A Stretch of Unpaired Purines in the Leader Region of Simian Immunodeficiency Virus (SIV) Genomic RNA is Critical for its Packaging into Virions

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.

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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

Journal of Virology ◽

10.1128/jvi.75.1.115-124.2001 ◽

2001 ◽

Vol 75 (1) ◽

pp. 115-124 ◽

Cited By ~ 14

Author(s):

Jason L. Yovandich ◽

Elena N. Chertova ◽

Brad P. Kane ◽

Tracy D. Gagliardi ◽

Julian W. Bess ◽

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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.

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