A minimal genome simian foamy virus type 1 vector system with efficient gene transfer

Foamy viruses have several inherent features for the opportunity to develop efficient and versatile vectors for gene therapy. We have constructed a series of vectors and helper plasmids based on simian foamy virus type 1 (SFV-1) to establish the minimum vector genome required for efficient gene transduction. To characterize the efficiency of gene transduction by these vectors, the green fluorescent protein (GFP) coding sequence is linked to the human cytomegalovirus immediate gene promoter. Several deletion analyses of SFV-1 vectors revealed that the minimum genome with efficient GFP transduction contained the 5' untranslated region extending to the first 637 nucleotides of the gag gene, a 596 nucleotides of pol sequence from position 3137-3733, the 3' pol region at position 5200-5693, the 3' end polypurine tract, and the 3' LTR. An additional 1131 nucleotides can be removed from the 3' end LTR without affecting the efficiency of vector transduction. SFV-1 vector can therefore accommodate a minimum 8930 base-size heterologous DNA fragment. Furthermore, the efficiency of SFV-1 vector transduction was analyzed using different packaging plasmids. GFP transduction with packaging plasmid that contained the 5' R-U5 region of the LTR was compared with helper plasmids that had deletions in this region except for 22 nucleotides (positions 21-41), the first 61, 77, or 140 nucleotides of the R of the LTR. Transduction efficiencies were significantly reduced with the deletion mutations implicating that for optimum SFV-1 vector productions a packaging construct that includes the 5' R-U5 is required.     

Genomic organization and expression of simian foamy virus type 3 (SFV-3).

The complete nucleotide sequence of simian foamy virus type 3 (SFV-3) strain LK-3, isolated from an African green monkey, was determined. In addition to translation frames representing the gag, pol, and env genes, two open reading frames are located in the region between the env gene and the 3' long terminal repeat (LTR). Both SFV-3 and SFV-1 encode two open reading frames between env and the 3' LTR, whereas HFV encodes three open reading frames in this region. Northern blot analysis of cell cultures infected with SFV-3 revealed subgenomic RNAs for these open reading frames. The protease of SFV-3 is encoded by the pol gene in contrast to HFV which encodes the protease in the gag gene. Notably, the pol gene of SFV-3 in the +1 translational frame relative to the gag gene; this observation is in agreement with SFV-1, but differs for HFV and all other retrovirus genomes reported. Thus, gag-pol precursors of the SFVs appear to be expressed by a +1 frameshift. Nucleotide and deduced amino acid alignments of SFV-3, SFV-1, and HFV revealed an unexpected homology pattern; highest homologies are observed in the pol and env genes but low homologies are noted in the gag genes and the additional open reading frames. Analysis of phylogenetic trees confirms the classification of foamy viruses as a subfamily of retroviruses, distinct from the lentiviruses and oncoviruses.     

The critical role of proximal gag sequences in feline immunodeficiency virus genome encapsidation

Retroviral RNA encapsidation is mediated by specific interactions between viral Gag proteins and cis-acting packaging sequences in genomic RNA. Feline immunodeficiency virus (FIV) RNA encapsidation determinants have been shown to be discrete and noncontinuous, comprising one region at the 5' end of the genomic mRNA (R-U5) and another region that mapped within the proximal 311 nt of gag. To aid comparative understanding of lentiviral encapsidation and refinement of FIV vector systems, we used RNase protection assays (RPAs) of cellular and virion RNAs to investigate in detail the gag element. mRNAs of subgenomic vectors as well as of full-length molecular clones were optimally packaged into viral particles and resulted in high-titer FIV vectors when they contained only the proximal 230 nucleotides (nt) of gag. Further 3' truncations of gag sequences progressively diminished encapsidation and transduction. Deletion of the initial ninety 5' nt of the gag gene abolished mRNA packaging, demonstrating that this segment is indispensable for encapsidation. Focusing further on this proximal sequence, we found that a deletion of only 13 nt at the 5' end of gag impaired encapsidation of subgenomic vector and proviral RNAs.     

Sequences within both the 5' untranslated region and the gag gene are important for efficient encapsidation of Mason-Pfizer monkey virus RNA

It has previously been shown that the 5' untranslated leader region (UTR), including about 495 bp of the gag gene, is sufficient for the efficient encapsidation and propagation of Mason-Pfizer monkey virus (MPMV) based retroviral vectors. In addition, a deletion upstream of the major splice donor, SD, has been shown to adversely affect MPMV RNA packaging. However, the precise sequence requirement for the encapsidation of MPMV genomic RNA within the 5' UTR and gag remains largely unknown. In this study, we have used a systematic deletion analysis of the 5' UTR and gag gene to define the cis-acting sequences responsible for efficient MPMV RNA packaging. Using an in vivo packaging and transduction assay, our results reveal that the MPMV packaging signal is primarily found within the first 30 bp immediately downstream of the primer binding site. However, its function is dependent upon the presence of the last 23 bp of the 5' UTR and approximately the first 100 bp of the gag gene. Thus, sequences that affect MPMV RNA packaging seem to reside both upstream and downstream of the major splice donor with the downstream region responsible for the efficient functioning of the upstream primary packaging determinant.     

Role of distal zinc finger of nucleocapsid protein in genomic RNA dimerization of human immunodeficiency virus type 1; no role for the palindrome crowning the R-U5 hairpin

Genomic RNA isolated from HIV-1 variously mutated in nucleocapsid protein (NC) was characterized by nondenaturing gel electrophoresis. Mutations in the C-terminal, the N-terminal, and the linker regions had no effect on genomic RNA dimerization [they are R7R10K11S, P31L, R32G, S3(32-34), and K59L], while a C36S/C39S mutation in the distal zinc knuckle (Cys-His box or zinc finger) inhibited genome dimerization as much as disrupting the kissing-loop domain. The four mutations which inhibited tRNA(Lys3) genomic placement (i.e., the in vivo placement of tRNA(Lys3) on the primer binding site) had no effect on genome dimerization. Among five mutations which inhibited genome packaging, four had no effect on genome dimerization. Thus the N-terminal and linker regions of NC control genome packaging/tRNA(Lys3) placement (two processes which do not require mature NC) but have little influence on genome dimerization and 2-base extension of tRNA(Lys3) (two processes which are likely to require mature NC). It has been suggested, based on electron microscopy, that the AAGCUU82 palindrome crowning the R-U5 hairpin stimulates genomic RNA dimerization. To test this hypothesis, we deleted AGCU81 from wild-type viruses and from viruses bearing a disrupted kissing-loop hairpin or kissing-loop domain; in another mutant, we duplicated AGCU81. The loss of AGCU81 reduced dimerization by 2.5 +/- 4%; its duplication increased it by 3 +/- 6%. Dissociation temperature was left unchanged. We reach two conclusions. First, the palindrome crowning the R-U5 hairpin has no impact on HIV-1 genome dimerization. Second, genomic RNA dimerization is differentially influenced by NC sequence: it is Zn finger dependent but independent of the basic nature of the N-terminal and linker subdomains. We propose that the NC regions implicated in 2-base extension of tRNA(Lys3) are required for a second (maturation) step of tRNA placement. Genome dimerization and mature tRNA placement would then become two RNA-RNA interactions sharing similar NC sequence requirements.     

Simian foamy virus type 1 (SFV-1) induces apoptosis

Foamy virus infection causes cytopathology in several cell types from different species. The mechanism of cell killing by foamy viruses is not known. In this report, the mechanism of cell death induced by simian foamy virus type 1 (SFV-1) infection was investigated in fibroblast and lymphoid derived cells lines. Infected L-929 (fibroblast) and Raji (B cell) cells showed chromatin condensation, chromatin cleavage into nucleosome oligomers, and ultrastructural changes consistent with apoptosis. These data suggest that SFV-1 induced apoptotic cell death in different cell lines from different species. The degree of apoptotic cell death in both L-929 and Raji cell lines correlated with increased virus replication. Apoptosis, therefore, is one mechanism by which SFV-1 causes cell death.     

Deletion of stem-loop 3 is compensated by second-site mutations within the Gag protein of human immunodeficiency virus type 1

Encapsidation of human immunodeficiency virus type 1 (HIV-1) RNA involves specific interactions between viral Gag proteins and viral RNA elements located at the 5' untranslated region (UTR). These RNA elements are termed packaging (psi) or encapsidation (E) signals and mainly comprise the stem-loop 1 (SL1) and SL3 RNA structures. We have previously shown that deletion of the SL1 sequences is compensated by second-site mutations within Gag. Similar studies are now extended to SL3 and the results demonstrate that deletion of this RNA structure is rescued by two point mutations, i.e., A11V in p2 and I12V in nucleocapsid (NC). These two compensatory mutations are different from those associated with the rescue of SL1 deletion, suggesting that SL1 and SL3 may bind to different residues of Gag during viral RNA packaging. Analysis of virion-derived RNA in native agarose gels shows that deletion of SL3 leads to decreases in both viral RNA packaging and dimerization. These defects are corrected by the compensatory mutations A11V and I12V. Yet, defects in viral RNA dimerization at an early stage that were caused by the SL3 deletion in the context of a viral protease-negative mutation cannot be overcome by these two suppressor mutations. Therefore, the positive effects of A11V and I12V on dimerization of the SL3-deleted RNA must have taken place at the maturation stage.     

The flavivirus-conserved penta-nucleotide in the 3' stem-loop of the West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation

A reporting replicon of West Nile virus (WN) was used to distinguish between the function of the 3' untranslated region (UTR) in viral translation and RNA replication. Deletions of various regions of the 3' UTR of the replicon did not significantly affect viral translation, but abolished RNA replication. A systematic mutagenesis showed that the flavivirus-conserved penta-nucleotide (5'-CACAG-3' located at the top of the 3' stem-loop of the genome) requires a specific sequence and structure for WN RNA synthesis, but not for viral translation. (i) Basepair structure and sequence at the 1st position of the penta-nucleotide are critical for RNA replication. (ii) The conserved nucleotides at the 2nd, 3rd, and 5th positions, but not at the 4th position of the penta-nucleotide, are essential for RNA synthesis. (iii) The nucleotide U (which is partially conserved in the genus Flavivirus) immediately downstream of the penta-nucleotide is not essential for viral replication.     

Deficient dimerization of human immunodeficiency virus type 1 RNA caused by mutations of the u5 RNA sequences

The human immunodeficiency virus type 1 (HIV-1) virion contains two copies of genomic RNA that are noncovalently attached along a region at their 5' ends, in which two contact sites have been observed by electron microscopy. One of these sites is believed to be the stem-loop 1 (SL1) sequence which serves as the dimerization initiation site (DIS), and the other site, closer to the 5' end of the viral RNA, may involve the R or U5 RNA sequences. In this study, we present biochemical evidence showing that alteration of the U5 RNA sequence in the context of full-length viral RNA leads to diminished dimerization of virion RNA. In particular, two stretches of GU-rich sequences, which are located at nucleotides (nt) 99 to 108 and nt 112 to 123 within U5, were either deleted or substituted with exogenous sequences. The mutated viruses thus generated all exhibited deficient RNA dimerization. This dimerization deficit was not corrected by second-site mutations that preserved local RNA structures, such as the poly(A) hairpin, and was overcome to only a limited extent by compensatory mutations within Gag; these mutations were identified after long-term culture of the relevant mutant viruses in permissive cell lines and were able to restore viral infectiousness and RNA packaging to wild-type levels. Therefore, these GU sequences do not regulate RNA dimerization by the formation of local secondary structures nor by the maintenance of efficient viral RNA packaging; instead, they may mediate direct RNA-RNA interactions in the dimer structure. In contrast, mutation of palindrome 5'-AAGCUU-3', which resides within R and crowns the poly(A) hairpin, did not affect either RNA dimerization or RNA packaging.     

Sequences in the 5' leader of Mason-Pfizer monkey virus which affect viral particle production and genomic RNA packaging: development of MPMV packaging cell lines.

We used a series of deletion mutations in the 5' untranslated region of the prototype D type retrovirus, Mason-Pfizer Monkey Virus (MPMV), to analyse RNA encapsidation. A region was identified upstream of the major splice donor which reduced particle production but had a proportionally greater effect on RNA packaging. A small deletion downstream of the splice donor had little effect on RNA production and caused no significant packaging defect. A large deletion encompassing the end of the primer binding site down to the splice donor had a dramatic effect, disrupting viral protein synthesis. Stable cell lines were produced containing packaging-defective virus. These first-generation packaging cell lines were used to package and transfer an MPMV-based vector.     

Mutational analysis of the predicted secondary RNA structure of the Mason-Pfizer monkey virus packaging signal

The 5' end of the Mason-Pfizer monkey virus (MPMV) genomic RNA has been predicted to fold into a complex stem/loop structure that is thought to play a role in specific RNA encapsidation. In this study, we used a set of mutations that either abrogated or recreated the first four stem loops predicted within the 5' untranslated region (5' UTR) for effects on RNA packaging. Test of these mutations in our biological assay revealed that only stem loop 1 (SL1) was important for the packaging potential of MPMV, while mutations in none of the other stem loops affected packaging significantly. Interestingly, it was the primary sequence of SL1 RNA and not its secondary structure that affected packaging since compensatory mutations that reformed SL1 were unable to restore the packaging efficiency of the retroviral vector. Additionally, our mutational analysis reveals that stem loop 4, predicted to be the major packaging determinant of MPMV, does not seem to have a significant role in packaging. Finally, results of the biological effects of the structural mutations are discussed in relation to their effects on the folding potential of the various stem loops.     

Structures required for poly(A) tail-independent translation overlap with, but are distinct from, cap-independent translation and RNA replication signals at the 3' end of Tobacco necrosis virus RNA

Tobacco necrosis necrovirus (TNV) RNA lacks both a 5' cap and a poly(A) tail but is translated efficiently, owing in part to a Barley yellow dwarf virus (BYDV)-like cap-independent translation element (BTE) in its 3' untranslated region (UTR). Here, we identify sequence downstream of the BTE that is necessary for poly(A) tail-independent translation in vivo by using RNA encoding a luciferase reporter gene flanked by viral UTRs. Deletions and point mutations caused loss of translation that was restored by adding a poly(A) tail, and not by adding a 5' cap. The two 3'-proximal stem-loops in the viral genome contribute to poly(A) tail-independent translation, as well as RNA replication. For all necroviruses, we predict a conserved 3' UTR secondary structure that includes the BTE at one end of a long helical axis and the stem-loops required for poly(A) tail-independent translation and RNA replication at the other end. This work shows that a viral genome can harbor distinct cap- and poly(A) tail-mimic sequences in the 3' UTR.     

Mutations that alter a repeated ACCA element located at the 5' end of the Potato virus X genome affect RNA accumulation

The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.     

Nucleotide sequence analysis of the chicken gene c-mil, the progenitor of the retroviral oncogene v-mil

The nucleotide sequence of the chicken gene c-mil was determined within and around all regions homologous to the oncogene v-mil of avian retrovirus MH2. The regions of homology to the previously determined v-mil sequence, ranging in size from 28 to 177 base pairs (bp), are distributed over 14 kilobase pairs (kbp) of the chicken genome and are organized in 11 exons. All exon-intron boundaries of c-mil, except the 5' boundary of exon 1 and the 3' boundary of exon 11, were unambiguously defined by the identification of consensus splice donor and acceptor sites precisely at positions where homology to v-mil ceases or resumes. The homology to v-mil starts within the coding sequence of exon 1 and ends within the 3' untranslated region of exon 11, 12 nucleotides downstream from the nonsense codon terminating the large open reading frame shared between c-mil and v-mil. The c-mil and v-mil sequences differ at only 7 out of 1153 nucleotide positions, and the predicted sequences of v-mil and c-mil proteins differ by one conservative and four nonconservative substitutions among 379 amino acid residues. Hence, the carboxy-terminal domains of the MH2 gag-mil hybrid protein and of the putative c-mil protein are very similar. However, the amino-terminal domain of the cellular protein is possibly encoded by additional 5' c-mil sequences not present in the transduced v-mil oncogene, while that of the MH2 hybrid protein is encoded by viral gag sequences. The sequence analysis also revealed that c-mil and c-myc derived sequences are immediately adjacent on the MH2 genome carrying both the v-mil and the v-myc oncogene. Hence, transduction of c-mil into MH2 involved recombination, at the 3' site, with either the c-myc locus or a previously transduced v-myc gene, and, at the 5' site, with gag sequences of the transducing virus. At both sites, no significant homologies were found between the sequence elements involved in the recombination.     

Feline foamy virus-mediated marker gene transfer: identification of essential genetic elements and influence of truncated and chimeric proteins

Retroviral vectors derived from foamy or spumaretroviruses are considered promising tools for targeted gene delivery and vaccination purposes. In order to fully exploit this potential, we identified essential cis-acting sequences on the feline foamy virus (FFV) genome by constructing and analyzing a series of FFV-based replication-deficient vector genomes. Cis-acting sequences essentially required for marker gene transfer were found to be localized at two sites on the FFV genome: (i) in the 5'-untranslated region and close to the gag ATG and (ii) in the central part of the pol gene. The presence of two cis-acting sequences and their relative location on the FFV genome are similar but not identical to the functionally corresponding elements described for simian and primate foamy viruses.     

The T12I mutation within the SP1 region of Gag restricts packaging of spliced viral RNA into human immunodeficiency virus type 1 with mutated RNA packaging signals and mutated nucleocapsid sequence

Specific packaging of human immunodeficiency virus type 1 (HIV-1) RNA is attributable to the high affinity of nucleocapsid (NC) sequence of Gag for the cis-acting RNA packaging signals located within the 5' un-translated region (5' UTR). Interestingly, we have previously reported that the T12I mutation (named MP2) within SP1 of Gag prevented incorporation of spliced viral RNA into mutated viruses that lacked the stem-loop 1 (SL1) RNA element (also named dimerization initiation site, DIS), suggesting a role for the SP1 sequence in viral RNA packaging. In this study, we have further tested this activity of MP2 in the context of a variety of mutations that affect viral RNA incorporation. The results showed that MP2 was able to effectively restrict packaging of spliced viral RNA into viruses containing either NC mutations R10A and K11A or mutated 5' UTR sequence, such as DeltaGU3 that lacked the 112-GUCUGUUGUGUG-123 sequence of U5, D1 that was deleted of a 27 nt fragment immediately downstream of the primer binding site (PBS), Delta(306-325) that had the SL3 RNA element removed and MD2 that was missing the 328-GGAG-331 sequence. As a result, MP2 contributed increased infectivity to the related viruses. Therefore, the MP2 mutation demonstrates a distinct role in HIV-1 RNA packaging that is neither pertained to the specific viral RNA packaging signal nor to the NC sequence.     

The 5′ untranslated region as a pathogenicity determinant of Semliki Forest virus in mice

An investigation of the role of the 5′ untranslated region (UTR) of Semliki Forest virus (SFV) in determining pathogenicity in infected mice was carried out by constructing 5′ UTR chimeras. Analysis of 5′ UTR sequences showed nucleotide differences between virulent and avirulent strains at positions 21, 35 and 42. Reciprocal chimeras incorporating these changes were constructed from avirulent CA7 and rA7[74], and virulent SFV-4 virus, derived from infectious clones, and avirulent A7 and A7[74] plaque-purified stock virus. Survival rates and neuropathology in intranasally (i.n.) infected mice were analysed. While no statistically significant difference between rates of RNA synthesis was detected between strains in cell culture, an increase in survival of infected mice and a reduction in the severity of brain lesions was observed on substitution of the 5′ UTR from a stock avirulent virus into an infectious clone where the remainder of the genome was derived from avirulent virus. However, substitution of a 5′ UTR from an avirulent stock virus into an infectious clone where the remainder of the genome was from virulent virus did not affect virulence. These results and other studies suggest that control of virulence is polygenic, and that the SFV 5′ UTR acts as a pathogenicity determinant in synergy with other determinants in the genome.     

RNA packaging signal of human immunodeficiency virus type 1.

Cells infected with a recombinant vaccinia virus carrying the gag and pol regions of the human immunodeficiency virus type 1 genome (Vac-gag/pol) released human immunodeficiency virus (HIV)-like particles containing HIV-specific RNA. However, cells infected with another recombinant vaccinia, Vac-gag/pol-dP, derived through the deletion of an 85-base region (nucleotide positions 679-763) of the HIV genome between the primer binding site and the gag initiation codon of Vac-gag/pol, produced HIV-like particles devoid of the HIV-specific RNA. This 85-base deletion was suggested to cause the collapse of a stable stem-loop structure of 46 bases (751-796) around the gag initiation codon. To examine the role of the stem-loop structure in the packaging of RNAs, we constructed a vaccinia vector plasmid that carried this 46-base sequence followed by the Sendai virus nucleocapsid (NP) gene. When both Vac-gag/pol-dP and this plasmid were introduced into cells, HIV-like particles released from the cells contained the NP gene RNA. However, another vaccinia vector plasmid, which carried the 46-base sequence in the midst of the NP gene, could not supply RNA for incorporation into HIV-like particles. Computer analysis of this plasmid sequence suggested that the 46-base sequence cannot form the stem-loop structure. These findings suggest that the stem-loop structure formed by the 46-base sequence is crucial as a packaging signal.