The complete genomic sequence of American hop latent virus (AHLV; genus Carlavirus) was determined. The genome consists of 8,601 nucleotides plus a 3′-polyadenylate tail. The genome encompasses six potential open reading frames (ORF) in the positive sense, and their organization is typical of other carlaviruses. Analysis of the coat protein coding sequence at both the nucleic acid level and the amino acid level indicates that AHLV is only remotely related to the other carlaviruses known to infect common hop. Polyclonal antibodies were produced against the bacterially expressed coat protein of AHLV. These antibodies differentiated between AHLV and other carlaviruses of hop

A Gramstat

AN Adams

AT Segwagwe

DB Dunn

G Probasco

GD Foster

KC Eastwell

Kenneth C. Eastwell

Keri L. Druffel

MJ Adams

SJ Pethybridge

English

PubMed

Springer - Publisher Connector

Complete genome organization of American hop latent virus and its relationship to carlaviruses

Crossref

Archives of Virology

ANNOTATED SEQUENCE RECORD
Complete genome organization of American hop latent virus
and its relationship to carlaviruses
Kenneth C. Eastwell • Keri L. Druffel
Received: 9 December 2011 / Accepted: 17 March 2012 / Published online: 17 April 2012
 The Author(s) 2012. This article is published with open access at Springerlink.com
Abstract The complete genomic sequence of American
hop latent virus (AHLV; genus Carlavirus) was determined.
The genome consists of 8,601 nucleotides plus a 30-poly-
adenylate tail. The genome encompasses six potential open
reading frames (ORF) in the positive sense, and their orga-
nization is typical of other carlaviruses. Analysis of the coat
protein coding sequence at both the nucleic acid level and the
amino acid level indicates that AHLV is only remotely
related to the other carlaviruses known to infect common
hop. Polyclonal antibodies were produced against the bac-
terially expressed coat protein of AHLV. These antibodies
differentiated between AHLV and other carlaviruses of hop.
American hop latent virus (AHLV) (order Tymovirales,
family Betaflexiviridae, genus Carlavirus) [1] is one of
three carlaviruses that infect hop plants (Humulus lupulus
L.). Mixed infections in hop of AHLV with other carlavi-
ruses hop mosaic virus (HpMV) and hop latent virus
(HpLV) are common [8], even in plants that do not express
overt symptoms. All three of the carlaviruses known to
infect hop are transmitted in a non-persistent manner by the
damson hop aphid, Myzus (Phorodon) humuli, and it is
presumed that this is the major route of natural
transmission in the field [2]. Although natural spread in
commercial hop plantings can be quite rapid, experimental
aphid transmission of these carlaviruses is inefficient. In
spite of the shared insect vectors, surveys of hop plantings
indicate that AHLV generally occurs at a lower frequency
than the other carlaviruses [8].
AHLV was first detected in commercial hops growing in
North America [10] in plants that were also infected with
other carlaviruses; AHLV was subsequently differentiated
and further characterized in Europe in 1982 [2]. A com-
parative study examined the effects of HpMV, HpLV and
AHLV on the cultivar Chinook [9]. AHLV reduced cone
yield by 14 %, resulting in a reduction in the total yield of
alpha acids by 12 %. A significant but slight impact was
recorded on other agronomic traits such as plant height and
lateral length. In general, the consequences of infection by
carlaviruses on hop cone brewing characteristics appear to
mimic changes associated with advanced maturity. The
objective of the study reported herein is to develop a
greater knowledge of the viruses infecting hop plants.
Initial cloning and sequencing of AHLV was performed
from partially purified virus from Chenopodium quinoa
mechanically inoculated with a virus isolate from H. lupulus
‘Galena’. After a preliminary genome sequence was estab-
lished for the virus maintained in C. quinoa (GenBank
JQ728538), the complete genomic sequence of AHLV was
derived directly from H. lupulus ‘Bittergold’. AHLV was
enriched from hop leaf extracts by treatment with bentonite
followed by polyethylene glycol precipitation [4]. Viral
RNA was precipitated after proteinase K digestion of the
partially purified virions [11]. The complete genome was
cloned and sequenced using a combination of cDNA cloning
and 50-RACE (Invitrogen, Carlsbad, CA) as described pre-
viously [5], and the sequence was deposited in the public
database (GenBank JQ245696).
The nucleotide sequences presented in this report were deposited in
the GenBank database as accessions JQ245696 and JQ728538.
K. C. Eastwell (&)
Department of Plant Pathology, Washington State University,
IAREC, 24106 North Bunn Road, Prosser, WA 99350, USA
e-mail: keastwell@wsu.edu
K. L. Druffel
Department of Plant Pathology, Washington State University,
P.O. Box 646430, Pullman, WA 99164-6430, USA
123
Arch Virol (2012) 157:1403–1406
DOI 10.1007/s00705-012-1312-0
The complete coat protein coding sequence was ampli-
fied from ‘Bittergold’ using primers AHLVcpF: 50-CCT
TTTTTTTTCCCATATGTCGAACGTTGAAAGG-30and
AHLVcpR: 50-TTATGGATCCTCAGTGCGCTTGTCGA
AACTC-30. The primers were extended (underlined
sequence) to allow digestion of the amplified product with
NdeI and BamHI and it was then ligated into plasmid
pET15b (Novagen: San Diego, CA). E. coli RosettaTM
 HpLV
 HpMV
 AcLV
 PotVM
 NCLV
 CPMMV
 DVS
 CVB
 PhlVB
 PhlVS
 PotLV
 AHLV
 PVS
 PVP
 LNRSV
 BlScV
 HdCMV
 LSV
 MjMV
 PLV
 KLV
 PopMV
 HeNNV-J
 HeNNV-G5
 RCVMV
 GarLV
 CVNV
 ButMV
 SPCFV
 NSV
 ASPV
 AOPRV
100
100
100
95
100
99
98
100
100
87
98
90
99
99
85
82
91
71
 NCLV
 PVM
 AcLV
 HpMV
 HpLV
 CapLV
 CPMMV
 DVS
 CVB
 PhlVB
 HVS
 PhlVS
 PotLV
 AHLV
 LNRSV
 PVP
 PVS
 HdCMV
 BlScV
 LSV
 PLV
 KLV
 MjMV
 SPCFV
 PopMV
 HeMV
 NSV
 NeLV
 HeNNV-G5
 HeNNV-J
 ButMV
 CLV
 CVNV
 GarCLV
 PeSV
 RCVMV
 GarLV
 SLV
 AOPRV
 ASPV
100
100
64
85
98
83
85
98
98
98
98
73
78
80
92
69
62
94
85
63
65
98
A B
Fig. 1 Cladogram of the predicted amino acid sequences from the
replicase (A), and coat protein (B) of members of the genus
Carlavirus. The phylogenetic tree was generated using the maxi-
mum-likelihood method in the MEGA5 analysis package. Branch
significance was evaluated by constructing 1,000 trees in bootstrap
analysis, and the bootstrap values ([60 %) are shown. The sequences
used were derived from the genomic sequences of Aconitum latent
virus (AcLV, gi: 14251190), American hop latent virus (AHLV, gi:
JQ245696), blueberry scorch virus (BlScV, gi: 19919919), butterbur
mosaic virus (ButMV, gi: 269954638), chrysanthemum virus B
(CVB, gi: 154814487), coleus vein necrosis virus (CVNV, gi:
156616260), cowpea mild mottle virus (CPMMV, gi: 313122763),
daphne virus S (DVS, gi: 94971809), garlic latent virus (GarLV, gi:
20143432), Helleborus net necrosis virus (HeNNV-G5, gi:
222509601 and HeNNV-J, gi: 349584640), hop latent virus (HpLV,
gi: 10314012), hop mosaic virus (HpMV, gi: 171473627), hydrangea
chlorotic mottle virus (HdCMV, gi: 242346753), Kalanchoe latent
virus (KLV, gi: 254667440), Ligustrum necrotic ringspot virus
(LNRSV, gi: 166851938), lily symptomless virus (LSV, gi:
37674199), Mirabilis jalapa mottle virus (MjMV, gi: 352749693),
narcissus common latent virus (NCLV, gi: 110835685), narcissus
symptomless virus (NSV, gi: 116734779), Passiflora latent virus
(PLV, gi: 113195533), phlox virus B (PhlVB, gi: 160700642), phlox
virus S (PhlVS, gi: 145651764), poplar mosaic virus (PopMV, gi:4
1057345), potato latent virus (PLV, gi: 211998869), potato virus M
(PVM, gi: 72257063), potato virus P (PVP, gi: 156603882), potato
virus S (PVS, gi: 71849435), red clover vein mosaic virus (RCVMV,
gi: 224923880), and sweet potato chlorotic fleck virus (SPCFV, gi:
56692800). In addition, the following carlaviruses were included in
the analysis of coat protein sequences: caper latent virus (CapLV, gi:
333034471), cole latent virus (CLV, gi: 33517421), garlic common
latent virus (GarCLV, gi: 3077682), Helenium virus S (HVS, gi:
221446), Helleborus mosaic virus (HeMV, gi: 222142735), Nerine
latent virus (NeLV, gi: 298504072), pea streak virus (PeSV, gi:
13811416), shallot latent virus (SLV, gi: 3077693). The correspond-
ing sequences of apple stem pitting virus (ASPV, gi: 19744938) and
African oil palm ringspot virus (AOPRV, gi: 226202432) were used
as out-groups
1404 K. C. Eastwell, K. L. Druffel
123
competent cells (Novagen) were transformed with the
plasmids, and proteins were expressed following the
manufacturer’s recommendations. Fusion proteins pro-
duced with the N-terminal His-tag were isolated and con-
centrated using a Ni column (ProBondTM Resin;
Invitrogen) according to manufacturer’s instructions. New
Zealand white rabbits were immunized with four injections
of 400 lg each of the resulting protein, and antibodies were
evaluated by indirect ELISA with absorbance values
determined two hours after substrate addition.
The complete genome of AHLV from ‘Bittergold’
consists of 8,601 nucleotides (nt) plus a poly-adenylated
tract at the 30-terminus. The genome contains six potential
open reading frames (ORFs) organized in a pattern typical
of members of the genus Carlavirus. ORF1 spans nt
positions 82 to 6015 and encodes a protein of 1,977 amino
acids (aa) with a predicted molecular mass of 224.4 kDa.
This corresponds to the largest protein produced through in
vitro translation of isolated virus RNA [6]. The predicted
protein contains domains characteristic of virus replicases,
including a viral methyl-transferase domain (aa residues 43
to 359) involved in mRNA capping. A 50 cap structure was
not detected by the methods used in this study but is
inferred by the similarity of AHLV to other members of the
genus Carlavirus and by in vitro translation data [6]. Other
signature conserved domains include the RNA-dependent
polymerase family domain (aa residues 1589 to 1967), a
carlavirus-specific endopeptidase (aa residues 998 to
1086), and a viral RNA helicase (aa 1172 to 1300). The
replicase polyfunctional protein of AHLV shows 36 to
42 % identity to nt sequences and 35 to 48 % identity to aa
sequences of the known carlaviruses (Fig. 1A). This is
consistent with the position of AHLV as a member of a
distinct species within the genus Calarvirus [1].
Overlapping ORFs 2, 3 and 4 span nt positions 6068 to
6772, 6741 to 7061 and 7034 to 7234, respectively, and
encode proteins of 234 aa (26.2 kDa), 106 aa (11.3 kDa)
and 66 aa (7.3 kDa) with homology to the triple gene block
proteins of virus superfamily 1.
ORF5 of the AHLV genome encodes a capsid protein
with a molecular mass of 36.0 kDa based on the predicted
aa sequence. This mass is consistent with a protein
produced in earlier wheat germ in vitro translation exper-
iments and precipitated by virion-specific antibodies [6].
The molecular mass of the AHLV coat protein is within the
range of 31–36 kDa anticipated for members of the genus
Carlavirus and contains two highly conserved domains
typical of carlaviruses and potexviruses. Antibodies pro-
duced to the bacterially-expressed coat protein of AHLV
reacted preferentially with AHLV-infected hop leaf
extracts (3.6 times above non-infected hop tissue), while
there was no significant reaction to HpLV or HpMV
(1.2 times above non-infected hop samples). This is similar
to serological data produced with antibodies against puri-
fied virions [3]. Whereas HpLV and HpMV share distant
antigenic similarities with each other and with many other
carlaviruses, AHLV is only remotely serologically related
to other carlaviruses, if at all [3]. This distinction is
reflected by phylogenetic analysis of the coat protein
sequences from carlaviruses (Fig. 1B). The predicted aa
sequences of the coat proteins of HpMV and HpLV share
66 % identity with each other, but only 37 % and 38 %
identity to AHLV, respectively. Moreover, the coat protein
of AHLV contains 323 aa, while those of HpMV and
HpLV contain 307 and 306 aa, respectively. When com-
pared to the coat proteins of other carlaviruses, the coat
protein of AHLV shows 14 to 47 % nt sequence identity
and 26 to 49 % aa identity.
There is no current information regarding possible
strains of AHLV. However, the two AHLV isolates
determined in this study share overall sequence identity of
98 % at the nt level. The coat protein coding sequences
share 99 % nt identity and 98 % aa identity.
ORF6 of AHLV encodes a 114-aa protein (13.1 kDa)
with homology to the carlavirus putative nucleic-acid-
binding protein, including the highly conserved cysteine
residue motif, consistent with the presence of a nucleic-
acid-binding zinc finger [7].
These data clearly demonstrate the position of AHLV as
a member of a unique species of the genus Carlavirus [1]
and reflect the biological data that suggest that it is distantly
related to the other carlaviruses associated with hop plants.
This study provides the foundation of further investigation
into the differentiation of AHLV into strains and their role
in the pathology of disease associated with hop.
Acknowledgments This study was supported in part by the
Department of Plant Pathology, College of Agricultural, Human, and
Natural Resource Sciences Agricultural Research Center Project No.
WNP00754, Washington State University, Pullman, WA,
99164-6240. Antibody production was performed under I.A.C.U.C.
protocol ASAF #03744. Funding from the Hop Research Council and
the Washington Hop Commission is gratefully acknowledged. Pre-
submission review and comments by Dr. Tefera Mekuria are
appreciated.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
References
1. Adams MJ, Candresse T, Hammond J, Kreuze JF, Martelli GP,
Namba S, Pearson MN, Ryu KH, Saldarelli P, Yoshikawa N
(2012) Family Betaflexiviridae. In: King AMQ, Adams MJ,
Carstens EB, Lefkowitz EJ (eds) Virus taxonomy: ninth report of
the international committee on taxonomy of viruses. Elsevier
Academic Press, London, pp 920–941
Complete genome organization of American hop latent virus 1405
123
2. Adams AN, Barbara DJ (1982) Host range, purification and some
properties of two carlaviruses from hop (Humulus lupulus): hop
latent and American hop latent. Ann Appl Biol 101:483–494
3. Adams AN, Barbara DJ (1983) The use of F(ab0)2-based ELISA
to detect serological relationships among carlaviruses. Ann Appl
Biol 101:495–500
4. Dunn DB, Hitchborn JH (1965) The use of bentonite in the
purification of plant viruses. Virology 25:171–192
5. Eastwell KC, Dutoit LJ, Druffel KL (2009) Helleborus net
necrosis virus: a new Carlavirus associated with ‘black death’ of
Helleborus spp. Plant Dis 93:332–338
6. Foster GD, Mills PR (1991) Cell-free translation of American
hop latent virus RNA. Virus Genes 5:327–334
7. Gramstat A, Courtpozanis A, Rohde W (1990) The 12 kDa
protein of potato virus M displays properties of a nucleic acid-
binding regulatory protein. FEBS Lett 276:34–38
8. Pethybridge SJ, Nelson ME, Eastwell KC, Klein RE, Kenny ST,
Wilson CR (2001) Incidence, spatial distribution, and associa-
tions of viruses in hop gardens of Washington State. Plant Dis
86:661–665
9. Probasco G, Murphey JM (1996) The effects of hop viruses on
brewing and agronomic characteristics in the hop variety
Chinook. MBAA Tech Quart 33:160–165
10. Probasco G, Skotland CB (1976) Host range and general prop-
erties of the third rod-shaped virus found in hops. Proc Am
Phytopath Soc 3:319
11. Segwagwe AT, Putnam ML, Druffel KL, Pappu HR, Eastwell KC
(2008) Molecular characterization of a new tymovirus from
Diascia ornamental plants. Arch Virol 153:1495–1503
1406 K. C. Eastwell, K. L. Druffel
123


Cell-free translation of American hop latent virus RNA. Virus Genes 5:327–334

Family Betaﬂexiviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy: ninth report of the international committee on taxonomy of viruses.

Helleborus net necrosis virus: a new Carlavirus associated with ‘black death’ of Helleborus spp.

Host range and general properties of the third rod-shaped virus found in hops.

Host range, puriﬁcation and some properties of two carlaviruses from hop (Humulus lupulus): hop latent and American hop latent.

Incidence, spatial distribution, and associations of viruses in hop gardens of Washington State.

Molecular characterization of a new tymovirus from Diascia ornamental plants.

The 12 kDa protein of potato virus M displays properties of a nucleic acidbinding regulatory protein.

The effects of hop viruses on brewing and agronomic characteristics in the hop variety Chinook.

The use of bentonite in the puriﬁcation of plant viruses.

The use of F(ab0)2-based ELISA to detect serological relationships among carlaviruses.

file:///data/remote/core/dit/data/Springer-OA/pdf/01e/aHR0cDovL2xpbmsuc3ByaW5nZXIuY29tLzEwLjEwMDcvczAwNzA1LTAxMi0xMzEyLTAucGRm.pdf

Complete genome organization of American hop latent virus and its relationship to carlaviruses

Abstract

Similar works

Full text

Available Versions

Springer - Publisher Connector

Crossref

Springer - Publisher Connector