The Experts below are selected from a list of 177 Experts worldwide ranked by ideXlab platform
Nobuyuki Yoshikawa - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement Protein.
Virology, 2008Co-Authors: Hajime Yaegashi, Akihiro Tamura, Masamichi Isogai, Nobuyuki YoshikawaAbstract:Apple chlorotic leaf spot virus 50 kDa movement Protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.
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Interference of Long-Distance Movement of Grapevine berry inner necrosis virus in Transgenic Plants Expressing a Defective Movement Protein of Apple chlorotic leaf spot virus.
Phytopathology, 2006Co-Authors: Nobuyuki Yoshikawa, Y. Saitou, A. Kitajima, T Chida, N. Sasaki, Masamichi IsogaiAbstract:ABSTRACT Transgenic Nicotiana occidentalis plants expressing a movement Protein (P50) and partially functional deletion mutants (DeltaA and DeltaC) of the Apple chlorotic leaf spot virus (ACLSV) showed resistance to Grapevine berry inner necrosis virus (GINV). The resistance is highly effective and GINV was below the level of detection in both inoculated and uninoculated upper leaves. In contrast, GINV accumulated in inoculated and uninoculated leaves of nontransgenic (NT) plants and transgenic plants expressing a dysfunctional mutant (DeltaG). On the other hand, in some plants of a transgenic plant line expressing a deletion mutant (DeltaA', deletion of the C-terminal 42 amino acids), GINV could spread in inoculated leaves, but not move into uninoculated leaves. In a tissue blot hybridization analysis of DeltaA'-plants inoculated with GINV, virus could be detected in leaf blade, midribs, and petiole of inoculated leaves, but neither in stems immediately above inoculated leaves nor in any tissues of uninoculated leaves. Immunohistochemical analysis of GINV-inoculated leaves of DeltaA'-plants showed that GINV could invade into phloem parenchyma cells through bundle sheath of minor veins, suggesting that the long-distance transport of GINV might be inhibited between the phloem cells and sieve element (and/or within sieve element) rather than bundle sheath-phloem interfaces. Immunogold electron microscopy using an anti-P50 antiserum showed that P50 accumulated on the parietal layer of sieve elements and on sieve plates. The results suggested that resistance in P50-transgenic plants to GINV is due to the interference of both long-distance and cell-to-cell movement of the virus.
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Transgenic Nicotiana occidentalis Plants Expressing the 50-kDa Protein of Apple chlorotic leaf spot virus Display Increased Susceptibility to Homologous Virus, but Strong Resistance to Grapevine berry inner necrosis virus.
Phytopathology, 2000Co-Authors: Nobuyuki Yoshikawa, S. Gotoh, M. Umezawa, N. Satoh, H. Satoh, Tsuyoshi Takahashi, T. Ito, K. YoshidaAbstract:ABSTRACT The 50-kDa Protein (P50) encoded by the open reading frame 2 of Apple chlorotic leaf spot virus (ACLSV), a putative movement Protein, was expressed in transgenic Nicotiana occidentalis plants. P50 in transgenic plants was mainly detected in a modified form in the cell wall fraction, similar to that in infected leaves. The P50-expressing plants (P50 plants) complemented the systemic spread of the P50-defective mutants of an infectious cDNA clone of ACLSV (pCLSF), indicating that P50 in transgenic plants was functional. Severity of symptoms was greatly enhanced and accumulation of virus in upper leaves was increased in P50 plants inoculated with pCLSF or ACLSV compared with that in nontransgenic control plants (NT plants). Conversely, transgenic plants expressing the coat Protein of ACLSV (CP plants) showed a significant delay in symptom development and a reduction of virus accumulation. However, most P50 plants inoculated with Grapevine berry inner necrosis virus (GINV), another species of the genus Trichovirus, neither developed obvious symptoms nor supported virus accumulation in inoculated or upper leaves. In contrast, systemic symptoms developed and virus accumulated equally in NT and CP plants inoculated with GINV. After inoculation with Apple stem grooving virus or Apple stem pitting virus, there was no difference in symptom development and virus accumulation among P50, CP, and NT plants. Our results indicate that transgenic plants expressing a functional P50 were more susceptible to homologous virus and, on the contrary, showed strong resistance to the heterologous virus GINV.
Lev P. Ovchinnikov - One of the best experts on this subject based on the ideXlab platform.
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The Major Messenger RibonucleoProtein Particle Protein P50 (YB-1) Promotes Nucleic Acid Strand Annealing
The Journal of biological chemistry, 2001Co-Authors: Maxim A. Skabkin, Valentina Evdokimova, Adri A. M. Thomas, Lev P. OvchinnikovAbstract:P50, a member of the Y-box binding transcription factor family, is tightly associated with eukaryotic mRNAs and is responsible for general translational regulation. Here we show that P50, in addition to its previously described ability to melt mRNA secondary structure, is capable of promoting rapid annealing of complementary nucleic acid strands. P50 accelerates annealing of RNA and DNA duplexes up to 1500-fold within a wide range of salt concentrations and temperatures. Phosphorylation of P50 selectively inhibits DNA annealing. Moreover, P50 catalyzes strand exchange between double-stranded and single-stranded RNAs yielding a product bearing a more extended double-stranded structure. Strikingly, P50 displays both RNA-melting and -annealing activities in a dose-dependent manner; a relatively low amount of P50 promotes formation of RNA duplexes, whereas an excess of P50 causes unwinding of double-stranded forms. Our results suggest that the alteration of nucleic acid conformation is a basic mechanism of the P50-dependent regulation of gene expression.
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Interaction of the universal mRNA-binding Protein, P50, with actin: a possible link between mRNA and microfilaments
Journal of Cell Science, 1999Co-Authors: P. V. Ruzanov, Valentina Evdokimova, N. L. Korneeva, J. W. B. Hershey, Lev P. OvchinnikovAbstract:We have shown previously that P50 is the most abundant Protein associated with a variety of eukaryotic mRNAs and exhibits about 98% amino acid sequence identity to mammalian Y-box binding transcription factors. The dual function of P50 in the cell as a regulator of both transcription and translation has been suggested. To gain insight into the role of P50 in these processes, we performed the yeast two-hybrid screen to identify P50 molecular partners. Here we report the identification of actin as a P50-interacting Protein. Coimmunoprecipitation of P50 and actin from HeLa extracts as well as in vitro binding studies indicate specificity and a high affinity for the interaction between P50 and actin. Interestingly, P50 binding to actin is affected by mRNA; binding was observed at a low P50/mRNA ratio and was greatly reduced at higher ratios. Since the P50/mRNA ratio appears to be important for mRNA translatability, we speculate that P50 can regulate the attachment of mRNA to the actin network depending on its translational activity. Using immunofluorescence, we show that P50 binds to actin filaments in permeabilized cells and causes actin fibers to bundle in vitro. Together, these findings support the view that P50 may play an important role in mRNA transport, anchoring, and localization on actin filaments in the cell.
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Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated Protein, P50.
The international journal of biochemistry & cell biology, 1999Co-Authors: Valentina Evdokimova, Lev P. OvchinnikovAbstract:Abstract P50, the major core Protein of messenger ribonucleoProtein particles (mRNPs), is a universal Protein found exclusively in association with different mRNA species in the cytoplasm of somatic mammalian cells. Furthermore, P50 is the most abundant and tightly bound Protein within both inactive mRNPs and active mRNPs derived from polysomes, although the latter contain a lower level of P50. Recent experiments have revealed that, depending on the P50 to mRNA ratio, P50 may either act as a repressor or an activator of Protein synthesis. On the other hand, P50 exhibits about 98% amino acid sequence identity to mammalian transcription factors that bind specifically to Y-box containing DNA. Thus, it is a counterpart of the Y-box binding Proteins which are found in bacteria, plants and animals, exhibiting multiple biological activities ranging from transcriptional regulation of a wide variety of genes to `masking' mRNA activity in germinal cells. This review summarizes our current knowledge of P50 structure and function. It also discusses the biological roles of P50 and related Proteins in gene expression and describes the likely mechanisms of their action.
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The major cytoplasmic mRNP Protein, P50, is required for efficient mRNA translation in vitro
Biokhimiia (Moscow Russia), 1996Co-Authors: Elizaveta A. Kovrigina, Dmitry Nashchekin, Valentina Evdokimova, Lev P. OvchinnikovAbstract:The major cytoplasmic mRNP Protein of somatic cells, P50, is the member of the Y-box-binding transcription factor family and can control gene expression at two levels including mRNA transcription and translation. It has been demonstrated that P50 is responsible for the inactive state of mRNA within free mRNPs. In this work, we show that the Y-box-containing DNA (Y-DNA) predominantly binds to P50 in rabbit reticulocyte lysates and causes translation inhibition of the endogenous and exogenous globin mRNA and prokaryotic beta-galactosidase mRNA. Preincubation of Y-DNA with purified P50 prevents the inhibition. Inhibition of Protein biosynthesis by the Y-DNA is not due to the degradation or functional inactivation of mRNA. The inhibition is associated with the decay of polyribosomes and dissociation of a newly synthesized Protein from the ribosomes. The data indicate that Y-DNA inhibits Protein biosynthesis predominantly at the initiation stage and that P50 is an essential component of the translation initiation apparatus.
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The Major Protein of Messenger RibonucleoProtein Particles in Somatic Cells Is a Member of the Y-box Binding Transcription Factor Family
The Journal of biological chemistry, 1995Co-Authors: Valentina Evdokimova, Chia-lin Wei, Albert S. Sitikov, Peter N. Simonenko, Oleg A. Lazarev, Konstantin S. Vasilenko, Valentin A. Ustinov, John W.b. Hershey, Lev P. OvchinnikovAbstract:Abstract A cDNA encoding the major core Protein, P50, of cytoplasmic messenger ribonucleoProtein particles (mRNPs) of somatic cells was cloned from a rabbit reticulocyte cDNA library. From the derived 324-amino acid sequence, P50 is identified as a member of the Y-box binding transcription factor family. The Protein was earlier described as a repressor of globin mRNA translation. These findings suggest that P50 may affect Protein biosynthesis at two levels: mRNA transcription in the nucleus and mRNA translation in the cytoplasm. Together with recently published results showing that masked mRNA in germ cells also is associated with Proteins of the Y-box binding Protein family, the present finding indicates that these Proteins are universal core Proteins responsible for the formation of cytoplasmic mRNPs in eukaryotes. Highly purified P50 forms large 18 S homomultimeric complexes with a molecular mass of about 800 kilodaltons and melts RNA secondary structure. This suggests that P50 may affect translation by changing the overall structure of the mRNA.
Masamichi Isogai - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement Protein.
Virology, 2008Co-Authors: Hajime Yaegashi, Akihiro Tamura, Masamichi Isogai, Nobuyuki YoshikawaAbstract:Apple chlorotic leaf spot virus 50 kDa movement Protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.
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Apple chlorotic leaf spot virus 50 kDa movement Protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana.
Journal of General Virology, 2007Co-Authors: Hajime Yaegashi, Masamichi Isogai, Tsubasa Takahashi, Takashi Kobori, Satoshi T. Ohki, Nobu YoshikawaAbstract:Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa Protein (P216) involved in replication, a 50 kDa movement Protein (P50) and a 21 kDa coat Protein (CP). In this study, it was investigated whether these Proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent Protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these Proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.
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Interference of Long-Distance Movement of Grapevine berry inner necrosis virus in Transgenic Plants Expressing a Defective Movement Protein of Apple chlorotic leaf spot virus.
Phytopathology, 2006Co-Authors: Nobuyuki Yoshikawa, Y. Saitou, A. Kitajima, T Chida, N. Sasaki, Masamichi IsogaiAbstract:ABSTRACT Transgenic Nicotiana occidentalis plants expressing a movement Protein (P50) and partially functional deletion mutants (DeltaA and DeltaC) of the Apple chlorotic leaf spot virus (ACLSV) showed resistance to Grapevine berry inner necrosis virus (GINV). The resistance is highly effective and GINV was below the level of detection in both inoculated and uninoculated upper leaves. In contrast, GINV accumulated in inoculated and uninoculated leaves of nontransgenic (NT) plants and transgenic plants expressing a dysfunctional mutant (DeltaG). On the other hand, in some plants of a transgenic plant line expressing a deletion mutant (DeltaA', deletion of the C-terminal 42 amino acids), GINV could spread in inoculated leaves, but not move into uninoculated leaves. In a tissue blot hybridization analysis of DeltaA'-plants inoculated with GINV, virus could be detected in leaf blade, midribs, and petiole of inoculated leaves, but neither in stems immediately above inoculated leaves nor in any tissues of uninoculated leaves. Immunohistochemical analysis of GINV-inoculated leaves of DeltaA'-plants showed that GINV could invade into phloem parenchyma cells through bundle sheath of minor veins, suggesting that the long-distance transport of GINV might be inhibited between the phloem cells and sieve element (and/or within sieve element) rather than bundle sheath-phloem interfaces. Immunogold electron microscopy using an anti-P50 antiserum showed that P50 accumulated on the parietal layer of sieve elements and on sieve plates. The results suggested that resistance in P50-transgenic plants to GINV is due to the interference of both long-distance and cell-to-cell movement of the virus.
Hajime Yaegashi - One of the best experts on this subject based on the ideXlab platform.
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Transient expression of the coat Protein of Apple chlorotic leaf spot virus inhibits the viral RNA accumulation in Nicotiana occidentalis
Julius-Kühn-Archiv, 2010Co-Authors: Hajime Yaegashi, N. YoshikawaAbstract:The coat Protein of Apple chlorotic leaf spot virus (ACLSV-CP) plays a crucial role in infectivity and efficient viral RNA accumulation in host cells (J. Gen. Virol, 88, 2007). In this study, the effect of ACLSV-CP on viral RNA accumulation in Nicotiana occidentalis was investigated. The CP, CPm40 (an amino acid (aa) substitution of Ala to Ser at aa position 40), CPm75 (a substitution of Phe to Tyr at aa position 75), and CPm40m75 (two aa substitutions at positions 40 and 75) of ACLSV (P205) were transiently expressed in N. occidentalis leaves by agroinfiltration. Immunoblot analysis showed that CP and CPm40m75 accumulated in infiltrated tissues, in contrast to CPm40 and CPm75 which were not detected, suggesting that the stable accumulation of CP is important for effective viral RNA accumulation. However, co-agroinfiltration of an infectious ACLSV cDNA clone (pBICLSF) or pBICLSF-based CP mutants (pBICLCPm40, pBICLCPm75, and pBICLCPm40m75) with a vector expressing CP (pBE2113-CP) showed no viral genomic RNA accumulations were found in any leaves infiltrated with these constructs. The inhibition of ACLSV-RNA accumulation was found only in leaves co-expressed with CP Protein, but not with a frame-shift mutant of CP, a movement Protein (P50), and a frame-shift mutant of P50. Keywords : Apple chlorotic leaf spot virus , coat Protein, Protein stability, coat Protein mediated resistance (CP-MR), agroinfiltration
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Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement Protein.
Virology, 2008Co-Authors: Hajime Yaegashi, Akihiro Tamura, Masamichi Isogai, Nobuyuki YoshikawaAbstract:Apple chlorotic leaf spot virus 50 kDa movement Protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.
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Apple chlorotic leaf spot virus 50 kDa movement Protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana.
Journal of General Virology, 2007Co-Authors: Hajime Yaegashi, Masamichi Isogai, Tsubasa Takahashi, Takashi Kobori, Satoshi T. Ohki, Nobu YoshikawaAbstract:Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa Protein (P216) involved in replication, a 50 kDa movement Protein (P50) and a 21 kDa coat Protein (CP). In this study, it was investigated whether these Proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent Protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these Proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.
Wenming Zhao - One of the best experts on this subject based on the ideXlab platform.
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Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging
Journal of Virology, 2020Co-Authors: Wenming Zhao, Charbel Akkawi, Marylène Mougel, Susan RossAbstract:ApolipoProtein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) Protein to counteract APOBEC3 Proteins. Vif induces degradation of APOBEC3G and other APOBEC3 Proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like Protein. Here, we show that the MLV P50 Protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo. Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a Protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.
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murine leukemia virus P50 Protein counteracts apobec3 by blocking its packaging
Journal of Virology, 2020Co-Authors: Wenming Zhao, Charbel Akkawi, Marylène Mougel, Susan R RossAbstract:ApolipoProtein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) Protein to counteract APOBEC3 Proteins. Vif induces degradation of APOBEC3G and other APOBEC3 Proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like Protein. Here, we show that the MLV P50 Protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a Protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode Proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a Protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.
