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Bovine leukemia virus G4 enhances virus production
Virus Research 238 (2017) 213–217
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Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Bovine leukemia virus G4 enhances virus production a,⁎
a
b
MARK c
Hironobu Murakami , Shotaro Asano , Jumpei Uchiyama , Reiichiro Sato , Masahiro Sakaguchib, Kenji Tsukamotoa a
Laboratory of Animal Health II, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan Laboratory of Veterinary Microbiology I, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan Laboratory of Farm Animal Internal Medicine, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Bovine leukemia virus Molecular clone Reverse genetics G4 Tax Virus production
The nonstructural G4 gene of bovine leukemia virus (BLV) has been thought to function in virus replication. However, the discovery of the AS1 gene on the antisense strand of the G4 gene has affected this interpretation. In this study, we investigated the function of G4 in virus production independent of the AS1 gene using a reverse genetic approach, and briefly examined the association of the G4 protein with Tax, which is also a nonstructural protein that promotes virus replication. First, we constructed a mutant molecular clone of BLV with a nonsense mutation in G4 that had a minimal effect on the AS1 gene. Comparison of the wild-type and mutant molecular clones indicated that the nonsense mutation resulted in a reduction of virus in the culture supernatant and accumulation of viral RNA (vRNA) in cells. Moreover, G4 and Tax expression in cells was shown to synergistically enhance virus production. Therefore, we suggest that G4 enhances virus production through abrogation of vRNA accumulation.
Bovine leukemia virus (BLV), which belongs to the family Retroviridae genus Deltaretrovirus, causes the development of a malignant lymphoma/leukemia in cattle, known as enzootic bovine leukosis (EBL). Although only a small percentage of BLV-infected cattle develop EBL, BLV infection also results in negative effects on the livestock industry by reducing lifetime milk production, reproductive efficiency and lifespan (Brenner et al., 1989; Nekouei et al., 2016; Schwartz and Levy, 1994). BLV randomly integrates into the genomic DNA of peripheral blood cells as a provirus and propagates in infected cattle (Gillet et al., 2007; Murakami et al., 2011). Although virus propagation in BLV-infected cattle is considered to be closely related to the pathogenesis of BLV infection and its transmissibility to other cattle (Jimba et al., 2010; Juliarena et al., 2016; Somura et al., 2014), the propagation mechanism of BLV is not fully understood. One of the important genes responsible for virus propagation and replication is G4, which is encoded on the sense strand of the BLV genome and produces a small nonstructural protein (105 amino acids, molecular weight 11.6 kDa) (Florins et al., 2006; Murakami et al., 2016; Willems et al., 1994). However, a recent transcriptomic study of BLV-infected cells showed that a putative gene, AS1, is encoded on the antisense strand of the G4 gene in the proviral genome (Durkin et al., 2016). Therefore, previous studies did not examine the function of G4 independent of that of AS1 protein. Moreover, it is uncertain whether
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Corresponding author. E-mail address: [email protected] (H. Murakami).
http://dx.doi.org/10.1016/j.virusres.2017.07.005 Received 14 April 2017; Received in revised form 19 June 2017; Accepted 4 July 2017 Available online 06 July 2017 0168-1702/ © 2017 Elsevier B.V. All rights reserved.
G4 is related to the nonstructural protein Tax, which plays a critical role in virus replication through activation of viral transcription (Aida et al., 2013; Derse, 1987). In this study, we investigated the role of G4 in virus production using a reverse genetic approach with minimal influence on the AS1 gene, and briefly examined the importance in virus production of the G4 protein combined with the viral transcriptional activator Tax. A BLV molecular clone and plasmid were prepared to examine the association of G4 with virus production. The molecular clone pBLVAN903, which was previously constructed from a provirus derived from a cow that developed EBL (Murakami et al., 2016), was used in this study. The genome of this BLV molecular clone was designed to impair G4 function with the least possible effect on AS1. Because amino acids 48–51 of G4 (RLPL; Fig. 1) seem to be important for virus production (Murakami et al., 2016), a nonsense mutation upstream of the 48th codon of the G4 gene will impair G4 protein function. However, the induction of such a nonsense mutation also results in a missense mutation in the AS1 gene. To ensure that this missense mutation had the least possible influence on AS1, the cytosines at nucleotide positions 7122 and 7123 were replaced with adenine and guanine, respectively (Fig. 1). These changes induced nonsense and missense mutations at the 42nd codon of the G4 gene and at the 31st codon of the AS1 gene, respectively. The missense mutation of the AS1 gene caused an amino acid substitution from glycine to alanine at the 31st residue of the AS1
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H. Murakami et al.
Fig. 1. Mutation induced in the G4 gene of BLV molecular clone pBLV-AN903. The site of the induced mutation in BLV molecular clone pBLV-AN903. The top panel shows the map of the BLV proviral genome. On the genome map, the right- and the left-facing arrows indicate the genes encoded on the sense and antisense strands, respectively. The induced mutation site overlaps the G4 and AS1 genes. The bottom panel shows the DNA and protein sequences around the mutation site of the molecular clone. The sequences important for virus production are shown in bold gray characters. The induced mutation codon is shown in bold black characters. The asterisk indicates the stop codon. The wild-type molecular clone pBLV-AN903 and its mutant molecular clone pBLV-AN903-G4S42X are shown in the upper and lower columns, respectively.
cDNA was synthesized from the vRNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. The qPCR was performed using primers and a probe against the polymerase gene using the cDNA as a template. All statistical analyses in this study were performed using the statistical software R, version 3.3.3 (R Core Team, 2017). First, we measured the virus titers produced from pBLV-AN903G4S42X and pBLV-AN903-transfected 293T and HeLa cells. The viruses released from the 293T cells caused the formation of larger and more distinct syncytia than those released from the HeLa cells used in our previous study (Murakami et al., 2016) (Supplementary Fig. S1A, B). Regarding virus production, the supernatants from the pBLV-AN903G4S42X-transfected 293T and HeLa cells formed significantly fewer than those from pBLV-AN903-transfected 293T and HeLa cells (Fig. 2A, Supplementary Fig. S1C). Therefore, 293T and HeLa cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X showed similar tends for virus production, which demonstrated that the mutation in pBLVAN903-G4S42X resulted in low virus production in both cell types. In addition, these results showed that the syncytium assay was improved by using 293T cells. Therefore, further studies used 293T cells for analysis of virus replication. To determine whether the G4 gene was responsible for virus production, the virus production by the molecular clones was further examined in cells expressing G4 and AS1 proteins. First, the G4 and AS1 genes fused with a hemagglutinin (HA) tag sequence at their 3′ end were cloned into the pCAGGS expression vector (Niwa et al., 1991) using cDNA synthesized from fetal ovine kidney cells persistently infected with BLV (FLK-BLV) (Van Der Maaten and Miller, 1975), and pCAGGS-G4-HA and pCAGGS-AS1-HA were prepared. The G4 and AS1 proteins of pBLV-AN903 showed 99.05% and 98.85% identity, respectively, to that derived from FLK-BLV. Using these plasmids together with the molecular clones, a complementation experiment was performed. The molecular clones and pTK-Luc together with pCAGGS-G4-HA or with empty vector pCAGGS
protein. Because glycine and alanine are structurally similar and apolar amino acids, this missense mutation of the AS1 gene was considered to have minimal effect on the function of the AS1 protein. This mutation was induced in pBLV-AN903 using an In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan) to construct a mutant molecular clone, pBLV-AN903G4S42X (Fig. 1). As a control for the transfection efficiency of the clones when conducting experiments on virus replication, we also prepared pTK-Luc, in which the Renilla luciferase gene of pRL-TK (Toyo Ink, Tokyo, Japan) was replaced with the firefly luciferase gene. Virus replication in culture supernatant and the level of viral RNA (vRNA) in cells were normalized using luciferase activity to correct for the influence of transfection efficiency. To determine whether virus production was affected by the mutation in pBLV-AN903, we examined virus production using a syncytium assay or quantitative polymerase chain reaction (qPCR). First, pBLVAN903 or pBLV-AN903-G4S42X together with pTK-Luc were transfected into 293T or HeLa cells. At 24 h posttransfection, the cells were washed twice with Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan), and the medium was replaced with fresh growth medium (10% fetal bovine serum, 90% DMEM). At 48 h posttransfection, the supernatants of the transfected cells were centrifuged to remove cell debris and were used for a syncytium assay and qPCR as described previously (Murakami et al., 2016). The syncytium assay was performed as follows. Briefly, CC81 cells were cultured with the serially-diluted supernatants in the presence of polybrene (Sigma-Aldrich, St. Louis, MO, USA) at 4 μg/ml in growth media (5% fetal bovine serum and 10% of 2.95% tryptose phosphate broth added to Eagle’s minimal essential medium) until confluent. Supernatants from both 293T and HeLa cells transfected with pBLV-AN903 and those transfected with pBLV-AN903-G4S42X formed syncytia in the CC81 cell line. For qPCR, cell debris was removed from the supernatants of the transfected cells, which were then used for vRNA extraction using a High Pure Viral Nucleic Acid Kit (Roche, Penzberg, Germany), according to the manufacturer’s instructions. 214
Virus Research 238 (2017) 213–217
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Relative vRNA copy number
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pBLV-AN903 -G4S42X
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0.5
0 AS1-HA
-
+
pBLV-AN903
Relative vRNA expression
2.0
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***
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pBLV-AN903 -G4S42X
***
NS
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0 G4-HA
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pBLV-AN903
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+
pBLV-AN903 -G4S42X
NS
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-
pBLV-AN903
E
pBLV-AN903
***
NS
1.0
pBLV-AN903 -G4S42X
**
3.0
pBLV-AN903 -G4S42X
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1.5
0 G4-HA
Relative vRNA copy number
**
+
1.0
**
NS
-
Fig. 2. Virus production and vRNA expression of 293T cells transfected with molecular clones pBLV-AN903 and pBLV-AN903-G4S42X. (A) Infectious virus production from 293T cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X. Virus production from 293T cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X expressing (B) G4-HA or (C) AS1-HA protein. The amounts of infectious virus shown as relative syncytiumforming units (SFU) and relative vRNA copy number were measured by syncytium-assay (left) and qPCR (right), respectively. (D) vRNA expression in 293T cells transfected with molecular clones pBLV-AN903 and pBLV-AN903-G4S42X. vRNA expression in cells transfected with molecular clones pBLV-AN903 and pBLV-AN903-G4S42X expressing (E) G4-HA or (F) AS1-HA protein. For RNA extraction of the transfected cells, at 24 h posttransfection, the cells were washed with DMEM. At 48 h posttransfection, total RNA was extracted from the cells using RNeasy Plus mini kit (Qiagen) according to the manufacturer’s instructions, and then cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo) using the extracted RNA as a template. To measure vRNA expression in cells, qPCR was performed using primers against polymerase and beta-actin genes, described in the text and supplementary material, respectively. The bars and error bars indicate the mean with standard error of triplicate experiments. Significant differences are indicated by single, double and triple asterisks (P < 0.05, P < 0.01 and P < 0.001, respectively, A and D: t-test; B, C, E and F: one-way analysis of variance). “NS” indicates that the difference is not significant.
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Relative vRNA copy number
H. Murakami et al.
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pBLV-AN903
The results showed that virus production from pBLV-AN903-transfected cells was not enhanced by G4-HA expression. However, the level of virus released from the 293T cells transfected with pBLV-AN903G4S42X was significantly higher in G4-HA-expressing cells than in
were transfected into 293T cells. The expression of the G4 protein fused with an HA tag (G4-HA) in the transfected 293T cells was confirmed by immunoblotting (Supplementary Fig. S2A). Virus production from the transfected cells was examined using the syncytium assay and qPCR. 215
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Fig. 3. Analysis of virus production in cells overexpressing G4 and Tax. Infectious virus was measured by a syncytium assay (left) and qPCR (right) after pBLV-AN903-G4S42X transfection. The bars and error bars indicate the mean with standard error of triplicate experiments. Significant differences are indicated by single, double and triple asterisks (P < 0.05, P < 0.01 and P < 0.001, respectively, one-way analysis of variance).
99.68% identical to that derived from FLK-BLV. The pBLV-AN903-G4S42X and pTK-Luc together with pCAGGS/ pCAGGS-G4-HA and pBApo-EF1α-Puro/pBApo-EF1α-Tax-Flag were transfected into 293T cells. The expression of G4-HA and Tax-Flag proteins in the transfected 293T cells was confirmed by immunoblotting (Supplementary Fig. S3). The virus production from the transfected 293T cells was measured using the syncytium assay and qPCR. The results showed that the virus production from the Tax-Flag or G4-HA expressing cells was significantly higher than that from the control cells (i.e., the cells with no expression of Tax-Flag and G4-HA). This result suggested that G4 functions to promote virus production independently of Tax, because Tax could promote virus production when G4 function was impaired. However, the virus production from the cells expressing both G4-HA and Tax-Flag was significantly higher than that from the cells expressing Tax-Flag/G4-HA or control cells (Fig. 3), although it was unclear whether G4 worked synergistically or additively with Tax to promote virus production. To determine the type of interaction between G4 and Tax, the above data were analyzed using a two-way factorial analysis of variance. According to this statistical method (Grenier et al., 2016), P > 0.05 and P < 0.05 are interpreted as indicating “additive” and “synergistic”, respectively. The analysis determined that the P values in the syncytium assay and qPCR data were 0.04848 and 7.019 × 10−5, respectively, both < 0.05. Therefore, we considered that the G4-HA and Tax-Flag protein acted synergistically to enhance virus production, meaning that the G4 protein is an important positive regulator of virus production. In addition, the result supported that the function of G4 different from that of Tax because the factors that have different modes of action usually show synergistic rather than additive effects. The present study demonstrated that the BLV G4 protein is a positive regulator of virus production. In addition, it suggested that mode of action of G4 is related to virus release, which differs from effect of Tax. In support of this, the upregulation of virus production by G4 was synergistically enhanced by Tax overexpression. These results suggested that the G4 protein was likely to function at a different phase of the BLV life cycle from the Tax protein. In addition, it has been shown that the G4 and Tax protein are localized in the mitochondria and nucleus, respectively, indicating that they may work independently at different sites in the cell (Lefebvre et al., 2002; Twizere et al., 2003). The different mechanisms by which G4 and Tax enhance virus production appear to be critical for optimal production of BLV, because impairment of only G4 function caused inefficient virus production in vitro in this study, and has been shown to lead to inefficient virus propagation in vivo (Florins et al., 2006; Willems et al., 1994). Moreover, recent studies have identified new BLV genotypes and several amino acid substitutions in G4 (Lee et al., 2016; Polat et al., 2016; Polat et al., 2017), indicating that polymorphisms of G4 could change the levels of pathology and transmissibility of BLV. To prevent the economic loss caused by BLV infection, cattle infected with BLV with high transmissibility and/or pathology could be quarantined rapidly if there was a good diagnostic tool to predict BLV transmissibility and pathology. Unfortunately, there is no useful tool
pCAGGS-transfected 293T cells (Fig. 2B). These results implied that the induced nonsense mutation could impair G4 function related to virus production through the production of a truncated form of G4 (Fig. 1; 41 aa). In addition, the results showed that, in contrast to the function of Tax, a positive regulator of virus transcription (Willems et al., 1987), G4 functioned as an enhancer of virus production. Next, the molecular clones and pTK-Luc with pCAGGS-AS1-HA or the empty vector pCAGGS were transfected into 293T cells. The expression of the AS1 protein fused with an HA tag (AS1-HA) in the 293T cells was confirmed by immunoblotting (Supplementary Fig. S2B), and virus production from the transfected cells was examined using the syncytium assay and qPCR. The results showed that there was no significant difference in virus production between cells with and without the AS1-HA protein for both pBLV-AN903 and pBLV-AN903-G4S42X transfection (Fig. 2C). Therefore, the low virus production shown in the previous experiment was considered to be induced by the nonsense mutation in the G4 gene rather than by the missense mutation on the AS1 gene, further suggesting that the G4 protein is an enhancing regulator of virus production. To investigate how G4 enhanced virus production, vRNA expression in cells was measured using qPCR. First, the level of vRNA in pBLVAN903-transfected 293T cells was less than that in pBLV-AN903G4S42X-transfected cells (Fig. 2D). This showed that the nonsense mutation resulted in the accumulation of vRNA in cells. Next, to examine whether the accumulation of vRNA was related to G4, we compared the vRNA expression in molecular clones together with empty vector pCAGGS, pCAGGS-G4-HA or pCAGGS-AS1-HA-transfected 293T cells. The expression of G4-HA and AS1-HA mRNA in the transfected 293T cells was confirmed by reverse-transcription PCR (RTPCR) (Supplementary Fig. S2A, B). The results showed that pCAGGS transfection did not affect the level of vRNA in cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X (Fig. 2E, F). However, G4-HA expression abrogated the accumulation of vRNA in pBLV-AN903G4S42X-transfected cells (Fig. 2E). In contrast, AS-1-HA expression did not affect the accumulation of vRNA in pBLV-AN903-G4S42X-transfected cells (Fig. 2F). These results suggested that G4 enhances virus production through assisting virus release. This is in contrast to the mode of action of Tax, which is known to be a powerful positive regulator of virus production via its transactivating activity (Willems et al., 1987). It was unclear whether G4 cooperated with the other positive regulator of virus production, Tax. We hypothesized that G4 promoted virus production through abrogation of the accumulation of vRNA enhanced by Tax. To test this hypothesis, virus production from 293T cells transfected with pBLV-AN903 was measured under conditions where G4 and/or Tax were overexpressed. To perform this experiment, plasmids for G4 and Tax overexpression were prepared. To overexpress the G4 protein, the pCAGGS-G4-HA plasmid described above was used. To overexpress Tax, the Tax gene fused with a Flag-tag sequence at its 3′ end was cloned into an expression vector pBApo-EF1α-Puro (Takara Bio), using cDNA synthesized from FLK-BLV cells, to prepare the plasmid pBApo-EF1α-Tax-Flag. The Tax protein of pBLV-AN903 was 216
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presently available to distinguish such potentially highly transmissible and pathogenic BLV strains. Therefore, it is important to increase knowledge about the associations between BLV variants and their biological traits. We believe that BLV genome analysis will contribute to the prevention of the spread of BLV infection in the future. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgment This work was partially supported by JSPS KAKENHI Grant Number 17K15385. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2017.07.005. References Aida, Y., Murakami, H., Takahashi, M., Takeshima, S.N., 2013. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Front. Microbiol. 4, 328. Brenner, J., Van-Haam, M., Savir, D., Trainin, Z., 1989. The implication of BLV infection in the productivity, reproductive capacity and survival rate of a dairy cow. Vet. Immunol. Immunopathol. 22 (3), 299–305. Derse, D., 1987. Bovine leukemia virus transcription is controlled by a virus-encoded trans-acting factor and by cis-acting response elements. J. Virol. 61 (8), 2462–2471. Durkin, K., Rosewick, N., Artesi, M., Hahaut, V., Griebel, P., Arsic, N., Burny, A., Georges, M., Van den Broeke, A., 2016. Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs. Retrovirology 13 (1), 33. Florins, A., Gillet, N., Asquith, B., Debacq, C., Jean, G., Schwartz-Cornil, I., Bonneau, M., Burny, A., Reichert, M., Kettmann, R., Willems, L., 2006. Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep. J. Virol. 80 (24), 11998–12008. Gillet, N., Florins, A., Boxus, M., Burteau, C., Nigro, A., Vandermeers, F., Balon, H., Bouzar, A.B., Defoiche, J., Burny, A., Reichert, M., Kettmann, R., Willems, L., 2007. Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human. Retrovirology 4, 18. Grenier, B., Dohnal, I., Shanmugasundaram, R., Eicher, S.D., Selvaraj, R.K., Schatzmayr, G., Applegate, T.J., 2016. Susceptibility of broiler chickens to coccidiosis when fed
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Virus Research journal homepage: www.elsevier.com/locate/virusres
Short communication
Bovine leukemia virus G4 enhances virus production a,⁎
a
b
MARK c
Hironobu Murakami , Shotaro Asano , Jumpei Uchiyama , Reiichiro Sato , Masahiro Sakaguchib, Kenji Tsukamotoa a
Laboratory of Animal Health II, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan Laboratory of Veterinary Microbiology I, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan Laboratory of Farm Animal Internal Medicine, School of Veterinary Medicine, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Bovine leukemia virus Molecular clone Reverse genetics G4 Tax Virus production
The nonstructural G4 gene of bovine leukemia virus (BLV) has been thought to function in virus replication. However, the discovery of the AS1 gene on the antisense strand of the G4 gene has affected this interpretation. In this study, we investigated the function of G4 in virus production independent of the AS1 gene using a reverse genetic approach, and briefly examined the association of the G4 protein with Tax, which is also a nonstructural protein that promotes virus replication. First, we constructed a mutant molecular clone of BLV with a nonsense mutation in G4 that had a minimal effect on the AS1 gene. Comparison of the wild-type and mutant molecular clones indicated that the nonsense mutation resulted in a reduction of virus in the culture supernatant and accumulation of viral RNA (vRNA) in cells. Moreover, G4 and Tax expression in cells was shown to synergistically enhance virus production. Therefore, we suggest that G4 enhances virus production through abrogation of vRNA accumulation.
Bovine leukemia virus (BLV), which belongs to the family Retroviridae genus Deltaretrovirus, causes the development of a malignant lymphoma/leukemia in cattle, known as enzootic bovine leukosis (EBL). Although only a small percentage of BLV-infected cattle develop EBL, BLV infection also results in negative effects on the livestock industry by reducing lifetime milk production, reproductive efficiency and lifespan (Brenner et al., 1989; Nekouei et al., 2016; Schwartz and Levy, 1994). BLV randomly integrates into the genomic DNA of peripheral blood cells as a provirus and propagates in infected cattle (Gillet et al., 2007; Murakami et al., 2011). Although virus propagation in BLV-infected cattle is considered to be closely related to the pathogenesis of BLV infection and its transmissibility to other cattle (Jimba et al., 2010; Juliarena et al., 2016; Somura et al., 2014), the propagation mechanism of BLV is not fully understood. One of the important genes responsible for virus propagation and replication is G4, which is encoded on the sense strand of the BLV genome and produces a small nonstructural protein (105 amino acids, molecular weight 11.6 kDa) (Florins et al., 2006; Murakami et al., 2016; Willems et al., 1994). However, a recent transcriptomic study of BLV-infected cells showed that a putative gene, AS1, is encoded on the antisense strand of the G4 gene in the proviral genome (Durkin et al., 2016). Therefore, previous studies did not examine the function of G4 independent of that of AS1 protein. Moreover, it is uncertain whether
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Corresponding author. E-mail address: [email protected] (H. Murakami).
http://dx.doi.org/10.1016/j.virusres.2017.07.005 Received 14 April 2017; Received in revised form 19 June 2017; Accepted 4 July 2017 Available online 06 July 2017 0168-1702/ © 2017 Elsevier B.V. All rights reserved.
G4 is related to the nonstructural protein Tax, which plays a critical role in virus replication through activation of viral transcription (Aida et al., 2013; Derse, 1987). In this study, we investigated the role of G4 in virus production using a reverse genetic approach with minimal influence on the AS1 gene, and briefly examined the importance in virus production of the G4 protein combined with the viral transcriptional activator Tax. A BLV molecular clone and plasmid were prepared to examine the association of G4 with virus production. The molecular clone pBLVAN903, which was previously constructed from a provirus derived from a cow that developed EBL (Murakami et al., 2016), was used in this study. The genome of this BLV molecular clone was designed to impair G4 function with the least possible effect on AS1. Because amino acids 48–51 of G4 (RLPL; Fig. 1) seem to be important for virus production (Murakami et al., 2016), a nonsense mutation upstream of the 48th codon of the G4 gene will impair G4 protein function. However, the induction of such a nonsense mutation also results in a missense mutation in the AS1 gene. To ensure that this missense mutation had the least possible influence on AS1, the cytosines at nucleotide positions 7122 and 7123 were replaced with adenine and guanine, respectively (Fig. 1). These changes induced nonsense and missense mutations at the 42nd codon of the G4 gene and at the 31st codon of the AS1 gene, respectively. The missense mutation of the AS1 gene caused an amino acid substitution from glycine to alanine at the 31st residue of the AS1
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Fig. 1. Mutation induced in the G4 gene of BLV molecular clone pBLV-AN903. The site of the induced mutation in BLV molecular clone pBLV-AN903. The top panel shows the map of the BLV proviral genome. On the genome map, the right- and the left-facing arrows indicate the genes encoded on the sense and antisense strands, respectively. The induced mutation site overlaps the G4 and AS1 genes. The bottom panel shows the DNA and protein sequences around the mutation site of the molecular clone. The sequences important for virus production are shown in bold gray characters. The induced mutation codon is shown in bold black characters. The asterisk indicates the stop codon. The wild-type molecular clone pBLV-AN903 and its mutant molecular clone pBLV-AN903-G4S42X are shown in the upper and lower columns, respectively.
cDNA was synthesized from the vRNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Osaka, Japan), according to the manufacturer’s instructions. The qPCR was performed using primers and a probe against the polymerase gene using the cDNA as a template. All statistical analyses in this study were performed using the statistical software R, version 3.3.3 (R Core Team, 2017). First, we measured the virus titers produced from pBLV-AN903G4S42X and pBLV-AN903-transfected 293T and HeLa cells. The viruses released from the 293T cells caused the formation of larger and more distinct syncytia than those released from the HeLa cells used in our previous study (Murakami et al., 2016) (Supplementary Fig. S1A, B). Regarding virus production, the supernatants from the pBLV-AN903G4S42X-transfected 293T and HeLa cells formed significantly fewer than those from pBLV-AN903-transfected 293T and HeLa cells (Fig. 2A, Supplementary Fig. S1C). Therefore, 293T and HeLa cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X showed similar tends for virus production, which demonstrated that the mutation in pBLVAN903-G4S42X resulted in low virus production in both cell types. In addition, these results showed that the syncytium assay was improved by using 293T cells. Therefore, further studies used 293T cells for analysis of virus replication. To determine whether the G4 gene was responsible for virus production, the virus production by the molecular clones was further examined in cells expressing G4 and AS1 proteins. First, the G4 and AS1 genes fused with a hemagglutinin (HA) tag sequence at their 3′ end were cloned into the pCAGGS expression vector (Niwa et al., 1991) using cDNA synthesized from fetal ovine kidney cells persistently infected with BLV (FLK-BLV) (Van Der Maaten and Miller, 1975), and pCAGGS-G4-HA and pCAGGS-AS1-HA were prepared. The G4 and AS1 proteins of pBLV-AN903 showed 99.05% and 98.85% identity, respectively, to that derived from FLK-BLV. Using these plasmids together with the molecular clones, a complementation experiment was performed. The molecular clones and pTK-Luc together with pCAGGS-G4-HA or with empty vector pCAGGS
protein. Because glycine and alanine are structurally similar and apolar amino acids, this missense mutation of the AS1 gene was considered to have minimal effect on the function of the AS1 protein. This mutation was induced in pBLV-AN903 using an In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan) to construct a mutant molecular clone, pBLV-AN903G4S42X (Fig. 1). As a control for the transfection efficiency of the clones when conducting experiments on virus replication, we also prepared pTK-Luc, in which the Renilla luciferase gene of pRL-TK (Toyo Ink, Tokyo, Japan) was replaced with the firefly luciferase gene. Virus replication in culture supernatant and the level of viral RNA (vRNA) in cells were normalized using luciferase activity to correct for the influence of transfection efficiency. To determine whether virus production was affected by the mutation in pBLV-AN903, we examined virus production using a syncytium assay or quantitative polymerase chain reaction (qPCR). First, pBLVAN903 or pBLV-AN903-G4S42X together with pTK-Luc were transfected into 293T or HeLa cells. At 24 h posttransfection, the cells were washed twice with Dulbecco’s modified Eagle’s minimal essential medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan), and the medium was replaced with fresh growth medium (10% fetal bovine serum, 90% DMEM). At 48 h posttransfection, the supernatants of the transfected cells were centrifuged to remove cell debris and were used for a syncytium assay and qPCR as described previously (Murakami et al., 2016). The syncytium assay was performed as follows. Briefly, CC81 cells were cultured with the serially-diluted supernatants in the presence of polybrene (Sigma-Aldrich, St. Louis, MO, USA) at 4 μg/ml in growth media (5% fetal bovine serum and 10% of 2.95% tryptose phosphate broth added to Eagle’s minimal essential medium) until confluent. Supernatants from both 293T and HeLa cells transfected with pBLV-AN903 and those transfected with pBLV-AN903-G4S42X formed syncytia in the CC81 cell line. For qPCR, cell debris was removed from the supernatants of the transfected cells, which were then used for vRNA extraction using a High Pure Viral Nucleic Acid Kit (Roche, Penzberg, Germany), according to the manufacturer’s instructions. 214
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Fig. 2. Virus production and vRNA expression of 293T cells transfected with molecular clones pBLV-AN903 and pBLV-AN903-G4S42X. (A) Infectious virus production from 293T cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X. Virus production from 293T cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X expressing (B) G4-HA or (C) AS1-HA protein. The amounts of infectious virus shown as relative syncytiumforming units (SFU) and relative vRNA copy number were measured by syncytium-assay (left) and qPCR (right), respectively. (D) vRNA expression in 293T cells transfected with molecular clones pBLV-AN903 and pBLV-AN903-G4S42X. vRNA expression in cells transfected with molecular clones pBLV-AN903 and pBLV-AN903-G4S42X expressing (E) G4-HA or (F) AS1-HA protein. For RNA extraction of the transfected cells, at 24 h posttransfection, the cells were washed with DMEM. At 48 h posttransfection, total RNA was extracted from the cells using RNeasy Plus mini kit (Qiagen) according to the manufacturer’s instructions, and then cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo) using the extracted RNA as a template. To measure vRNA expression in cells, qPCR was performed using primers against polymerase and beta-actin genes, described in the text and supplementary material, respectively. The bars and error bars indicate the mean with standard error of triplicate experiments. Significant differences are indicated by single, double and triple asterisks (P < 0.05, P < 0.01 and P < 0.001, respectively, A and D: t-test; B, C, E and F: one-way analysis of variance). “NS” indicates that the difference is not significant.
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The results showed that virus production from pBLV-AN903-transfected cells was not enhanced by G4-HA expression. However, the level of virus released from the 293T cells transfected with pBLV-AN903G4S42X was significantly higher in G4-HA-expressing cells than in
were transfected into 293T cells. The expression of the G4 protein fused with an HA tag (G4-HA) in the transfected 293T cells was confirmed by immunoblotting (Supplementary Fig. S2A). Virus production from the transfected cells was examined using the syncytium assay and qPCR. 215
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Fig. 3. Analysis of virus production in cells overexpressing G4 and Tax. Infectious virus was measured by a syncytium assay (left) and qPCR (right) after pBLV-AN903-G4S42X transfection. The bars and error bars indicate the mean with standard error of triplicate experiments. Significant differences are indicated by single, double and triple asterisks (P < 0.05, P < 0.01 and P < 0.001, respectively, one-way analysis of variance).
99.68% identical to that derived from FLK-BLV. The pBLV-AN903-G4S42X and pTK-Luc together with pCAGGS/ pCAGGS-G4-HA and pBApo-EF1α-Puro/pBApo-EF1α-Tax-Flag were transfected into 293T cells. The expression of G4-HA and Tax-Flag proteins in the transfected 293T cells was confirmed by immunoblotting (Supplementary Fig. S3). The virus production from the transfected 293T cells was measured using the syncytium assay and qPCR. The results showed that the virus production from the Tax-Flag or G4-HA expressing cells was significantly higher than that from the control cells (i.e., the cells with no expression of Tax-Flag and G4-HA). This result suggested that G4 functions to promote virus production independently of Tax, because Tax could promote virus production when G4 function was impaired. However, the virus production from the cells expressing both G4-HA and Tax-Flag was significantly higher than that from the cells expressing Tax-Flag/G4-HA or control cells (Fig. 3), although it was unclear whether G4 worked synergistically or additively with Tax to promote virus production. To determine the type of interaction between G4 and Tax, the above data were analyzed using a two-way factorial analysis of variance. According to this statistical method (Grenier et al., 2016), P > 0.05 and P < 0.05 are interpreted as indicating “additive” and “synergistic”, respectively. The analysis determined that the P values in the syncytium assay and qPCR data were 0.04848 and 7.019 × 10−5, respectively, both < 0.05. Therefore, we considered that the G4-HA and Tax-Flag protein acted synergistically to enhance virus production, meaning that the G4 protein is an important positive regulator of virus production. In addition, the result supported that the function of G4 different from that of Tax because the factors that have different modes of action usually show synergistic rather than additive effects. The present study demonstrated that the BLV G4 protein is a positive regulator of virus production. In addition, it suggested that mode of action of G4 is related to virus release, which differs from effect of Tax. In support of this, the upregulation of virus production by G4 was synergistically enhanced by Tax overexpression. These results suggested that the G4 protein was likely to function at a different phase of the BLV life cycle from the Tax protein. In addition, it has been shown that the G4 and Tax protein are localized in the mitochondria and nucleus, respectively, indicating that they may work independently at different sites in the cell (Lefebvre et al., 2002; Twizere et al., 2003). The different mechanisms by which G4 and Tax enhance virus production appear to be critical for optimal production of BLV, because impairment of only G4 function caused inefficient virus production in vitro in this study, and has been shown to lead to inefficient virus propagation in vivo (Florins et al., 2006; Willems et al., 1994). Moreover, recent studies have identified new BLV genotypes and several amino acid substitutions in G4 (Lee et al., 2016; Polat et al., 2016; Polat et al., 2017), indicating that polymorphisms of G4 could change the levels of pathology and transmissibility of BLV. To prevent the economic loss caused by BLV infection, cattle infected with BLV with high transmissibility and/or pathology could be quarantined rapidly if there was a good diagnostic tool to predict BLV transmissibility and pathology. Unfortunately, there is no useful tool
pCAGGS-transfected 293T cells (Fig. 2B). These results implied that the induced nonsense mutation could impair G4 function related to virus production through the production of a truncated form of G4 (Fig. 1; 41 aa). In addition, the results showed that, in contrast to the function of Tax, a positive regulator of virus transcription (Willems et al., 1987), G4 functioned as an enhancer of virus production. Next, the molecular clones and pTK-Luc with pCAGGS-AS1-HA or the empty vector pCAGGS were transfected into 293T cells. The expression of the AS1 protein fused with an HA tag (AS1-HA) in the 293T cells was confirmed by immunoblotting (Supplementary Fig. S2B), and virus production from the transfected cells was examined using the syncytium assay and qPCR. The results showed that there was no significant difference in virus production between cells with and without the AS1-HA protein for both pBLV-AN903 and pBLV-AN903-G4S42X transfection (Fig. 2C). Therefore, the low virus production shown in the previous experiment was considered to be induced by the nonsense mutation in the G4 gene rather than by the missense mutation on the AS1 gene, further suggesting that the G4 protein is an enhancing regulator of virus production. To investigate how G4 enhanced virus production, vRNA expression in cells was measured using qPCR. First, the level of vRNA in pBLVAN903-transfected 293T cells was less than that in pBLV-AN903G4S42X-transfected cells (Fig. 2D). This showed that the nonsense mutation resulted in the accumulation of vRNA in cells. Next, to examine whether the accumulation of vRNA was related to G4, we compared the vRNA expression in molecular clones together with empty vector pCAGGS, pCAGGS-G4-HA or pCAGGS-AS1-HA-transfected 293T cells. The expression of G4-HA and AS1-HA mRNA in the transfected 293T cells was confirmed by reverse-transcription PCR (RTPCR) (Supplementary Fig. S2A, B). The results showed that pCAGGS transfection did not affect the level of vRNA in cells transfected with pBLV-AN903 and pBLV-AN903-G4S42X (Fig. 2E, F). However, G4-HA expression abrogated the accumulation of vRNA in pBLV-AN903G4S42X-transfected cells (Fig. 2E). In contrast, AS-1-HA expression did not affect the accumulation of vRNA in pBLV-AN903-G4S42X-transfected cells (Fig. 2F). These results suggested that G4 enhances virus production through assisting virus release. This is in contrast to the mode of action of Tax, which is known to be a powerful positive regulator of virus production via its transactivating activity (Willems et al., 1987). It was unclear whether G4 cooperated with the other positive regulator of virus production, Tax. We hypothesized that G4 promoted virus production through abrogation of the accumulation of vRNA enhanced by Tax. To test this hypothesis, virus production from 293T cells transfected with pBLV-AN903 was measured under conditions where G4 and/or Tax were overexpressed. To perform this experiment, plasmids for G4 and Tax overexpression were prepared. To overexpress the G4 protein, the pCAGGS-G4-HA plasmid described above was used. To overexpress Tax, the Tax gene fused with a Flag-tag sequence at its 3′ end was cloned into an expression vector pBApo-EF1α-Puro (Takara Bio), using cDNA synthesized from FLK-BLV cells, to prepare the plasmid pBApo-EF1α-Tax-Flag. The Tax protein of pBLV-AN903 was 216
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presently available to distinguish such potentially highly transmissible and pathogenic BLV strains. Therefore, it is important to increase knowledge about the associations between BLV variants and their biological traits. We believe that BLV genome analysis will contribute to the prevention of the spread of BLV infection in the future. Conflict of interest The authors have no conflicts of interest to declare. Acknowledgment This work was partially supported by JSPS KAKENHI Grant Number 17K15385. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2017.07.005. References Aida, Y., Murakami, H., Takahashi, M., Takeshima, S.N., 2013. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus. Front. Microbiol. 4, 328. Brenner, J., Van-Haam, M., Savir, D., Trainin, Z., 1989. The implication of BLV infection in the productivity, reproductive capacity and survival rate of a dairy cow. Vet. Immunol. Immunopathol. 22 (3), 299–305. Derse, D., 1987. Bovine leukemia virus transcription is controlled by a virus-encoded trans-acting factor and by cis-acting response elements. J. Virol. 61 (8), 2462–2471. Durkin, K., Rosewick, N., Artesi, M., Hahaut, V., Griebel, P., Arsic, N., Burny, A., Georges, M., Van den Broeke, A., 2016. Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs. Retrovirology 13 (1), 33. Florins, A., Gillet, N., Asquith, B., Debacq, C., Jean, G., Schwartz-Cornil, I., Bonneau, M., Burny, A., Reichert, M., Kettmann, R., Willems, L., 2006. Spleen-dependent turnover of CD11b peripheral blood B lymphocytes in bovine leukemia virus-infected sheep. J. Virol. 80 (24), 11998–12008. Gillet, N., Florins, A., Boxus, M., Burteau, C., Nigro, A., Vandermeers, F., Balon, H., Bouzar, A.B., Defoiche, J., Burny, A., Reichert, M., Kettmann, R., Willems, L., 2007. Mechanisms of leukemogenesis induced by bovine leukemia virus: prospects for novel anti-retroviral therapies in human. Retrovirology 4, 18. Grenier, B., Dohnal, I., Shanmugasundaram, R., Eicher, S.D., Selvaraj, R.K., Schatzmayr, G., Applegate, T.J., 2016. Susceptibility of broiler chickens to coccidiosis when fed
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