No abstract

The packaging determinants of feline immunodeficiency virus (FIV) consist of two discontinuous core regions, extending from R to ϳ150 bp of the 5 untranslated region and the first ϳ100 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.A series of recent studies have suggested that the packaging determinants of feline immunodeficiency virus (FIV) are complex and multipartite, consisting of at least two discontinuous core regions, one located upstream of the major splice donor sequence from R/U5 at the 5Ј end to the first ϳ150 bp of the 5Ј untranslated region (UTR), while the other is within the first 100 bp of gag (2, 3, 6, 7). To determine whether the region intervening between the core determinants was required for maintaining the stability of the FIV packaging signal, we modified our previously described vector, MB15 (2), and constructed a series of transfer vectors, AG002 to AG004, that maintained the R/U5 and 100 bp of gag but kept only the first 90, 120, or 150 bp of the 5Ј UTR, generating incremental deletions between the core packaging determinants (Fig. 1). In a second series, AG013 to AG015, the deletions were replaced with heterologous sequences of the same lengths to further determine whether the deleted/substituted region had a role at the structural level or acted only as a spacer (Fig. 1).The two series of mutant vector RNAs were tested for their ability to be packaged by FIV proteins, using our well-established in vivo packaging assay along with TR394, which contains the entire 270-bp UTR with a 333-bp gag sequence (1-3). The amount of transfer vector RNA packaged into viral particles was analyzed by reverse transcriptase PCR (RT-PCR) (10) for variable cycle numbers, and the resulting products were Southern blotted and hybridized using an R/U5 probe. All mutant vector RNAs, either with deletions or deletion/ substitution, were packaged into the virus particles but with different efficiencies, while the control vectors, MB15 and TR394, were packaged at similar levels ( Fig. 2A). This was despite the fact that all cultures produced similar levels of viral particles and the transfection efficiencies were within twofold of each other ( Fig. 2B and C). Since the PCRs were conducted across the deleted or deleted/substituted region, the size of the PCR fragment varied with each construct, confirming that correct packaging constructs were being expressed in each transfection.To determine the packaging efficiencies of the various constructs accurately, RT-PCRs were repeated using primers within the R/U5 region that would result in same-sized fragme...

Avian cell lines derived from germinal crescent primordial germ cells and gonadal gonocytes with long-term proliferative capacity in vitro and their subsequent rates of colonization and germline transmission are described. In general, male cultures proliferate more rapidly than female cultures although both can be developed into cell lines of >2 × 10(6) cells, at which time, they can be grown indefinitely and a cell bank can be established. All the cell lines injected into embryos transmitted through the germline with the percentage of germline transmission of both male and female cell lines varying from single digits to the high 90s. The derivation of these primordial germ cell and gonadal cell lines and the subsequent robustness of germline transmission validates these cells as suitable for establishment of lines of chickens bearing novel genetic modifications.

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. The 5′ and 3′ sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem–loop (SL2) and a small palindromic stem–loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8–5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5–3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.

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.