SHAPE analysis of the FIV Leader RNA reveals a structural switch potentially controlling viral packaging and genome dimerization.

Feline immunodeficiency virus (FIV) infects many species of cat, and is related to HIV, causing a similar pathology. High-throughput selective 2' hydroxyl acylation analysed by primer extension (SHAPE), a technique that allows structural interrogation at each nucleotide, was used to map the secondary structure of the FIV packaging signal RNA. Previous studies of this RNA showed four conserved stem-loops, extensive long-range interactions (LRIs) and a small, palindromic stem-loop (SL5) within the gag open reading frame (ORF) that may act as a dimerization initiation site (DIS), enabling the virus to package two copies of its genome.

Reciprocal cross-packaging of primate lentiviral (HIV-1 and SIV) RNAs by heterologous non-lentiviral MPMV proteins.

Retroviral RNA packaging signal (ψ) allows the preferential packaging of genomic RNA into virus particles through its interaction with the nucleocapsid protein. The specificity of this interaction came into question when it was shown that primate retroviruses, such as HIV-1, could cross-package RNA from its simian cousin, SIV, and vice versa and that feline retrovirus, FIV could cross-package RNA from a distantly related primate retrovirus, MPMV. To study the generality of this phenomenon further, we determined whether there is a greater packaging restriction between the lentiviral class of retroviruses (HIV-1 and SIV) and a non-lentivirus, MPMV.

Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure.

A G-C-rich palindromic structural motif and a stretch of single-stranded purines are required for optimal packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA.

During retroviral RNA packaging, two copies of genomic RNA are preferentially packaged into the budding virus particles whereas the spliced viral RNAs and the cellular RNAs are excluded during this process. Specificity towards retroviral RNA packaging is dependent upon sequences at the 5' end of the viral genome, which at times extend into Gag sequences. It has earlier been suggested that the Mason-Pfizer monkey virus (MPMV) contains packaging sequences within the 5' untranslated region (UTR) and Gag.

Cross-packaging of genetically distinct mouse and primate retroviral RNAs.

Background: The mouse mammary tumor virus (MMTV) is unique from other retroviruses in having multiple viral promoters, which can be regulated by hormones in a tissue specific manner. This unique property has lead to increased interest in studying MMTV replication with the hope of developing MMTV based vectors for human gene therapy. However, it has recently been reported that related as well as unrelated retroviruses can cross-package each other's genome raising safety concerns towards the use of candidate retroviral vectors for human gene therapy.

Role of a heterologous retroviral transport element in the development of genetic complementation assay for mouse mammary tumor virus (MMTV) replication.

The mouse mammary tumor virus (MMTV) is a type B retrovirus that is unique from other retroviruses in having multiple "tissue specific" and "hormone inducible" promoters. This unique feature has lead to the increasing interest in studying the biology of MMTV replication with the ultimate goal of developing MMTV based vectors for potentially targeted human gene therapy. In this report, we describe, for the first time, the establishment of an in vivo genetic complementation assay to study various aspects of MMTV replication.

The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop.

Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA.

Both the 5' and 3' LTRs of FIV contain minor RNA encapsidation determinants compared to the two core packaging determinants within the 5' untranslated region and gag.

This study was undertaken to address the role of feline immunodeficiency virus (FIV) long terminal repeats (LTR) as potential packaging determinants. A number of studies in the recent past have clearly demonstrated that the core packaging determinants of FIV reside within at least two distinct regions at the 5' end of the viral genome, from R in the 5' LTR to approximately 150 bp within the 5' untranslated region (5' UTR) and within the first 100 bp of gag; however, there have been conflicting observations as to the role of the LTR regions in packaging and whether they contain the principal packaging determinants of FIV. Using a semi-quantitative RT-PCR approach on heterologous non-viral vector RNAs in an in vivo packaging assay, this study demonstrates that the principal packaging determinants of FIV reside within the first 150 bp of 5' UTR and 100 bp of gag (the two core regions) and not the viral 5' LTR.

Sequences intervening between the core packaging determinants are dispensable for maintaining the packaging potential and propagation of feline immunodeficiency virus transfer vector RNAs.

The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to approximately 150 bp of the 5' untranslated region and the first approximately 100 bp of gag. However, the role of sequences intervening between the core regions in packaging has not been clear. A mutational analysis was conducted to determine whether the intervening sequences played a role in FIV RNA packaging, using an in vivo packaging assay complemented with semiquantitative reverse transcriptase PCR.

Relative activity of the feline immunodeficiency virus promoter in feline and primate cell lines.

The feline immunodeficiency virus (FIV) long terminal repeat (LTR), compared with some primate lentiviral LTRs, is quite a strong basal promoter. However, it seems to be highly species-specific in function and generally not very efficient in cells of non-feline origin. This study systematically explored the function of the FIV LTR in simian Cos cells compared with its activity in feline and human cells.

Close proximity of the MPMV CTE to the polyadenylation sequences is important for efficient function in the subgenomic context.

The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) is a short cis-acting sequence element critical for virus gene expression. Analogous to the Rev/Rev Responsive Element (RRE) of primate lentiviruses, CTE allows the nucleocytoplasmic transport of unspliced viral mRNAs. In fact, CTE can functionally replace Rev/RRE in the genomic context and has been used successfully in the expression of viral and cellular genes from expression vectors as well.