnel
Number of
Personnel
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1.00
Post Doctoral Associate
$3,500
12.00
$42,000
20.2%
$8,484
$50,484
$0
1.00
Graduate Students
$2,000
12.00
$24,000
13.3%
$3,192
$27,192
$0
0.00
Undergraduate Students
$0
4.6%
$0
$0
$0
0.00
Secretarial/Clerical
$0
48.5%
$0
$0
$0
0.00
Non-Student Hourly
$0
12.0%
$0
$0
$0
Select one
$0
0.0%
$0
$0
$0
Select one
$0
0.0%
$0
$0
$0
Select one
$0
0.0%
$0
$0
$0
Total Other Personnel
$77,676
Total Other Personnel
Year 1 Totals
Fringes
$11,676
Salaries
$66,000
Worksheet for Project Budget - Salaries & Wages
Year 2
Senior Personnel
Prefix
Middle
Init.
First Name
Last Name
Classification
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
2
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
3
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
4
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
5
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
6
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
7
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
8
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Senior Personnel
$0
Total Senior Personnel
Other Personnel
Number of
Personnel
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1.00
Post Doctoral Associate
$3,500
12.00
0.00
0.00
$42,000
20.2%
$8,484
$50,484
$0
$0
1.00
Graduate Students
$2,000
12.00
0.00
0.00
$24,000
13.3%
$3,192
$27,192
$0
$0
0.00
Undergraduate Students
$0
0.00
0.00
0.00
$0
4.6%
$0
$0
$0
$0
0.00
Secretarial/Clerical
$0
0.00
0.00
0.00
$0
48.5%
$0
$0
$0
$0
0.00
Non-Student Hourly
$0
0.00
0.00
0.00
$0
12.0%
$0
$0
$0
$0
0.00
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0.00
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0.00
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Other Personnel
$77,676
Year 2 Totals
Fringes
$11,676
Salaries
$66,000
Total Other Personnel
Worksheet for Project Budget - Salaries & Wages
Year 3
Senior Personnel
Prefix
Middle
Init.
First Name
Last Name
Classification
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
2
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
3
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
4
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
5
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
6
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
7
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
8
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Senior Personnel
$0
Total Senior Personnel
Other Personnel
Number of
Personnel
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1.00
Post Doctoral Associate
$3,500
12.00
0.00
0.00
$42,000
20.2%
$8,484
$50,484
$0
$0
1.00
Graduate Students
$2,000
12.00
0.00
0.00
$24,000
13.3%
$3,192
$27,192
$0
$0
0.00
Undergraduate Students
$0
0.00
0.00
0.00
$0
4.6%
$0
$0
$0
$0
0.00
Secretarial/Clerical
$0
0.00
0.00
0.00
$0
48.5%
$0
$0
$0
$0
0.00
Non-Student Hourly
$0
0.00
0.00
0.00
$0
12.0%
$0
$0
$0
$0
0.00
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0.00
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0.00
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Other Personnel
$77,676
Year 3 Totals
Fringes
$11,676
Salaries
$66,000
Total Other Personnel
Worksheet for Project Budget - Salaries & Wages
Year 4
Senior Personnel
Prefix
Middle
Init.
First Name
Last Name
Classification
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
2
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
3
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
4
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
5
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
6
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
7
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
8
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Senior Personnel
$0
Total Senior Personnel
Other Personnel
Number of
Personnel
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
0.00
Post Doctoral Associate
$0
0.00
0.00
0.00
$0
20.2%
$0
$0
$0
$0
0.00
Graduate Students
$0
0.00
0.00
0.00
$0
13.3%
$0
$0
$0
$0
0.00
Undergraduate Students
$0
0.00
0.00
0.00
$0
4.6%
$0
$0
$0
$0
0.00
Secretarial/Clerical
$0
0.00
0.00
0.00
$0
48.5%
$0
$0
$0
$0
0.00
Non-Student Hourly
$0
0.00
0.00
0.00
$0
12.0%
$0
$0
$0
$0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Other Personnel
$0
Year 4 Totals
Fringes
$0
Salaries
$0
Total Other Personnel
Worksheet for Project Budget - Salaries & Wages
Year 5
Senior Personnel
Prefix
Middle
Init.
First Name
Last Name
Classification
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
1
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
2
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
3
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
4
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
5
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
6
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
7
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
8
0
0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Senior Personnel
$0
Total Senior Personnel
Other Personnel
Number of
Personnel
Base Salary/
Month
Cal Month
Acad.
Month
Summer
Month
Requested
Salary
Fringe Rate
%
Fringe Benefit $
Federal Fund
Requested $
Cost-Shared /
Matching Salary
Fringe Benefit $
0.00
Post Doctoral Associate
$0
0.00
0.00
0.00
$0
20.2%
$0
$0
$0
$0
0.00
Graduate Students
$0
0.00
0.00
0.00
$0
13.3%
$0
$0
$0
$0
0.00
Undergraduate Students
$0
0.00
0.00
0.00
$0
4.6%
$0
$0
$0
$0
0.00
Secretarial/Clerical
$0
0.00
0.00
0.00
$0
48.5%
$0
$0
$0
$0
0.00
Non-Student Hourly
$0
0.00
0.00
0.00
$0
12.0%
$0
$0
$0
$0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
0
0
Select one
$0
0.00
0.00
0.00
$0
0.0%
$0
$0
$0
$0
Total Other Personnel
$0
Year 5 Totals
Fringes
$0
Salaries
$0
Cumulative Totals - All Years
Fringes
$35,028
Salaries
$198,000
Total Other Personnel
rate is based on the
al Senior Personnel
tal Other Personnel
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
al Senior Personnel
tal Other Personnel
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
al Senior Personnel
tal Other Personnel
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
al Senior Personnel
tal Other Personnel
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
al Senior Personnel
tal Other Personnel
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
Cost-Shared /
Matching Funds
$
$0
$0
$0
$0
$0
$0
$0
$0
$0
Scroll to the Right to enter Cost
Sharing amounts.
Worksheet for Project Budget - Equipment, Travel, Participant/Trainee Support Costs
Equipment Description
Year 1
Year 2
Year 3
Year 4
Year 5
Year 1
Year 2
Year 3
Year 4
Year 5
Funds
Requested
Funds
Requested
Funds
Requested
Funds
Requested
Funds
Requested
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
List items and dollar amount for each item exceeding $5,000
Equipment item
1
Fluroescent Microscope and supplies
$30,000
2
3
4
5
6
7
8
9
10
Total funds requested for all eqiupment listed
$30,000
Travel
1
Domestic Travel Costs (incl. Canada, Mexico and U.S. Possessions
$1,000
$1,000
$1,000
2
Foreign Travel Costs
$2,000
$2,000
$2,000
$3,000
$3,000
$3,000
Total Travel Cost
Participant/Trainee Support Costs (for NSF only)
1
Tuition/Fees/Health Insurance
2
Stipend
3
Travel
4
Subsistence
5
Other
Total Participant/Trainee Support Costs
# of Participants/Trainees
Running Total for Cumulative Budget Amount =
$
482,789.17
Worksheet for Project Budget - Equipment, Travel, Participant/Trainee Support Costs
Other Direct Costs
Year 1
Funds
Requested
1
Materials and Supplies
2
Publication Costs
3
Consultant Services
4
ADP/Computer Services
5
Subawards/Consortium/Contractual Costs
6
Equipment or Facility Rental/User Fees
7
Alterations and Renovations
8
Graduate Student Tuition
Year 2
Funds
Requested
Year 3
Funds
Requested
Year 4
Funds
Requested
Year 5
Funds
Requested
$20,000
$20,600
$21,218
$1,500
$1,545
$1,591
$10,000
$5,000
$1,000
$1,030
$1,061
$9,482
$10,043
$10,133
Summer
Summer 2011
Summer 2012
Summer 2012
Summer 2013
Summer 2014
Fall
Fall 2012
Fall 2013
Fall 2012
Fall 2013
Fall 2014
Spring
Spring 2012
Spring 2013
Spring 2012
Spring 2013
Spring 2014
Summer
Summer 2011
Summer 2012
Summer 2013
Summer 2013
Summer 2014
Fall
Fall 2011
Fall 2013
Fall 2013
Fall 2013
Fall 2014
Spring
Spring 2012
Spring 2013
Spring 2013
Spring 2013
Spring 2014
Summer
Summer 2010
Summer 2011
Summer 2012
Summer 2013
Summer 2014
Fall
Fall 2010
Fall 2011
Fall 2012
Fall 2013
Fall 2014
Spring
Spring 2010
Spring 2011
Spring 2012
Spring 2013
Spring 2014
Summer
Summer 2010
Summer 2011
Summer 2012
Summer 2013
Summer 2014
Fall
Fall 2010
Fall 2011
Fall 2012
Fall 2013
Fall 2014
Spring
Spring 2010
Spring 2011
Spring 2012
Spring 2013
Spring 2014
Total Other Direct Costs
$41,982
$38,218
$34,003
Total All Direct Costs
$152,658
$118,894
$114,679
select 1/4-time/1/2-time 1/2-time
>>
NON-Engineering Students Only
Year 2
Year 3
Year 4
Year 5
PhD Students
(enter no. of
students per term)
Masters
Students (enter
no. of students
per term)
Year 1
1
1
1
1
1
1
1
1
1
Engineering Students Only
8
Year 2
Year 3
Year 4
Year 5
Masters
Students (enter
no. of students
per term)
Year 1
PhD Students
(enter no. of
students per term)
Year 1
9
other
10
other
Year 2
Year 3
Year 4
Year 5
Year 1
Year 2
Year 3
Year 4
Year 5
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Replace "Subcontract #" with the
name of the proposed subcontract
recipient.
Scroll to the Right to enter Cost
Sharing amounts.
Worksheet for Project Budget - Subcontracts
For each subcontract, you will need to submit the following with the GoldSheet:
1. Transmittal letter from the Sponsored Research Office: http://www.ospa.iastate.edu/proposal/docs/TransmittalLetter.doc
2. Statement of Work
3. Separate subcontractor's budget
4. Budget Justification
If you have questions regarding subcontracts, please contact Tammy Polaski, 515-294-5225.
Year 1
Funds
Requested
Subcontract 1 - Direct Costs
Subcontract 1 - Indirect Costs
Subcontract 2 - Direct Costs
Subcontract 2 - Indirect Costs
Subcontract 3 - Direct Costs
Subcontract 3 - Indirect Costs
Subcontract 4 - Direct Costs
Subcontract 4 - Indirect Costs
Subcontract 5 - Direct Costs
Subcontract 5 - Indirect Costs
Subcontract 6 - Direct Costs
Subcontract 6 - Indirect Costs
Subcontract 7 - Direct Costs
Subcontract 7 - Indirect Costs
Subcontract 8 - Direct Costs
Subcontract 8 - Indirect Costs
Subcontract 9 - Direct Costs
Subcontract 9 - Indirect Costs
Subcontract 10 - Direct Costs
Subcontract 10 - Indirect Costs
Total All Direct Costs
Total All Indirect Costs
Total All Subcontract Costs
Year 2
Funds
Requested
Year 3
Funds
Requested
Year 4
Funds
Requested
Year 5
Funds
Requested
Year 1
Year 2
Year 3
Year 4
Year 5
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Cost-Sharing /
Matching Funds
Worksheet for Project Budget - Cumulative Budget
A
Senior/Key Personnel
B
Other Personnel
Total Number other Personnel
Year 1
Year 1
Year 2
Year 2
Year 3
Year 3
Year 4
Year 4
Year 5
Year 5
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$77,676
$0
$77,676
$0
$77,676
$0
$0
$0
$0
$0
2.00
2.00
2.00
0.00
Total
Total
Total
Funds Requested
Cost-Sharing /
Matching Funds
Cost of Project (all
sources)
$0
$0
$0
$233,028
$0
$233,028
0.00
Total Salary, Wages and Fringe Benefits
$77,676
$0
$77,676
$0
$77,676
$0
$0
$0
$0
$0
$233,028
$0
$233,028
C
Equipment
$30,000
$0
$0
$0
$0
$0
$0
$0
$0
$0
$30,000
$0
$30,000
D
Travel
$3,000
$0
$3,000
$0
$3,000
$0
$0
$0
$0
$0
$9,000
$0
$9,000
Domestic
$1,000
$0
$1,000
$0
$1,000
$0
$0
$0
$0
$0
$3,000
$0
$3,000
Foreign
$2,000
$0
$2,000
$0
$2,000
$0
$0
$0
$0
$0
$6,000
$0
$6,000
E
Participant/Trainee Support Costs
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
1
Tuition/Fees/Health Insurance
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
2
Stipends
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
3
Travel
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
4
Subsistence
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
5
Other
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
6
Number of Participants/Trainees
F
Other Direct Costs
$41,982
$0
$38,218
$0
$34,003
$0
$0
$0
$0
$0
$114,203
$0
$114,203
1
Materials and Supplies
$20,000
$0
$20,600
$0
$21,218
$0
$0
$0
$0
$0
$61,818
$0
$61,818
2
Publication Costs
$1,500
$0
$1,545
$0
$1,591
$0
$0
$0
$0
$0
$4,636
$0
$4,636
3
Consultant Services
$10,000
$0
$5,000
$0
$0
$0
$0
$0
$0
$0
$15,000
$0
$15,000
4
ADP/Computer Services
$1,000
$0
$1,030
$0
$1,061
$0
$0
$0
$0
$0
$3,091
$0
$3,091
5
Subawards/Consortium/Contractual Costs
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
6
Equipment or Facility Rental/User Fees
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
7
Alterations and Renovations
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
8
Graduate Student Tuition
$9,482
$0
$10,043
$0
$10,133
$0
$0
$0
$0
$0
$29,658
$0
$29,658
9
other
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
10
other
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$152,658
$0
$118,894
$0
$114,679
$0
$0
$0
$0
$0
$386,231
$0
$386,231
$38,165
$0
$29,723
$0
$28,670
$0
$0
$0
$0
$0
$96,558
$0
$96,558
$190,823
$0
$148,617
$0
$143,349
$0
$0
$0
$0
$0
$482,789
$0
$482,789
Percent
Requested
Percent CostShared
100.00%
0.00%
G
Direct Costs
H
Indirect Costs
I
Total Direct and Indirect Costs
J
Fee
0.00
Rate
25.00%
Base Amount > >
If this is an NIH Modular application, this is your modular amount
each year (must be in multiple of $25,000) >>
Salary
Fringe
$
152,658
0.00
$
-
0.00
$
118,894
0.00
$
-
0.00
$
114,679
0.00
$
-
0.00
$
-
0.00
$
-
0.00
$
-
0.00
$
-
$152,658
$118,894
$114,679
$0
$0
$386,231
$66,000
$11,676
$66,000
$11,676
$66,000
$11,676
$0
$0
$0
$0
$198,000
$35,028
100.00%