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. 2006 Dec 21:3:96.
doi: 10.1186/1742-4690-3-96.

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239

James B Whitney  1 , Mark A Wainberg
Affiliations

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239

James B Whitney et al. Retrovirology. .

Abstract

Background: The 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIVmac239 leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC).

Results: Regions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC.

Conclusion: In the leader region of SIVmac239, stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIVmac239 and RNA dimerization.

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Figures

Figure 1
Figure 1
Nucleotide position of deletion mutations within the leader sequence and the secondary structure of SIVmac239 leader RNA. A. Denotes the size and nucleotide position of deletion mutations located upstream (pU) or downstream (pD) of the major SD. All nucleotide deletions are relative to the transcriptional initiation site (1+) based on the sequence of the wild type clone of SIVmac239 [50]. B. Secondary structure was adapted from published information [13, 51]. All hairpin motifs are labeled according to their putative function and/or after comparable elements encoded by HIV-1/HIV-2 leader sequences. The following motifs are shown in bold type: the putative DIS palindrome at position +417-422, the splice donor (SD) at position +462, and the Gag initiation codon at position +533.
Figure 2
Figure 2
Thermodynamic folding analysis of resolved RNA SIVmac239 leader structures. Shown are predicted free-energy minimized structures from the SIVmac239 wild-type leader (I.) and attenuated mutants pUΔ19 (II.) and pDΔ60 (III.) [51]. Fig. I, arrows labeled a, b, c, and d denote SL1, SL2, SL3 and SL4, respectively, in the SIVmac239 leader structures or their absence in deletion variants.
Figure 3
Figure 3
Both genomic RNA packaging efficiency and specificity are compromised in SIVmac239 by mutations within the leader region. A. Analysis of viral RNA packaging was conducted in triplicate in COS-7 cells. Shown are results from purified viral RNA preparations that were normalized on the basis of p27-CA ELISA. RNA packaging of various mutant constructs as a percent average of WT virus. The bars of the first lane for each sample represent total incorporated viral RNA. The second bar for each lane represents incorporated full-length viral RNA. B. Shown at the bottom is percent specific incorporation as a ratio of that obtained with the wild type full-length genome.
Figure 4
Figure 4
Non-denaturing Northern analysis of SIV RNA. Non-denaturing analysis of intra-virion RNA from transient transfections of mutant clones. Viral genomic RNA was isolated from virus particles after transfection of COS-7 cells with wild type or mutant plasmids. The relative mobility of dimers (D) and monomers (M) in 0.90% agarose are indicated. The plus (+) denotes the addition of RNase to preparations prior to electrophoresis. A. Shows non-denaturing RNA preparations from deletion mutants encompassing the region from nt +426 - +465. B. Shows non-denaturing RNA preparations from mutants encompassing the nt region +473 - +480. C. RNA preparations from mutants encompassing the region directly adjacent to the PBS, i.e. SD1, SD2, and SD3 deleted the of nucleotide regions +322-344, +398-418, and +345-397 respectively [13]. The adjacent panel shows thermal denaturing analysis of the SD1 mutant (Δ+322 - +344), comprising a 23-nucleotide deletion upstream of SL1 in comparison to dimer extracted from the wild-type virions.
Figure 5
Figure 5
Replication kinetics of various mutant constructs in CEMx174 cells and primary rhesus PBMC. Cells were infected with 10 ng viral equivalents and viral replication was monitored by RT assay of culture supernatant at multiple time points. Mock denotes infection with heat-denatured wild-type virus. All replication experiments were conducted in triplicate. A. Representative growth curves of viruses deleted between the DIS and the SD (i.e. nt +424-462) in CEMx174 cells. B. Replication of viruses deleted between the SD and the Gag AUG (nt +471-530) in CEMx174 cells. Viral replication was assessed in activated rhesus PBMCs using viral inocula normalized on the basis of p27-CA Ag. Growth curves were determined by p27-CA Ag ELISA of culture supernatant taken at multiple time points. All results are the average of duplicates. C. Growth curves of variants deleted within the nt region +424-462. D. Growth curves of variants deleted within the region +471-530. Mock infection denotes exposure of cells to heat-inactivated wild-type virus as a negative control. Note that the scales of the ordinates are logarithmic. The dashed line representing 0.01 ng of p27/ml indicates the threshold of sensitivity of the assay.
Figure 6
Figure 6
Analysis of protein expression and viral core ultrastructure of wild type and mutant viral particles. A. Viral replication analysis of mutated viruses TCID50 analysis of viral infectivity, scale of ordinate is logarithmic. B. Western analysis of wild type and mutant virus particles C. TEM of late (fixed 36 hr post-transfection) wild type and mutant particles were assessed and scored from multiple sections. Panel I, wild-type virus has typical size and core morphology. Panel II, the pUΔ19 mutant shows diminished production of viral particles, with altered core placement and morphology. Panel III, shows the pDΔ60 mutant; virus particle release less affected; particle condensation to a mature state is impaired. Bar size is .5 μM.
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References

    1. Aldovini A, Young RA. Mutations of RNA and protein sequences involved in human immunodeficiency virus type 1 packaging result in production of noninfectious virus. J Virol. 1990;64:1920–1926. - PMC - PubMed
    1. Berkowitz RD, Hammarskjold ML, Helga-Maria C, Rekosh D, Goff SP. 5' regions of HIV-1 RNAs are not sufficient for encapsidation: implications for the HIV-1 packaging signal. Virology. 1995;212:718–723. doi: 10.1006/viro.1995.1530. - DOI - PubMed
    1. Rein A. Retroviral RNA packaging: a review. Arch Virol Suppl. 1994;9:513–522. - PubMed
    1. McBride MS, Panganiban AT. Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo. J Virol. 1997;71:2050–2058. - PMC - PubMed
    1. Russell RS, Hu J, Beriault V, Mouland AJ, Laughrea M, Kleiman L, Wainberg MA, Liang C. Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA. J Virol. 2003;77:84–96. doi: 10.1128/JVI.77.1.84-96.2003. - DOI - PMC - PubMed

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