scholarly journals Alteration of Zinc-Binding Residues of Simian Immunodeficiency Virus p8NC Results in Subtle Differences in Gag Processing and Virion Maturation Associated with Degradative Loss of Mutant NC

2001 ◽  
Vol 75 (1) ◽  
pp. 115-124 ◽  
Author(s):  
Jason L. Yovandich ◽  
Elena N. Chertova ◽  
Brad P. Kane ◽  
Tracy D. Gagliardi ◽  
Julian W. Bess ◽  
...  
Keyword(s):  
Simian Immunodeficiency Virus ◽  
High Performance ◽  
Point Mutations ◽  
Particle Morphology ◽  
Reversed Phase ◽  
Significant Loss ◽  
Full Length ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Immunodeficiency Virus

ABSTRACT In all retroviruses analyzed to date (except for the spumaretroviruses), the Zn2+-coordinating residues of nucleocapsid (NC) perform or assist in crucial reactions necessary to complete the retrovirus life cycle. Six replication-defective mutations have been engineered in the two NC Zn2+ fingers (ZFs) of simian immunodeficiency virus [SIV(Mne)] that change or delete specific Zn2+-interacting Cys residues and were studied by using electron microscopy, reversed-phase high-performance liquid chromatography, immunoblotting, and RNA quantification. We focused on phenotypes of produced particles, specifically morphology, Gag polyprotein processing, and genomic RNA packaging. Phenotypes were similar among viruses containing a point or deletion mutation involving the same ZF. Mutations in the proximal ZF (ZF1) resulted in near-normal Gag processing and full-length genomic RNA incorporation and were most similar to wild-type (WT) virions with electron-dense, conical cores. Mutation of the distal ZF, as well as point mutations in both ZFs, resulted in more unprocessed Gag proteins than a deletion or point mutation in ZF1, with an approximate 30% reduction in levels of full-length genomic RNA in virions. These mutant virions contained condensed cores; however, the cores typically appeared less electron dense and more rod shaped than WT virions. Surprisingly, deletion of both ZFs, including the basic linker region between the ZFs, resulted in the most efficient Gag processing. However, genomic RNA packaging was ∼10% of WT levels, and those particles produced were highly abnormal with respect to size and core morphology. Surprisingly, all NC mutations analyzed demonstrated a significant loss of processed NC in virus particles, suggesting that Zn2+-coordinated NC is protected from excessive proteolytic cleavage. Together, these results indicate that Zn2+ coordination is important for correct Gag precursor processing and NC protein stability. Additionally, SIV particle morphology appears to be the result of proper and complete Gag processing and relies less on full-length genomic RNA incorporation, as dictated by the Zn2+ coordination in the ZFs of the NC protein.

scholarly journals The Simian Immunodeficiency Virus 5′ Untranslated Leader Sequence Plays a Role in Intracellular Viral Protein Accumulation and in RNA Packaging

2003 ◽  
Vol 77 (11) ◽  
pp. 6284-6292 ◽  
Author(s):  
Jignesh Patel ◽  
Shainn-Wei Wang ◽  
Elena Izmailova ◽  
Anna Aldovini
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Initiation Codon ◽  
Primer Binding Site ◽  
Deletion Mutants ◽  
Protein Accumulation ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Immunodeficiency Virus ◽  
Selective Incorporation

ABSTRACT 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.

scholarly journals Human Immunodeficiency Virus Types 1 and 2 Differ in the Predominant Mechanism Used for Selection of Genomic RNA for Encapsidation

1999 ◽  
Vol 73 (4) ◽  
pp. 3023-3031 ◽  
Author(s):  
Jane F. Kaye ◽  
Andrew M. L. Lever
Keyword(s):  
Human Immunodeficiency Virus ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Initiation Mechanism ◽  
Gag Protein ◽  
Gag Proteins ◽  
Viral Genomes ◽  
Immunodeficiency Virus

ABSTRACT Retroviral RNA encapsidation is a highly selective process mediated through recognition by the viral Gag proteins of cis-acting RNA packaging signals in genomic RNA. This RNA species is also translated, producing the viral gag gene products. The relationship between these processes is poorly understood. Unlike that of human immunodeficiency virus type 1 (HIV-1), the dominant packaging signal of HIV-2 is upstream of the major splice donor and present in both unspliced and spliced viral RNAs, necessitating additional mechanisms for preferential packaging of unspliced genomic RNA. Encapsidation studies of a series of HIV-2-based vectors showed efficient packaging of viral genomes only if the unspliced, encapsidated RNA expressed full-length Gag protein, including functional nucleocapsid. We propose a novel encapsidation initiation mechanism, providing selectivity, in which unspliced HIV-2 RNA is captured in cis by the Gag protein. This has implications for the use of HIV-2 and other lentiviruses as vectors.

scholarly journals An Intact U5-Leader Stem Is Important for Efficient Replication of Simian Immunodeficiency Virus

2001 ◽  
Vol 75 (23) ◽  
pp. 11924-11929 ◽  
Author(s):  
Yongjun Guan ◽  
Karidia Diallo ◽  
James B. Whitney ◽  
Chen Liang ◽  
Mark A. Wainberg
Keyword(s):  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Point Mutations ◽  
Large Deletion ◽  
Primer Binding Site ◽  
Replication Capacity ◽  
Biologically Relevant ◽  
Immunodeficiency Virus ◽  
Efficient Replication ◽  
Viral Replication Capacity

ABSTRACT Previous work has shown that four deletions in simian immunodeficiency virus (SIV), termed SD1a, SD1b, SD1c, and SD6, which eliminated sequences at nucleotide positions 322 to 362, 322 to 370, 322 to 379, and 371 to 379, respectively, located downstream of the primer binding site, impaired viral replication capacity to different extents. Long-term culturing of viruses containing the SD1a, SD1b, and SD6 deletions led to revertants that possessed wild-type replication kinetics. We now show that these revertants retained the original deletions in each case but that novel additional mutations were also present. These included a large deletion termed D1 (nt +216 to +237) within the U5 region that was shown to be biologically relevant to reversion of both the SD1a and SD1b constructs. In the case of SD6, two compensatory point mutations, i.e., A+369G, termed M1, located immediately upstream of the SD6 deletion, and C+201T, termed M2, within U5, were identified and could act either singly or in combination to restore viral replication. Secondary structure suggests that an intact U5-leader stem is important in SIV for infectiousness and that the additional mutants described played important roles in restoration of this motif.

scholarly journals Leader Sequences Downstream of the Primer Binding Site Are Important for Efficient Replication of Simian Immunodeficiency Virus

2000 ◽  
Vol 74 (19) ◽  
pp. 8854-8860 ◽  
Author(s):  
Yongjun Guan ◽  
James B. Whitney ◽  
Karidia Diallo ◽  
Mark A. Wainberg
Keyword(s):  
Binding Site ◽  
Simian Immunodeficiency Virus ◽  
Point Mutations ◽  
Significant Degree ◽  
Viral Rna ◽  
Site Directed Mutagenesis ◽  
Primer Binding Site ◽  
Replication Capacity ◽  
Stem Loop ◽  
Immunodeficiency Virus

ABSTRACT Simian immunodeficiency virus (SIV) infection of macaques is remarkably similar to that of human immunodeficiency virus type 1 (HIV-1) in humans, and the SIV-macaque system is a good model for AIDS research. We have constructed an SIV proviral DNA clone that is deleted of 97 nucleotides (nt), i.e., construct SD, at positions (+322 to +418) immediately downstream of the primer binding site (PBS) of SIVmac239. When this construct was transfected into COS-7 cells, the resultant viral progeny were severely impaired with regard to their ability to replicate in C8166 cells. Further deletion analysis showed that a virus termed SD1, containing a deletion of 23 nt (+322 to +344), was able to replicate with wild-type kinetics, while viruses containing deletions of 21 nt (+398 to +418) (construct SD2) or 53 nt (+345 to +397) (construct SD3) displayed diminished capacity in this regard. Both the SD2 and SD3 viruses were also impaired with regard to ability to package viral RNA, while SD1 viruses were not. The SD and SD3 constructs did not revert to increased replication ability in C8166 cells over 6 months in culture. In contrast, long-term passage of the SD2 mutated virus resulted in a restoration of replication capacity, due to the appearance of four separate point mutations. Two of these substitutions were located in leader sequences of viral RNA within the PBS and the dimerization initiation site (DIS), while the other two were located within two distinct Gag proteins, i.e., CA and p6. The biological relevance of three of these point mutations was confirmed by site-directed mutagenesis studies that showed that SD2 viruses containing each of these substitutions had regained a significant degree of viral replication capacity. Thus, leader sequences downstream of the PBS, especially the U5-leader stem and the DIS stem-loop, are important for SIV replication and for packaging of the viral genome.

scholarly journals Nucleocapsid and Matrix Protein Contributions to Selective Human Immunodeficiency Virus Type 1 Genomic RNA Packaging

1998 ◽  
Vol 72 (3) ◽  
pp. 1983-1993 ◽  
Author(s):  
Dexter T. K. Poon ◽  
Guangde Li ◽  
Anna Aldovini
Keyword(s):  
Human Immunodeficiency Virus ◽  
Amino Acid ◽  
Matrix Protein ◽  
Zinc Binding ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Binding Motifs ◽  
Binding Domains ◽  
Immunodeficiency Virus

ABSTRACT 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.

scholarly journals Effect of the Attenuating Deletion and of Sequence Alterations Evolving In Vivo on Simian Immunodeficiency Virus C8-Nef Function

1999 ◽  
Vol 73 (4) ◽  
pp. 2790-2797 ◽  
Author(s):  
Silke Carl ◽  
A. John Iafrate ◽  
Jacek Skowronski ◽  
Christiane Stahl-Hennig ◽  
Frank Kirchhoff
Keyword(s):  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Point Mutations ◽  
Residual Activity ◽  
Intermediate Phenotype ◽  
Cell Surface Expression ◽  
In Vitro Assays ◽  
Immunodeficiency Virus

ABSTRACT The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8nef-long terminal repeat region encompasses only 12 bp and is “repaired” in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmac nef variants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in the nef gene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.

scholarly journals Single-Laboratory Validation of a Modified Liquid Chromatographic Method with UV Detection for Determination of Trenbolone Residues in Bovine Liver and Muscle

2003 ◽  
Vol 86 (5) ◽  
pp. 916-924 ◽  
Author(s):  
James D MacNeil ◽  
Joann Reid ◽  
Constance D Neiser ◽  
Adrian C E Fesser
Keyword(s):  
High Performance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Internal Standard ◽  
Bovine Liver ◽  
Reversed Phase ◽  
Significant Loss ◽  
Feedlot Cattle ◽  
Uv Detection ◽  
Critical Control Points

Abstract Trenbolone acetate is a synthetic testosterone analog registered for use in a number of countries as a growth-promoting hormone, applied as an implant in the ears of feedlot cattle. The method is intended for the detection and quantitation of trace amounts of α- and β-trenbolone in bovine tissues (muscle, liver) by liquid chromatography (LC) with UV detection and eliminates the use of the structural analog, 19-nortestosterone, as an internal standard. Trenbolone residues are extracted from tissues that have been homogenized in sodium acetate with a 3-phase liquid–liquid extraction by adding a mixture of water–acetonitrile–dichloromethane– hexane, with trenbolone residues preferentially partitioned into the middle acetonitrile layer. The extract is passed through solid-phase extraction cartridges (both C18 and silica gel) using, respectively, methanol–water and acetone–toluene as eluents. Reversed-phase high-performance LC separation is performed, an octadecyl-bonded column with methanol–acetonitrile–water used as mobile phase for sample analysis. The limit of detection is 0.2 ng/g in muscle tissue and 0.6 ng/g in liver tissue, with coefficients of variation of 3.5–12.1% for α- and β-trenbolone at concentrations from 0.2 to 4.0 ng/g fortified in muscle and 3.3–26.0% from liver fortified at 0.6–10.0 ng/g. Absolute recoveries of 40–130% were observed, but the use of fortified matrix curves eliminated recovery correction. Critical control points were identified in a pH adjustment step and an evaporation step during method validation, which included ruggedness testing. Analysis of incurred tissues (bovine liver and muscle) stored at –20°C for over 25 weeks did not identify any significant loss of residues.

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