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Shinji Makino - One of the best experts on this subject based on the ideXlab platform.
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Roles of the Coding and Noncoding Regions of Rift Valley Fever Virus RNA Genome Segments in Viral RNA Packaging
Journal of virology, 2012Co-Authors: Shin Murakami, Krishna Narayanan, Kaori Terasaki, Shinji MakinoAbstract:ABSTRACT We characterized the RNA elements involved in the Packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5′-terminal 25 nucleotides of each RNA segment were equally competent for RNA Packaging and carried an RNA Packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA Packaging.
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Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal
Journal of Virology, 2003Co-Authors: Krishna Narayanan, Junko Maeda, Chun-jen Chen, Shinji MakinoAbstract:For any of the enveloped RNA viruses studied to date, recognition of a specific RNA Packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA Packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the Packaging signal, determines the selective RNA Packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral Packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-Packaging signal interaction led to specific Packaging of the Packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA Packaging mechanism for an enveloped virus, where the specific RNA Packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.
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cooperation of an RNA Packaging signal and a viral envelope protein in coronavirus RNA Packaging
Journal of Virology, 2001Co-Authors: Krishna Narayanan, Shinji MakinoAbstract:Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective Packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA Packaging signal is necessary and sufficient for Packaging RNA into MHV particles. The present study tested the possibility that the selective M protein-mRNA 1 interaction is due to the Packaging signal in mRNA 1. Regardless of the presence or absence of the Packaging signal, N protein bound to MHV defective interfering RNAs and intracellularly expressed non-MHV RNA transcripts to form ribonucleoprotein complexes; M protein, however, interacted selectively with RNAs containing the Packaging signal. Moreover, only the RNA that interacted selectively with M protein was efficiently packaged into MHV particles. Thus, it was the Packaging signal that mediated the selective interaction between M protein and viral RNA to drive the specific Packaging of RNA into virus particles. This is the first example for any RNA virus in which a viral envelope protein and a known viral RNA Packaging signal have been shown to determine the specificity and selectivity of RNA Packaging into virions.
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Characterization of Coronavirus DI RNA Packaging
Advances in experimental medicine and biology, 1998Co-Authors: Kyongmin Hwang Kim, Krishna Narayanan, Shinji MakinoAbstract:Studies of defective interfering (DI) RNAs of mouse hepatitis virus (MHV), suggest that a 69 nt-long Packaging signal, which is located about 20 kb from the 5’-end of the 31 kb-long MHV genomic RNA, is necessary and sufficient for MHV genomic RNA Packaging into MHV particles. We demonstrated that use of a low pH culture medium combined with subsequent ultrafiltration increased MHV infectivity about 60 times over MHV preparations grown in neutral medium. Using this virus concentration procedure, we successfully prepared DI particle-rich MHV preparations. Characterization of virus samples released from the cells infected with DI particle-rich MHV revealed that infectious MHV genomic RNA was not required for Packaging of DI RNAs. These data suggested that interaction of the DI Packaging signal with an unidentified region(s) of helper virus genomic RNA is unlikely, and therefore unlikely to facilitate the Packaging of MHV DI RNA into the MHV virion. Rather, both DI RNA and MHV genomic RNA probably use the Packaging signal for RNA Packaging.
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Murine coronavirus Packaging signal confers Packaging to nonviral RNA.
Journal of virology, 1997Co-Authors: K. Woo, Krishna Narayanan, Kyongmin Hwang Kim, M. Joo, Shinji MakinoAbstract:Studies of defective interfering (DI) RNAs of the murine coronavirus mouse hepatitis virus (MHV) suggest that a 69-nucleotide-long Packaging signal is necessary for MHV genomic RNA Packaging into MHV particles. In this study we showed that when RNA transcripts that consisted of a non-MHV sequence and the Packaging signal were expressed in MHV-infected cells, they were packaged into MHV particles. Those RNA transcripts that lacked the Packaging signal or those containing a mutated Packaging signal did not package efficiently. Thus, the presence of the Packaging signal was sufficient for RNA Packaging into MHV particles.
Anna Aldovini - One of the best experts on this subject based on the ideXlab platform.
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the simian immunodeficiency virus 5 untranslated leader sequence plays a role in intracellular viral protein accumulation and in RNA Packaging
Journal of Virology, 2003Co-Authors: Jignesh Patel, Anna Aldovini, Elena Izmailova, Shainn Wei WangAbstract:We investigated the role of 5′ untranslated leader sequences of simian immunodeficiency virus (SIVmac239) in RNA encapsidation and protein expression. A series of progressively longer deletion mutants was constructed with a common endpoint six nucleotides upstream of the gag initiation codon and another endpoint at the 3′ end of the primer binding site (PBS). We found that efficient intracellular Gag-Pol protein accumulation required the region between the PBS and splice donor (SD) site. Marked reduction of genomic RNA Packaging was observed with all the deletion mutants that involved sequences at both the 5′ and at the 3′ ends of the major SD site, and increased nonspecific RNA incorporation could be detected in these mutants. RNA encapsidation was affected only modestly by a deletion of 54 nucleotides at the 3′ end of the SD site when the mutant construct pΔ54 was transfected alone. In contrast, the amount of pΔ54 genomic RNA incorporated into particles was reduced more than 10-fold when this mutant was cotransfected with a construct specifying an RNA molecule with a wild-type Packaging signal. Therefore, we conclude that the 175 nucleotides located 5′ of the gag initiation codon are critical for efficient and selective incorporation of genomic RNA into virions. This location of the SIV Ψ element provides the means for efficient discrimination between viral genomic and spliced RNAs.
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Functional Analysis of the Murine Sarcoma Virus RNA Packaging Sequence
Journal of virology, 2002Co-Authors: Elena Izmailova, Anna AldoviniAbstract:We investigated the features of the Moloney murine sarcoma virus leader sequence necessary for RNA Packaging function by using a deletion analysis approach. We found that sequences that extend beyond those characterized genetically in previous reports are important for optimal Packaging efficiency. A fragment covering a minimum of four potential stem-loop structures is required for the shortest Packaging element compatible with gene transfer. Our results reveal the extent to which each of the segments of the Packaging sequence contribute to Packaging efficiency.
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Human immunodeficiency virus nucleocapsid protein polymorphisms modulate the infectivity of RNA Packaging mutants.
Virology, 2002Co-Authors: Paul Krogstad, Yongzhi Geng, Osvaldo Rey, Jude Canon, F. Javier Ibarrondo, Bradley Ackerson, Jignesh Patel, Anna AldoviniAbstract:The nucleocapsid protein (NC) of retroviruses is involved in viral RNA Packaging and initiation of reverse transcription. NC also mediates interactions between Gag and actin filaments. We found that residues at the amino terminus of NC are involved in efficient actin binding. When alanine residues were substituted for the arginine and lysine at positions 10 and 11 of NC in HIV(NL4-3), these mutations decreased actin binding but had only a modest effect on virus infectivity. A similarly mutated virus based on the HXB2 clone of HIV was not infectious. Mutational analysis of NL4-3 NC residues demonstrated that NC polymorphisms modulated the phenotype of NC mutations. Conservative amino acid differences between HXB2 and NL4-3 NCs were sufficient to explain the difference in infectivity of viruses carrying the R10A and K11A mutations.
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Nucleocapsid and matrix protein contributions to selective human immunodeficiency virus type 1 genomic RNA Packaging.
Journal of virology, 1998Co-Authors: D. T. K. Poon, Anna AldoviniAbstract:The nucleocapsid protein (NC) of retroviruses plays a major role in genomic RNA Packaging, and some evidence has implicated the matrix protein (MA) of certain retroviruses in viral RNA binding. To further investigate the role of NC in the selective recognition of genomic viral RNA and to address the potential contribution of MA in this process, we constructed chimeric and deletion human immunodeficiency virus type 1 (HIV-1) mutants that alter the NC or MA protein. Both HIV and mouse mammary tumor virus (MMTV) NC proteins have two zinc-binding domains and similar basic amino acid compositions but differ substantially in total length, amino acid sequence, and spacing of the zinc-binding motifs. When the entire NC coding sequence of HIV was replaced with the MMTV NC coding sequence, we found that the HIV genome was incorporated into virions at 50% of wild-type levels. Viruses produced from chimeric HIV genomes with complete NC replacements, or with the two NC zinc-binding domains replaced with MMTV sequences, preferentially incorporated HIV genomes when both HIV and MMTV genomes were simultaneously present in the cell. Viruses produced from chimeric MMTV genomes in which the MMTV NC had been replaced with HIV NC preferentially incorporated MMTV genomes when both HIV and MMTV genomes were simultaneously present in the cell. In contrast, viruses produced from chimeric HIV genomes containing the Moloney NC, which contains a single zinc-binding motif, were previously shown to preferentially incorporate Moloney genomic RNA. Taken together, these results indicate that an NC protein with two zinc-binding motifs is required for specific HIV RNA Packaging and that the amino acid context of these motifs, while contributing to the process, is less crucial for specificity. The data also suggest that HIV NC may not be the exclusive determinant of RNA selectivity. Analysis of an HIV MA mutant revealed that specific RNA Packaging does not require MA protein.
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Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA Packaging and infectivity.
Journal of virology, 1996Co-Authors: D. T. K. Poon, Anna AldoviniAbstract:Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA Packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA Packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA Packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA Packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA Packaging.
Krishna Narayanan - One of the best experts on this subject based on the ideXlab platform.
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Roles of the Coding and Noncoding Regions of Rift Valley Fever Virus RNA Genome Segments in Viral RNA Packaging
Journal of virology, 2012Co-Authors: Shin Murakami, Krishna Narayanan, Kaori Terasaki, Shinji MakinoAbstract:ABSTRACT We characterized the RNA elements involved in the Packaging of Rift Valley fever virus RNA genome segments, L, M, and S. The 5′-terminal 25 nucleotides of each RNA segment were equally competent for RNA Packaging and carried an RNA Packaging signal, which overlapped with the RNA replication signal. Only the deletion mutants of L RNA, but not full-length L RNA, were efficiently packaged, implying the possible requirement of RNA compaction for L RNA Packaging.
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Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal
Journal of Virology, 2003Co-Authors: Krishna Narayanan, Junko Maeda, Chun-jen Chen, Shinji MakinoAbstract:For any of the enveloped RNA viruses studied to date, recognition of a specific RNA Packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA Packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the Packaging signal, determines the selective RNA Packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral Packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-Packaging signal interaction led to specific Packaging of the Packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA Packaging mechanism for an enveloped virus, where the specific RNA Packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.
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cooperation of an RNA Packaging signal and a viral envelope protein in coronavirus RNA Packaging
Journal of Virology, 2001Co-Authors: Krishna Narayanan, Shinji MakinoAbstract:Murine coronavirus mouse hepatitis virus (MHV) produces a genome-length mRNA, mRNA 1, and six or seven species of subgenomic mRNAs in infected cells. Among these mRNAs, only mRNA 1 is efficiently packaged into MHV particles. MHV N protein binds to all MHV mRNAs, whereas envelope M protein interacts only with mRNA 1. This M protein-mRNA 1 interaction most probably determines the selective Packaging of mRNA 1 into MHV particles. A short cis-acting MHV RNA Packaging signal is necessary and sufficient for Packaging RNA into MHV particles. The present study tested the possibility that the selective M protein-mRNA 1 interaction is due to the Packaging signal in mRNA 1. Regardless of the presence or absence of the Packaging signal, N protein bound to MHV defective interfering RNAs and intracellularly expressed non-MHV RNA transcripts to form ribonucleoprotein complexes; M protein, however, interacted selectively with RNAs containing the Packaging signal. Moreover, only the RNA that interacted selectively with M protein was efficiently packaged into MHV particles. Thus, it was the Packaging signal that mediated the selective interaction between M protein and viral RNA to drive the specific Packaging of RNA into virus particles. This is the first example for any RNA virus in which a viral envelope protein and a known viral RNA Packaging signal have been shown to determine the specificity and selectivity of RNA Packaging into virions.
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Characterization of Coronavirus DI RNA Packaging
Advances in experimental medicine and biology, 1998Co-Authors: Kyongmin Hwang Kim, Krishna Narayanan, Shinji MakinoAbstract:Studies of defective interfering (DI) RNAs of mouse hepatitis virus (MHV), suggest that a 69 nt-long Packaging signal, which is located about 20 kb from the 5’-end of the 31 kb-long MHV genomic RNA, is necessary and sufficient for MHV genomic RNA Packaging into MHV particles. We demonstrated that use of a low pH culture medium combined with subsequent ultrafiltration increased MHV infectivity about 60 times over MHV preparations grown in neutral medium. Using this virus concentration procedure, we successfully prepared DI particle-rich MHV preparations. Characterization of virus samples released from the cells infected with DI particle-rich MHV revealed that infectious MHV genomic RNA was not required for Packaging of DI RNAs. These data suggested that interaction of the DI Packaging signal with an unidentified region(s) of helper virus genomic RNA is unlikely, and therefore unlikely to facilitate the Packaging of MHV DI RNA into the MHV virion. Rather, both DI RNA and MHV genomic RNA probably use the Packaging signal for RNA Packaging.
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Murine coronavirus Packaging signal confers Packaging to nonviral RNA.
Journal of virology, 1997Co-Authors: K. Woo, Krishna Narayanan, Kyongmin Hwang Kim, M. Joo, Shinji MakinoAbstract:Studies of defective interfering (DI) RNAs of the murine coronavirus mouse hepatitis virus (MHV) suggest that a 69-nucleotide-long Packaging signal is necessary for MHV genomic RNA Packaging into MHV particles. In this study we showed that when RNA transcripts that consisted of a non-MHV sequence and the Packaging signal were expressed in MHV-infected cells, they were packaged into MHV particles. Those RNA transcripts that lacked the Packaging signal or those containing a mutated Packaging signal did not package efficiently. Thus, the presence of the Packaging signal was sufficient for RNA Packaging into MHV particles.
Rodney S. Russell - One of the best experts on this subject based on the ideXlab platform.
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The T12I mutation within the SP1 region of Gag restricts Packaging of spliced viral RNA into human immunodeficiency virus type 1 with mutated RNA Packaging signals and mutated nucleocapsid sequence.
Virology, 2005Co-Authors: Bibhuti Bhusan Roy, Rodney S. Russell, Dan Turner, Chen LiangAbstract:Abstract Specific Packaging of human immunodeficiency virus type 1 (HIV-1) RNA is attributable to the high affinity of nucleocapsid (NC) sequence of Gag for the cis-acting RNA Packaging signals located within the 5′ un-translated region (5′ UTR). Interestingly, we have previously reported that the T12I mutation (named MP2) within SP1 of Gag prevented incorporation of spliced viral RNA into mutated viruses that lacked the stem-loop 1 (SL1) RNA element (also named dimerization initiation site, DIS), suggesting a role for the SP1 sequence in viral RNA Packaging. In this study, we have further tested this activity of MP2 in the context of a variety of mutations that affect viral RNA incorporation. The results showed that MP2 was able to effectively restrict Packaging of spliced viral RNA into viruses containing either NC mutations R10A and K11A or mutated 5′ UTR sequence, such as ΔGU3 that lacked the 112-GUCUGUUGUGUG-123 sequence of U5, D1 that was deleted of a 27 nt fragment immediately downstream of the primer binding site (PBS), Δ(306–325) that had the SL3 RNA element removed and MD2 that was missing the 328-GGAG-331 sequence. As a result, MP2 contributed increased infectivity to the related viruses. Therefore, the MP2 mutation demonstrates a distinct role in HIV-1 RNA Packaging that is neither pertained to the specific viral RNA Packaging signal nor to the NC sequence.
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a riboswitch regulates RNA dimerization and Packaging in human immunodeficiency virus type 1 virions
Journal of Virology, 2004Co-Authors: Marcel Ooms, Rodney S. Russell, Chen Liang, Hendrik Huthoff, Ben BerkhoutAbstract:The genome of retroviruses, including human immunodeficiency virus type 1 (HIV-1), consists of two identical RNA strands that are packaged as noncovalently linked dimers. The core Packaging and dimerization signals are located in the downstream part of the untranslated leader of HIV-1 RNA—the Ψ and the dimerization initiation site (DIS) hairpins. The HIV-1 leader can adopt two alteRNAtive conformations that differ in the presentation of the DIS hairpin and consequently in their ability to dimerize in vitro. The branched multiple-hairpin (BMH) structure folds the poly(A) and DIS hairpins, but these domains are base paired in a long distance interaction (LDI) in the most stable LDI conformation. This LDI-BMH riboswitch regulates RNA dimerization in vitro. It was recently shown that the Ψ hairpin structure is also presented differently in the LDI and BMH structures. Several detailed in vivo studies have indicated that sequences throughout the leader RNA contribute to RNA Packaging, but how these diverse mutations affect the Packaging mechanism is not known. We reasoned that these effects may be due to a change in the LDI-BMH equilibrium, and we therefore reanalyzed the structural effects of a large set of leader RNA mutations that were presented in three previous studies (J. L. Clever, D. Mirandar, Jr., and T. G. Parslow, J. Virol. 76: 12381-12387, 2002; C. Helga-Maria, M. L. Hammarskjold, and D. Rekosh, J. Virol. 73: 4127-4135, 1999; R. S. Russell, J. Hu, V. Beriault, A. J. Mouland, M. Laughrea, L. Kleiman, M. A. Wainberg, and C. Liang, J. Virol. 77: 84-96, 2003). This analysis revealed a strict correlation between the status of the LDI-BMH equilibrium and RNA Packaging. Furthermore, a correlation is apparent between RNA dimerization and RNA Packaging, and these processes may be coordinated by the same LDI-BMH riboswitch mechanism.
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sequences downstream of the 5 splice donor site are required for both Packaging and dimerization of human immunodeficiency virus type 1 RNA
Journal of Virology, 2003Co-Authors: Andrew J Mouland, Rodney S. Russell, Jing Hu, Veronique Beriault, Lawrence Kleiman, Mark A. WainbergAbstract:Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5′ splice donor site at nucleotide (nt) 289 have been shown to act as Packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a “kissing-loop” mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other Packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA Packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as Packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-Packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA Packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5′ splice donor site, are required for efficient RNA Packaging and dimerization.
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Sequences Downstream of the 5′ Splice Donor Site Are Required for both Packaging and Dimerization of Human Immunodeficiency Virus Type 1 RNA
Journal of virology, 2003Co-Authors: Rodney S. Russell, Andrew J Mouland, Mark A. Wainberg, Veronique Beriault, Lawrence Kleiman, Chen LiangAbstract:Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5′ splice donor site at nucleotide (nt) 289 have been shown to act as Packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a “kissing-loop” mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other Packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA Packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as Packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-Packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA Packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5′ splice donor site, are required for efficient RNA Packaging and dimerization.
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Translation of Pr55gag Augments Packaging of Human Immunodeficiency Virus Type 1 RNA in a Cis-Acting Manner
AIDS research and human retroviruses, 2002Co-Authors: Chen Liang, Rodney S. Russell, Mark A. WainbergAbstract:The full-length RNA of human immunodeficiency virus type 1 (HIV-1) serves both as a messenger (mRNA) to direct the translation of Pr55(gag) proteins and as genomic or viral particle RNA (vpRNA) to be packaged into virions. In this study, we have assessed a putative cis-acting effect of Pr55(gag) translation on HIV-1 RNA Packaging. To pursue this subject, we have measured the relative competence of two distinct types of HIV-1 RNA for being packaged by virus particles under conditions in which only one of them is permissive for production of Pr55(gag). Not surprisingly, wild-type BH10 RNA was packaged at far higher efficiency than that associated with mutant viral RNA that was deleted of RNA Packaging signals and incapable of Pr55(gag) production. However, when production of Pr55(gag) was eliminated from the wild-type BH10 viral RNA by insertion of stop codons either in matrix (MA) or in capsid (CA) sequences, regardless of retention of wild-type RNA Packaging signals, these Pr55(gag)-deficient viral RNAs were packaged at low levels similar to those observed with viral RNA species that lack RNA Packaging signals and are capable of Pr55(gag) generation. Moreover, loss of Pr55(gag) production did not affect stability of the relevant viral RNA; this observation rules out the possibility that lowered Packaging efficiency associated with Pr55(gag)-deficient HIV-1 RNA is a result of reduced RNA stability. Taken together, our data demonstrate that cis translation of Pr55(gag) is needed for efficient Packaging of HIV-1 RNA.
Mark A. Wainberg - One of the best experts on this subject based on the ideXlab platform.
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Recovery of fitness of a live attenuated simian immunodeficiency virus through compensation in both the coding and non-coding regions of the viral genome
Retrovirology, 2007Co-Authors: James B Whitney, Mark A. WainbergAbstract:We have analyzed a SIV deletion mutant that was compromised both in viral replication and RNA Packaging. Serial passage of this variant in two different T-cell lines resulted in compensatory reversion and the generation of independent groups of point mutations within each cell line. Within each group, single point mutations were shown to contribute to increased viral infectivity and the rescue of wild-type replication kinetics. The complete recovery of viral fitness ultimately correlated with the restoration of viral RNA Packaging. Consistent with the latter finding was the rescue of Pr^55 Gag processing, also restoring proper virus core morphology in mature virions. These seemingly independently arising groups of compensatory mutations were functionally interchangeable in regard to the recovery of wild type replication in rhesus PBMCs. These findings indicate that viral reversion that overcomes a genetic bottleneck is not limited to a single pathway, and illustrates the remarkable adaptability of lentiviruses.
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Sequences Downstream of the 5′ Splice Donor Site Are Required for both Packaging and Dimerization of Human Immunodeficiency Virus Type 1 RNA
Journal of virology, 2003Co-Authors: Rodney S. Russell, Andrew J Mouland, Mark A. Wainberg, Veronique Beriault, Lawrence Kleiman, Chen LiangAbstract:Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5′ splice donor site at nucleotide (nt) 289 have been shown to act as Packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a “kissing-loop” mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other Packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA Packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as Packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-Packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA Packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5′ splice donor site, are required for efficient RNA Packaging and dimerization.
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sequences downstream of the 5 splice donor site are required for both Packaging and dimerization of human immunodeficiency virus type 1 RNA
Journal of Virology, 2003Co-Authors: Andrew J Mouland, Rodney S. Russell, Jing Hu, Veronique Beriault, Lawrence Kleiman, Mark A. WainbergAbstract:Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5′ splice donor site at nucleotide (nt) 289 have been shown to act as Packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a “kissing-loop” mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other Packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA Packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as Packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-Packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA Packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5′ splice donor site, are required for efficient RNA Packaging and dimerization.
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Translation of Pr55gag Augments Packaging of Human Immunodeficiency Virus Type 1 RNA in a Cis-Acting Manner
AIDS research and human retroviruses, 2002Co-Authors: Chen Liang, Rodney S. Russell, Mark A. WainbergAbstract:The full-length RNA of human immunodeficiency virus type 1 (HIV-1) serves both as a messenger (mRNA) to direct the translation of Pr55(gag) proteins and as genomic or viral particle RNA (vpRNA) to be packaged into virions. In this study, we have assessed a putative cis-acting effect of Pr55(gag) translation on HIV-1 RNA Packaging. To pursue this subject, we have measured the relative competence of two distinct types of HIV-1 RNA for being packaged by virus particles under conditions in which only one of them is permissive for production of Pr55(gag). Not surprisingly, wild-type BH10 RNA was packaged at far higher efficiency than that associated with mutant viral RNA that was deleted of RNA Packaging signals and incapable of Pr55(gag) production. However, when production of Pr55(gag) was eliminated from the wild-type BH10 viral RNA by insertion of stop codons either in matrix (MA) or in capsid (CA) sequences, regardless of retention of wild-type RNA Packaging signals, these Pr55(gag)-deficient viral RNAs were packaged at low levels similar to those observed with viral RNA species that lack RNA Packaging signals and are capable of Pr55(gag) generation. Moreover, loss of Pr55(gag) production did not affect stability of the relevant viral RNA; this observation rules out the possibility that lowered Packaging efficiency associated with Pr55(gag)-deficient HIV-1 RNA is a result of reduced RNA stability. Taken together, our data demonstrate that cis translation of Pr55(gag) is needed for efficient Packaging of HIV-1 RNA.
