scholarly journals Functional Replacement of Nucleocapsid Flanking Regions by Heterologous Counterparts with Divergent Primary Sequences: Effects of Chimeric Nucleocapsid on the Retroviral Replication Cycle

2003 ◽  
Vol 77 (1) ◽  
pp. 754-761 ◽  
Author(s):  
William Fu ◽  
Wei-Shau Hu
Keyword(s):  
Virus Assembly ◽  
Leukemia Virus ◽  
Replication Cycle ◽  
Viral Rna ◽  
Rna Packaging ◽  
Gag Polyprotein ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis ◽  
Flanking Regions

ABSTRACT Nucleocapsid (NC) proteins in most retroviruses have a well-conserved Cys-His box(es) as well as more divergent flanking regions that are rich in basic residues. Mutations in the flanking regions can affect RNA packaging, virus assembly, and reverse transcription of the viral RNA. To gain a further understanding of the roles of NC flanking regions in the retroviral replication cycle, we generated and characterized chimeric gag-pol expression constructs derived from murine leukemia virus and spleen necrosis virus by replacing an NC flanking region from one virus with the counterpart from the other virus. We found that all four chimeras were able to generate virions, package viral RNA, and complete the viral replication cycle. Two chimeras had mild defects in virus assembly that correlated with a decrease in the isoelectric points of NCs, suggesting that the basic nature of NC is important in virus assembly. This finding indicates that, although the primary sequences of these flanking regions have little homology, the heterologous sequences are functional both as part of the Gag polyprotein and as processed NC protein.

scholarly journals Involvement of the Matrix and Nucleocapsid Domains of the Bovine Leukemia Virus Gag Polyprotein Precursor in Viral RNA Packaging

2003 ◽  
Vol 77 (17) ◽  
pp. 9431-9438 ◽  
Author(s):  
Huating Wang ◽  
Kendra M. Norris ◽  
Louis M. Mansky
Keyword(s):  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Limited Information ◽  
Rna Packaging ◽  
Alanine Scanning Mutagenesis ◽  
Basic Residue ◽  
Gag Polyprotein ◽  
Human T Cell ◽  
Polyprotein Precursor

ABSTRACT The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.

scholarly journals The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

1999 ◽  
Vol 73 (11) ◽  
pp. 9170-9177 ◽  
Author(s):  
Jeanine L. Certo ◽  
Timur O. Kabdulov ◽  
Michelle L. Paulson ◽  
Jeffrey A. Anderson ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Viral Vector ◽  
Leukemia Virus ◽  
Gene Products ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Necrosis Virus ◽  
Spleen Necrosis Virus ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimericgag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Ψ) and another viral vector RNA containing the SNV packaging signal (E). The chimericgag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Ψ. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLVpol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNVcis-acting elements are not ideal substrates for MLVpol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and othercis-acting elements.

scholarly journals cis-Acting Elements Important for Retroviral RNA Packaging Specificity

2002 ◽  
Vol 76 (10) ◽  
pp. 4950-4960 ◽  
Author(s):  
Benjamin E. Beasley ◽  
Wei-Shau Hu
Keyword(s):  
Leukemia Virus ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Core Element ◽  
Viral Packaging ◽  
Cis Acting ◽  
Spleen Necrosis ◽  
Flanking Regions ◽  
Packaging Signals

ABSTRACT Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Ψ and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Ψ. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5′-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.

scholarly journals Nonreciprocal Pseudotyping: Murine Leukemia Virus Proteins Cannot Efficiently Package Spleen Necrosis Virus-Based Vector RNA

1998 ◽  
Vol 72 (7) ◽  
pp. 5408-5413 ◽  
Author(s):  
Jeanine L. Certo ◽  
Betsy F. Shook ◽  
Philip D. Yin ◽  
John T. Snider ◽  
Wei-Shau Hu
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rna Structures ◽  
Packaging Signal ◽  
Necrosis Virus ◽  
Hairpin Rna ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis ◽  
Murine Leukemia

ABSTRACT It has been documented that spleen necrosis virus (SNV) can package murine leukemia virus (MLV) RNA efficiently and propagate MLV vectors to the same titers as it propagates SNV-based vectors. Although the SNV packaging signal (E) and MLV packaging signal (Ψ) have little sequence homology, similar double-hairpin RNA structures were predicted and supported by experimental evidence. To test whether SNV RNA can be packaged by MLV proteins, we modified an SNV vector to be expressed in an MLV-based murine helper cell line. Surprisingly, we found that MLV proteins could not support the replication of SNV vectors. The decrease in titer was approximately 2,000- to 20,000-fold in one round of retroviral replication. RNA analysis revealed that SNV RNA was not efficiently packaged by MLV proteins. RNA hybridization of the cellular and viral RNAs indicated that SNV RNA was packaged at least 25-fold less efficiently than MLV RNA, which was the sensitivity limit of the hybridization assay. The contrast between the MLV and SNV packaging specificity is striking. SNV proteins can recognize both SNV E and MLV Ψ, but MLV can recognize only MLV Ψ. This is the first demonstration of two retroviruses with nonreciprocal packaging specificities.

scholarly journals Retroviral Recombination Rates Do Not Increase Linearly with Marker Distance and Are Limited by the Size of the Recombining Subpopulation

1998 ◽  
Vol 72 (2) ◽  
pp. 1195-1202 ◽  
Author(s):  
Jeffrey A. Anderson ◽  
Ella Harvey Bowman ◽  
Wei-Shau Hu
Keyword(s):  
Homologous Recombination ◽  
Recombination Rate ◽  
Leukemia Virus ◽  
Recombination Rates ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis ◽  
Marker Distance ◽  
Retroviral Recombination ◽  
Previous Assumption

ABSTRACT Recombination occurs at high frequencies in all examined retroviruses. The previously determined homologous recombination rate in one retroviral replication cycle is 4% for markers 1.0 kb apart in spleen necrosis virus (SNV). This has often been used to suggest that approximately 30 to 40% of the replication-competent viruses with 7- to 10-kb genomes undergo recombination. These estimates were based on the untested assumption that a linear relationship exists between recombination rates and marker distances. To delineate this relationship, we constructed three sets of murine leukemia virus (MLV)-based vectors containing the neomycin phosphotransferase gene (neo) and the hygromycin phosphotransferase B gene (hygro). Each set contained one vector with a functionalneo and an inactivated hygro and one vector with a functional hygro and an inactivated neo. The two inactivating mutations in the three sets of vectors were separated by 1.0, 1.9, and 7.1 kb. Recombination rates after one round of replication were 4.7, 7.4, and 8.2% with markers 1.0, 1.9, and 7.1 kb apart, respectively. Thus, the rate of homologous recombination with 1.0 kb of marker distance is similar in MLV and SNV. The recombination rate increases when the marker distance increases from 1.0 to 1.9 kb; however, the recombination rates with marker distances of 1.9 and 7.1 kb are not significantly different. These data refute the previous assumption that recombination is proportional to marker distance and define the maximum recombining population in retroviruses.

scholarly journals Effects of Gag Mutation and Processing on Retroviral Dimeric RNA Maturation

2006 ◽  
Vol 80 (3) ◽  
pp. 1242-1249 ◽  
Author(s):  
William Fu ◽  
Que Dang ◽  
Kunio Nagashima ◽  
Eric O. Freed ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Viral Protein ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Protein Cleavage ◽  
Rna Packaging ◽  
Virus Rna ◽  
Rna Dimer ◽  
Rna Maturation ◽  
Hiv 1

ABSTRACT After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.

scholarly journals Spliced Spleen Necrosis Virus Vector RNA Is Not Encapsidated: Implications for Retroviral Replication and Vector Design

2004 ◽  
Vol 9 (4) ◽  
pp. 557-565 ◽  
Author(s):  
Adrienne Goodrich ◽  
Zahida Parveen ◽  
Ralph Dornburg ◽  
Matthias J Schnell ◽  
Roger J Pomerantz
Keyword(s):  
Virus Vector ◽  
Necrosis Virus ◽  
Spleen Necrosis Virus ◽  
Retroviral Replication ◽  
Spleen Necrosis

scholarly journals Unpaired Guanosines in the 5′ Untranslated Region of HIV-1 RNA Act Synergistically To Mediate Genome Packaging

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Olga A. Nikolaitchik ◽  
Xayathed Somoulay ◽  
Jonathan M. O. Rawson ◽  
Jennifer A. Yoo ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Binding Sites ◽  
Untranslated Region ◽  
Virus Assembly ◽  
Viral Rna ◽  
Rna Packaging ◽  
Genome Packaging ◽  
Stem Loop ◽  
Rna Genome ◽  
Genome Encapsidation ◽  
Hiv 1

ABSTRACT The viral protein Gag selects full-length HIV-1 RNA from a large pool of mRNAs as virion genome during virus assembly. Currently, the precise mechanism that mediates the genome selection is not understood. Previous studies have identified several sites in the 5′ untranslated region (5′ UTR) of HIV-1 RNA that are bound by nucleocapsid (NC) protein, which is derived from Gag during virus maturation. However, whether these NC binding sites direct HIV-1 RNA genome packaging has not been fully investigated. In this report, we examined the roles of single-stranded exposed guanosines at NC binding sites in RNA genome packaging using stable cell lines expressing competing wild-type and mutant HIV-1 RNAs. Mutant RNA packaging efficiencies were determined by comparing their prevalences in cytoplasmic RNA and in virion RNA. We observed that multiple NC binding sites affected RNA packaging; of the sites tested, those located within stem-loop 1 of the 5′ UTR had the most significant effects. These sites were previously reported as the primary NC binding sites by using a chemical probe reverse-footprinting assay and as the major Gag binding sites by using an in vitro assay. Of the mutants tested in this report, substituting 3 to 4 guanosines resulted in <2-fold defects in packaging. However, when mutations at different NC binding sites were combined, severe defects were observed. Furthermore, combining the mutations resulted in synergistic defects in RNA packaging, suggesting redundancy in Gag-RNA interactions and a requirement for multiple Gag binding on viral RNA during HIV-1 genome encapsidation. IMPORTANCE HIV-1 must package its RNA genome during virus assembly to generate infectious viruses. To better understand how HIV-1 packages its RNA genome, we examined the roles of RNA elements identified as binding sites for NC, a Gag-derived RNA-binding protein. Our results demonstrate that binding sites within stem-loop 1 of the 5′ untranslated region play important roles in genome packaging. Although mutating one or two NC-binding sites caused only mild defects in packaging, mutating multiple sites resulted in severe defects in genome encapsidation, indicating that unpaired guanosines act synergistically to promote packaging. Our results suggest that Gag-RNA interactions occur at multiple RNA sites during genome packaging; furthermore, there are functionally redundant binding sites in viral RNA.

scholarly journals Proper Processing of Avian Sarcoma/Leukosis Virus Capsid Proteins Is Required for Infectivity

2001 ◽  
Vol 75 (13) ◽  
pp. 6016-6021 ◽  
Author(s):  
Yan Xiang ◽  
Rebekah Thorick ◽  
Marcy L. Vana ◽  
Rebecca Craven ◽  
Jonathan Leis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Virus Assembly ◽  
Leukemia Virus ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Amino Acid Substitutions ◽  
Gag Polyprotein ◽  
Initial Cleavage ◽  
Avian Sarcoma

ABSTRACT The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430–6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.

Sign in / Sign up

Export Citation Format

Share Document