Presence of a Shared 5'-Leader Sequence in Ancestral Human and Mammalian Retroviruses and

50 Pages·2017·6.86 MB·English

JVI Accepted Manuscript Posted Online 2 August 2017
J. Virol. doi:10.1128/JVI.00829-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Presence of a Shared 5ʹ-Leader Sequence in Ancestral Human and Mammalian
2 Retroviruses and its Transduction into Feline Leukemia Virus.
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4 Junna Kawasaki1, Maki Kawamura2, Yoshiharu Ohsato3, Jumpei Ito1,4 and Kazuo
5 Nishigaki1,2, #
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7 1Laboratory of Molecular Immunology and Infectious Disease, Joint Faculty of a
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8 Veterinary Medicine, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515,
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9 Japan

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10 2Laboratory of Molecular Immunology and Infectious Disease, The United Graduate tp
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11 School of Veterinary Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi i.
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12 753-8515, Japan
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13 3Kahotechno Co., Ltd, 680-41 Iizuka, Fukuoka 820-0067, Japan g
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14 4Division of Human Genetics, Department of Integrated Genetics, National Institute of
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15 Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan u
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17 #Address correspondence to Kazuo Nishigaki, [email protected] 0
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18 b
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19 Running head: Shared sequence among ERVs and recombined exRV u
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21 Abstract: [#] 242 words + 149 words
22 Full text: [#] 10511 words
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25 Abstract
26 Recombination events induce significant genetic changes, and this process can result in
27 virus genetic diversity or in the generation of novel pathogenicity. We discovered a new
28 recombinant feline leukemia virus (FeLV) gag gene harboring an unrelated insertion,
29 termed the “X-region”, which was derived from FcERV-gamma4, a feline endogenous
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30 gammaretrovirus. The identified FcERV-gamma4 proviruses have lost their coding
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31 capability, but some can express their viral RNA in feline tissues. Although the a
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32 X-region-carrying recombinant FeLVs appeared to be replication-defective viruses, they
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33 were detected in 6.4% of tested FeLV-infected cats. All isolated recombinant FeLV

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34 clones commonly incorporated a middle part of FcERV-gamma4 5′-leader region as an p
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35 X-region. Surprisingly, a sequence corresponding to the portion contained in all i.
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36 X-regions is also present in at least 13 endogenous retroviruses (ERVs) observed in the
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37 cat, human, primate, and pig genomes. We termed this shared genetic feature the g
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38 “commonly-shared sequence” (CS-sequence). Despite our phylogenetic analysis
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39 indicating that all CS-sequence-carrying ERVs are classified as gammaretroviruses, no u
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40 obvious closeness was revealed among these ERVs. However, the Shannon entropy in
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41 the CS-sequence was lower than that in other parts of the provirus genome. Notably, the 0
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42 CS-sequence of human endogenous retrovirus T had 73.8% similarity with that of b
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43 FcERV-gamma4, and specific signals were detected in the human genome by southern u
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44 blot analysis using a probe for the FcERV-gamma4 CS-sequence. Our results provide an
45 interesting evolutionary history for CS-sequence circulation among several distinct
46 ancestral viruses and a novel recombined virus over a prolonged period.
47
48 Importance
49 Recombination among ERVs or modern viral genomes causes a rapid evolution of
50 retroviruses, and this phenomenon can result in the serious situation of viral disease
51 re-emergence. We identified a novel recombinant FeLV gag gene that contains an
52 unrelated sequence, termed the X-region. This region originated from 5ʹ-leader of
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53 FcERV-gamma4, a replication-incompetent feline ERV. Surprisingly, a sequence
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54 corresponding to the X-region is also present in the 5ʹ-portion of other ERVs, including a
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55 human endogenous retroviruses. Scattered copies of the ERVs carrying the unique
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56 genetic feature, here named the “commonly-shared sequence” (CS-sequence), were

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57 found in each host genome, suggesting that ancestral viruses may have captured and p
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58 maintained the CS-sequence. More recently, a novel recombinant FeLV hijacked the i.
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59 CS-sequence from inactivated FcERV-gamma4 as the X-region. Therefore, tracing the
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60 CS-sequences can provide unique models for not only the modern reservoir of new g
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61 recombinant viruses but also for the genetic features shared among ancient retroviruses.
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62 u
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63 Introduction
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64 Retroviruses are classified into exogenous retroviruses (exRVs) and endogenous 0
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65 retroviruses (ERVs) according to their manner of viral transmission. ExRVs are b
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66 horizontally transmitted among their hosts and induce malignant diseases in susceptible u
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67 hosts. In contrast, ERVs are remnants of ancestral retroviral infections of germ cells that
68 have thereafter been transmitted vertically from parent to offspring according to
69 Mendelian inheritance patterns (1, 2). ERVs occupy significant fractions of animal
70 genomes, for example 8–10% each of the human and mouse genomes (3, 4).
71 Paleovirology deals with the multistep events that make up the evolutionary
72 history of ERVs (5–7). Because of accumulated mutations or internal recombinations
73 that result in a solo long terminal repeat (LTR), almost all ERVs are inactivated;
74 however, the neutralization strategies and strengths among proviruses are diverse. For
75 example, some recently acquired ERVs retain replication competence and, therefore,
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76 can threaten host existence (8–12). Moreover, interactions among partially inactivated
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77 ERVs or modern viral genomes that occur through re-assortment or recombination a
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78 result in a rapid evolution of retroviruses, and this phenomenon can cause increasing
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79 genetic diversities or result in the serious occurrence of viral disease re-emergence

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80 (13–16). In contrast, some suitably inactivated ERVs gain a physiological function, p
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81 such as placentation or viral resistance, and eventually evolve into host genes (17, 18), i.
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82 though this phenomenon, known as “domestication”, only occurs infrequently.
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83 Gammaretroviruses can infect the vast majority of mammalian species, including g
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84 mice (11), pigs (19–22), koalas (12, 23), bats (24), and humans (25), and the abundant
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85 ERV copy numbers suggest the occurrence of repeated invasions into host genomes (26). u
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86 Comparative analyses using these many copies of “fossil” records containing human
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87 endogenous retroviruses (HERVs), which were identified from various mammalian 0
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88 species, help reconstruct the evolutionary history of ancient exRVs, such as retrovirus b
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89 cross-species transmission (27, 28). u
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90 Gammaretrovirus elements possess a simple genome consisting of the 5′-LTR, gag,
91 pol, env, 3′-LTR, and 5′-leader. The 5′-leader region is defined as the region between the
92 primer binding site (PBS) and the AUG codon of the gag gene, and this region regulates
93 essential steps for viral replication through encoding some universal signals, such as the
94 PBS, splicing donor, dimerization initiation signal, and packaging signal (29–31).
95 Moreover, some retrovirus 5′-leaders harbor an internal ribosome entry site (IRES) that
96 promotes the synthesis of viral proteins independently of the 5′-cap structure (32–34).
97 One unique genetic feature of gammaretroviruses is that their 5′-leader sequences
98 are longer than those of other retroviruses. Some gammaretroviruses, such as murine
99 leukemia virus (MLV) (35), feline leukemia virus (FeLV) (36), and koala retrovirus (23),
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100 encode additional open reading frames (ORFs) termed “glycoGag”. These additional
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101 ORFs are translated via CUG in the 5′-leader, and enhance the infectivity of the viruses a
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102 (35, 37–40).
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103 FeLV is an exRV belonging to the genus Gammaretrovirus. A recent

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104 epidemiological survey of FeLV infection in Japan detected FeLV in 12.2% of the 1,770 p
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105 tested cats (41). This virus is known to induce various pathologies in domestic cats i.
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106 (Felis silvestris catus), such as lymphoma, myelodysplastic syndrome, anaplastic
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107 anemia, acute myelogenous leukemia, and immune deficiency (42, 43). The g
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108 mechanisms by which this virus induces the multifarious symptoms of FeLV-associated
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109 diseases are still unclear; however, the genetic structure polymorphisms resulting from u
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110 substitutions or recombinations have led to changes in FeLV pathogenicity and
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111 unexpected symptoms (8, 44–48). 0
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112 Here, we discovered a novel recombinant FeLV gag gene containing an unrelated b
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113 sequence, termed the X-region. This region is homologous to a portion of the 5ʹ-leader u
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114 sequence of FcERV-gamma4, which is a feline ERV classified as a gammaretrovirus;
115 therefore, to analyze the genetic constitution of the recombinant FeLV, we characterized
116 FcERV-gamma4. Furthermore, during additional screening for sequences analogous to
117 the X-region, we unexpectedly discovered corresponding sequences in the 5ʹ-portions of
118 at least 13 mammalian endogenous gammaretroviruses. Thus, our results indicated an
119 interesting circulation of this unique genetic feature among ancestral retroviruses and a
120 modern recombined virus.
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122 Materials and Methods
123 Samples
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124 Chromosomal DNA for the isolation of FcERV-gamma4 provirus and a subsequent
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125 southern blot analysis was extracted from the blood of a female, specific-pathogen-free a
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126 (SPF) cat (described in previous work and here as Kyoto-SPF) (8). For our southern blot
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127 analysis, we additionally isolated chromosomal DNA from the whole blood of four SPF

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128 cats (cat1, cat2, cat3, and cat4), which were kindly provided by Drs. Hajime Tsujimoto p
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129 (The Tokyo University), Masaharu Hisasue (The Azabu University), Toshihiro Watari i.
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130 (The Nihon University), and Yasuyuki Endo (The Kagoshima University), respectively.
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131 Chromosomal DNA from HEK293T (49, 50), K562 (51), HT1080 (52), AH927 (53), g
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132 and Rat2 (54) cell lines were also extracted and used for the southern blot analysis. The
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133 DNA extractions were carried out as previously described (55). Chromosomal DNA u
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134 from blood samples that were voluntarily supplied by veterinary hospitals in Japan from
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135 FeLV-positive and FeLV-negative cats (8, 41) were also used in this study. Additionally, 0
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136 chromosomal DNAs from FeLV-infected cats were kindly supplied from Kahotechno b
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137 Co., Ltd. (Fukuoka, Japan). u
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138
139 Isolation of FcERV-gamma4 proviruses
140 First, nine loci of the FcERV-gamma4 group were identified from a reference genome of
141 Felis catus (ICGSC Felis_catus 6.2/felcat5) by using the BLAT program implemented
142 in the University of California, Santa Cruz (UCSC) Genome Browser
143 (http://genome.ucsc.edu/) (56). The proviruses were amplified from the chromosomal
144 DNA of a female cat (Kyoto-SPF) by performing PCR with specific primers and the
145 DNA polymerase KOD Fx Neo (Toyobo, Osaka, Japan). They were then cloned into
146 pCR-Blunt II-TOPO vectors (Invitrogen, Carlsbad, CA, USA). A portion of each
147 FcERV-gamma4 isolate from domestic cats that did not have a gamma4-X2 provirus
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148 was amplified by performing PCR with KOD Fx Neo (Toyobo) and primers Fe-406S
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149 and Fe-141R, both of which are specific for FcERV-gamma4 LTRs. The amplicons were a
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150 cloned into pCR-4Blunt-topo vectors (Invitrogen). Details of primers used for the
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151 cloning of nine FcERV-gamma4 proviruses will be provided on request.

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152 p
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153 Southern blotting i.
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154 Each chromosomal DNA sample (10 μg) was digested by BamHI, BglII, EcoRI, KpnI,
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155 and EcoRV because the restriction sites for each of these enzymes were present within g
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156 FcERV-gamma4. Digested DNA was separated by electrophoresis and transferred to a
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157 positively charged nylon membrane (GE Healthcare UK Ltd., Little Chalfont, UK). The u
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158 specific probe for the FcERV-gamma4 env gene was amplified by performing PCR
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159 using Fe-313S and Fe-283R primers and then labeled with digoxigenin (Roche 0
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160 Molecular Biochemicals, Mannheim, Germany). The membranes were hybridized with b
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161 the env probe for 16 h at 42 °C. After hybridization, the membranes were washed with u
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162 0.1% sodium dodecyl sulfate (SDS) and 2× saline sodium citrate (SSC) (low-stringency
163 buffer) at room temperature and then by 0.1% SDS and 1× SSC (high-stringency buffer)
164 at 68 °C. The bound probe was visualized by CSPD Chemiluminescent Substrate
165 (Roche Molecular Biochemicals). Zoo blot analyses were conducted by performing
166 electrophoresis on a total of 15 μg of chromosomal DNA per sample, each of which had
167 been isolated from cell lines and digested with EcoRI. The digoxigenin-labeled specific
168 probe for the FcERV-gamma4 5′-leader was amplified by performing PCR with Fe-86S
169 and Fe-519R primers. The membrane was hybridized at 37 °C for 16 h and washed with
170 the low-stringency buffer at room temperature followed by a wash with the
171 high-stringency buffer at 60 °C.
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172
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173 Investigation of insert polymorphism among FcERV-gamma4 proviruses a
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174 We randomly chose 64 cats with regard to sex, age, and geographical distribution
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175 in Japan and determined the status and the frequency of provirus integration based on a

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176 previously described strategy (8). Details of primers used for detection of proviruses p
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177 and their preintegration sites will be provided on request. i.
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178
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179 RT-PCR g
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180 The protocols used for the total RNA extraction from selected organs of a female cat
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181 (Kyoto-SPF) and the subsequent cDNA synthesis were performed as previously u
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182 described (18). To detect transcripts derived from FcERV-gamma4, RT-PCR was
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183 conducted using KOD Fx Neo (Toyobo) with Fe-406S and Fe-422R primers. The 0
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184 1.5-kbp and 1.2-kbp transcripts isolated from peripheral blood mononuclear cells were b
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185 cloned into pCR4Blunt-topo vectors (Invitrogen) and their sequences were determined. u
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186 Additionally, β-actin was also amplified as an internal control using primers
187 Hub-b-actin(DNA)s and Hub-b-actin(DNA)r, which were previously published (8).
188 The transcripts derived from full-length recombinant FeLV provirus (48-2fl) were
189 detected by performing RT-PCR using KOD Fx Neo (Toyobo) with primers Fe-222S
190 (specific for the FeLV LTR R region) and Fe-7R (specific for the FeLV LTR U3 region),
191 and their sequences were then determined. The splicing donor was ascertained via
192 sequencing of gamma4-X2 transcripts amplified by RT-PCR using Fe-406S and
193 Fe-141R. The cDNA were synthesized by using total RNA collected from NIH3T3 cells
194 (57) that had been transfected with a pCR4Blunt-topo vector (Invitrogen) containing
195 48-2fl or a pCR-Blunt II-TOPO vector (Invitrogen) containing gamma4-X2 provirus,
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196 respectively. The transfected cells were prepared by using Lipofectamine 3000
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197 (Invitrogen). The total RNA extraction was performed following a previously described a
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198 protocol (18), and the subsequent cDNA synthesis was performed by using a
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199 PrimeScript RT-PCR Kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s

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200 instructions. p
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201 i.
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202 Estimating the integrated age of FcERV-gamma4
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203 Because the mutations rates of FcERV-gamma4 LTRs are not able to be predicted, the g
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204 neutral substitution rates for felid lineages (1.8 × 10−9 substitutions/site/year) were used
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205 instead (58). The genetic distances between the 5′-LTR and 3′-LTR of each provirus u
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206 were calculated by following a Kimura 2-parameter model (59).
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207 0
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208 Phylogenetic and genetic analyses b
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209 Our phylogenetic trees were constructed by using the Repbase consensus sequences u
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210 (60) and the following sequences: Avian leukemia virus (ALV; NC_015116), Baboon
211 endogenous retrovirus (BaEV; D10032), Cervid endogenous retrovirus (CrERV;
212 JN592050), Duck infectious anemia virus (DIAV; KF313137), endogenous FeLV
213 (enFeLV; AY364318), Endogenous retrovirus domestic cat 10 and 14 (ERV-DC10, and
214 -14; AB674444 and AB674445, respectively), Friend MLV (F-MLV; NC_001362),
215 FeLV subgroup A (FeLV-A; FCVF6A and AB060732), Gibbon ape leukemia virus
216 (GaLV; NC001885), Human endogenous retrovirus HCML-ARV (HCML-ARV;
217 AAP06677), Jaagsiekte sheep retrovirus (JSRV; A27950), Koala retrovirus (KoRV;
218 AF151794), Moloney MLV (M-MLV; NC001501 and AF033811), Exogenous mouse
219 mammary tumor virus (MMTV; AF228552), Porcine endogenous retrovirus class C and
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220 E (PERV-C and -E; KC116221 and AF356697, respectively), RD114 retrovirus (RD114;
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221 EU030001), Reticuloendotheliosis virus A (REV-A; NC_006934) and Rhinolophus a
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222 ferrumequinum retrovirus (RfRV; JQ303225). All programs used for these phylogenetic
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223 analyses were packaged in MEGA6 (61). Alignments for each phylogenetic tree were

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224 conducted by using MUSCLE (62). Our phylogenetic trees were generated using the p
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225 maximum-likelihood (ML) method, and the robustness of each was evaluated by the i.
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226 bootstrap method (1000 times). The best-fit model for amino acid sequence or
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227 nucleotide evolution was selected on the basis of its Bayesian Information Criterion g
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228 score (BIC score); Pol in gammaretroviruses, rtREV+G (63); Env transmembrane in
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229 retrovirus, JTT+F+G (64); and FcERV-gamma4 5′-leader sequence, TN93+G (65). u
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230 To determine a consensus sequence of the 21-bp direct repeat, the direct repeats
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231 were aligned by MUSCLE and visualized using WebLogo (66). 0
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232 b
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233 Isolation of a recombinant FeLV gag gene u
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234 The specific PCR method used for surveying the exogenous FeLV gag gene was
235 previously established; it uses primers Fe-23S and Fe-48R corresponding to the 5′-LTR
236 and pol gene, respectively, and can detect the entire 2.3-kbp FeLV gag gene (67). We
237 conducted PCR on our FeLV-positive samples, and the resulting fragments were each
238 cloned into a pCR4Blunt-topo vector (Invitrogen). Alternatively, PCR primers Fe-23S