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. Our results revealed that primate lentiviral RNAs can be cross-packaged by primate non-lentiviral particles reciprocally, but the cross-packaged RNAs could not be propagated by the heterologous particles. Packaging of RNA in the context of both retroviral vectors as well as non-retroviral RNA containing SIV, HIV, and MPMV packaging determinants by each others proteins further confirmed the specificity of cross-packaging conferred by the packaging sequences. These results reveal the promiscuous nature of retroviral packaging determinants and raise caution against their wide spread presence on retroviral vectors to be used for human gene therapy.

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. These studies have also suggested that the packaging determinants of MPMV that lie in the UTR are bipartite and are divided into two regions both upstream and downstream of the major splice donor. However, the precise boundaries of these discontinuous regions within the UTR and the role of the intervening sequences between these dipartite sequences towards MPMV packaging have not been investigated. Employing a combination of genetic and structural prediction analyses, we have shown that region "A", immediately downstream of the primer binding site, is composed of 50 nt, whereas region "B" is composed of the last 23 nt of UTR, and the intervening 55 nt between these two discontinuous regions do not contribute towards MPMV RNA packaging. In addition, we have identified a 14-nt G-C-rich palindromic sequence (with 100% autocomplementarity) within region A that has been predicted to fold into a structural motif and is essential for optimal MPMV RNA packaging. Furthermore, we have also identified a stretch of single-stranded purines (ssPurines) within the UTR and 8 nt of these ssPurines are duplicated in region B. The native ssPurines or its repeat in region B when predicted to refold as ssPurines has been shown to be essential for RNA packaging, possibly functioning as a potential nucleocapsid binding site. Findings from this study should enhance our understanding of the steps involved in MPMV replication including RNA encapsidation process.

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. Therefore, using a trans complementation assay, we looked at the ability of MMTV RNA to be cross-packaged and propagated by an unrelated primate Mason-Pfizer monkey virus (MPMV) that has intracellular assembly process similar to that of MMTV. RESULTS: Our results revealed that MMTV and MPMV RNAs could be cross-packaged by the heterologous virus particles reciprocally suggesting that pseudotyping between two genetically distinct retroviruses can take place at the RNA level. However, the cross-packaged RNAs could not be propagated further indicating a block at post-packaging events in the retroviral life cycle. To further confirm that the specificity of cross-packaging was conferred by the packaging sequences (psi), we cloned the packaging sequences of these viruses on expression plasmids that generated non-viral RNAs. Test of these non-viral RNAs confirmed that the reciprocal cross-packaging was primarily due to the recognition of psi by the heterologous virus proteins. CONCLUSION: The results presented in this study strongly argue that MPMV and MMTV are promiscuous in their ability to cross-package each other's genome suggesting potential RNA-protein interactions among divergent retroviral RNAs proposing that these interactions are more complicated than originally thought. Furthermore, these observations raise the possibility that MMTV and MPMV genomes could also co-package providing substrates for exchanging genetic information.

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. In the assay described here, the function of MMTV Rem/RmRE regulatory pathway has been successfully substituted by a heterologous retroviral constitutive transport element (CTE) from Mason Pfizer Monkey Virus (MPMV) for mature MMTV particle production. Our results revealed that in the absence of MPMV CTE or Rem/RmRE, RNA transcribed from MMTV Gag-Pol expression plasmids were efficiently transported to the cytoplasm. However, the presence of CTE was indispensable for Gag-Pol protein expression. In addition, we report the development of MMTV based vectors in which the packageable RNA was transcribed either from MMTV LTR or from a chimeric LTR, which could successfully be packaged and propagated by particles produced from MMTV Gag-Pol expression plasmids containing a heterologous transport element. The role of MPMV CTE in the transport of MMTV transfer vector RNA was not found to be significant. Development of such an assay should not only shed light on how MMTV regulates its gene expression, but also should provide additional molecular tools for delineating the packaging determinants for MMTV, which is imperative for the development of novel vectors for targeted and inducible gene therapy.

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. Furthermore, it shows that in addition to the 5' LTR, the 3' LTR also contains packaging determinants, but of a less significant nature compared to the core packaging determinants. This study defines the relative contribution of the various regions implicated in FIV genomic RNA packaging, and reveals that like other primate lentiviruses, the packaging determinants of FIV are multipartite and spread out, an observation that has implications for safer and more streamlined design of FIV-based gene transfer vectors.

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. Our analyses reveal that the intervening sequences are dispensable not only for vector RNA packaging but also for propagation, confirming the discontinuous nature of the FIV packaging signal.