1 The University of Texas MD Anderson Cancer Center, Department of Veterinary Sciences, Bastrop, TX 78602, USA
2 Department of Medical Microbiology, Faculty of Medicine and Health Sciences (FMHS), The United Arab Emirates University, PO Box 17666, Al Ain, United Arab Emirates
Correspondence
Tahir Rizvi (at UAE University)
tarizvi{at}uaeu.ac.ae
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ABSTRACT |
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MAIN TEXT |
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To assess the requirement of sequences within the 5'UTR for packaging, we generated a series of transfer vectors containing various amounts of the 5'UTR in the presence of 333 bp of gag (Fig. 1A). The 5'UTR was deleted in 30 bp increments from upstream of the gag initiator codon (nt 627) to the U5/UTR junction at the 5' end of the primer binding site (PBS) (nt 357) using TR394, which has been shown to be packaged and propagated efficiently by FIV proteins (Browning et al., 2001
). The ability of RNA from transfer vectors MB19 to be packaged and propagated by the FIV packaging construct MB22, and the Env expression construct MD.G., were tested using our trans-complementation assay (Browning et al., 2001
).
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However, the packaging efficiency dropped about 6-fold compared with TR394 when RNA was packaged from MB6, the vector that contained only 90 bp out of 270 bp of the 5'UTR (Fig. 1B, panel III), and remained at about the same level (
4- to 6-fold reduction compared with TR394) irrespective of the remaining sequences in the 5'UTR (MB79). Thus, between 90 and 120 bp of the 5'UTR appear to be required for efficient packaging of FIV RNA. Interestingly, considerable packaging was observed with MB9, the vector that had lost even the PBS (Fig. 1B
, panel III). The packaging observed with MB9 could not have been due to non-specific packaging of any hygromycin mRNA from the SV40 promoter because, along with these vectors, we have tested our control plasmid, TR174, in the trans-complementation assay (data not shown). TR174 expressed the hygromycin gene from the SV40 promoter and used polyadenylation sequences from the 3'LTR for transcript termination. It lacked the sequences necessary for vector RNA packaging and propagation and did not package RNA into virions or give rise to hygromycin-resistant colonies (Hygr) with either human, simian or feline immunodeficiency viruses (HIV, SIV, FIV), or MasonPfizer monkey virus (MPMV) proteins when tested in the trans-complementation assay (Rizvi & Panganiban, 1993
; Browning et al., 2001
).
Successful transduction of most of the FIV 5'UTR deletion transfer vectors was indicated by the appearance of Hygr colonies (Fig. 1A). The overall viral titres observed in the trans-complementation assay correlated well with the packaging efficiency. However, deletion of the first 30 bp of the 5'UTR containing the PBS in MB9 resulted in a total loss of viral titre, despite only a 5·7-fold reduction in packaging. The complete absence of Hygr colonies despite considerable packaging was expected in MB9, since the PBS is required for reverse transcription, a step necessary for the successful propagation of the transfer vector. Thus, the coordinated loss of titre with incremental deletions in the 5'UTR and corresponding reductions in the packaging efficiency suggest that truncations of the 5'UTR continue to affect vector RNA packaging, which in turn affects vector RNA propagation proportionally.
To determine whether FIV gag sequences are important for the encapsidation process, we generated MTB, a construct that contains the entire 5'UTR in the absence of gag (Fig. 2A). Next, sequential amounts of the gag sequence were added to MTB after the 270 bp of the 5'UTR, from 30 bp of gag in MB10 to 210 bp in MB14 (Fig. 2A
). Tests of MTB and MB1014 in the in vivo packaging assay revealed that slightly lower levels of particles were produced in cultures expressing MTB, MB10 and MB13 (Fig. 2A
, panel IV). However, these levels were comparable with those expressed by the control vector, TR394. Slot-blot analysis of RNA isolated from purified virions revealed that there was a 3·6-fold loss of packaging in the absence of any gag sequences in MTB compared with TR394 (Fig. 2B
, panel III), when normalized to the amount of RNA loaded and the intracellular steady-state levels of transfer vector RNAs expressed (Fig. 2B
, panels I and II). This loss remained essentially the same (3·12-fold compared with TR394) in the presence of 30 bp of gag (MB10, Fig. 2B
, panel III). It was not until 90 bp of the gag sequence was present (MB11) that the packaging efficiency became comparable (less than 2-fold) with TR394 (Fig. 2B
, panel III), and remained essentially the same for vectors MB1214. The lower level of RNA packaged in MTB and MB10 was not due to lower transfection efficiency of these vectors, since MB13 and TR394 were expressed at about the same level or even lower and yet were packaged efficiently (Fig. 2B
, panels II and III).
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To study the relative contribution of sequences within the 5'UTR and gag towards packaging, we generated MB3038, which contained the same successive 5'UTR deletions made in MB19, but in the absence of gag (Fig. 3A). Tests of MB30 revealed a drastic 41-fold reduction in packaging efficiency compared with TR394, after normalization for RNA loading and intracellular steady-state levels of transfer vector RNAs (Fig. 3B
, panels I and II). MB30 lacked the last 30 bp of the 5'UTR in addition to gag sequences. This loss represented an approximately 11-fold greater reduction in packaging efficiency compared with MTB, the vector that also lacked gag sequences, but contained the entire 270 bp of the 5'UTR (Fig. 2
). The greater reduction in packaging efficiency, therefore, can be attributed to the loss of the last 30 bp of the 5'UTR in the absence of gag sequences, since only a 1·3-fold reduction in RNA packaging was observed with this deletion in the absence of gag, while deletions as much as 4-fold larger than this reduced packaging by only 2-fold in the presence of gag (see MB15, Fig. 1
). Thus, the last 30 bp of the 5'UTR are critical for the functioning of the FIV packaging determinant located at the beginning of the 5'UTR in the absence of gag sequences.
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Once again, reduction in genomic RNA incorporation into nascent virions observed in MB3038 was not completely abrogated and a constant low level of packaging was detectable, even in the absence of the entire 5'UTR and gag sequences (MB38, Fig. 3B). The presence of low levels of packaging, even in the absence of the entire 5'UTR and gag sequences, suggests that there are additional packaging determinants outside these sequences. We can exclude non-FIV sequences present on the vectors such as the human cytomegalovirus (hCMV) promoter, the SV-Hygr cassette and the constitutive transport element (CTE) as contributing to non-specific FIV RNA packaging, since the presence of these sequences on several other transfer vectors in the absence of any FIV sequences has not led to any FIV-specific RNA packaging by the structural proteins expressed by MB22 (Browning et al., 2001
). The only other FIV-specific sequences found in our vectors were the FIV LTR and
129 bp immediately upstream of the FIV 3'LTR. Therefore, these sequences could potentially harbour additional packaging determinants for genomic RNA encapsidation. A similar involvement of sequences of secondary importance to RNA packaging has been observed in other retroviruses, such as murine leukaemia virus (Yu et al., 2000
), Rous sarcoma virus (Sorge et al., 1983
) and HIV-1 (Kim et al., 1994
; McBride & Panganiban, 1996
, 1997
; McBride et al., 1997
; Harrich et al., 2000
).
Tests of MB30 in the in vivo transduction assay revealed that, despite the presence of almost the entire 5'UTR, the lack of gag sequences and the last 30 bp of the 5'UTR reduced the transduction efficiency by nearly 8-fold compared with TR394 (Fig. 3A). This pattern of progressive loss in titre continued with each incremental deletion in the 5'UTR until essentially no Hygr colonies were observed with MB38, the vector that contains no 5'UTR, including the PBS (Fig. 3A
). The continued drop in transduction efficiency was observed despite the fact that equal amounts of virions were produced in each of the transfected cultures (Fig. 3B
, panel IV). The essentially steady drop in viral titres also did not correlate with the reduction in the packaging efficiency, which oscillated between 16- and 48-fold depending on the deletion. Additionally, the drop in viral titres was accompanied by large standard deviations not observed with the 5'UTR deletion series in the absence of gag (Fig. 1
). The large standard deviations of viral titres along with the oscillation in packaging efficiency observed, once again, suggested that the absence of gag and the last 30 bp of the 5'UTR variably affected the formation of some structural element(s) at the 5' end of the UTR important for vector RNA packaging and propagation.
In short, the present study reveals that, similar to the of complex retroviruses, the
of FIV is multipartite, consisting of at least two discontinuous core elements, one residing within the first 90120 bp of the 5'UTR upstream of the major SD (Fig. 1
), while the other resides in the first 90 bp of the gag ORF (Fig. 2
). The two core elements are equally important and simultaneously required for packaging, since deletion of either element alone reduces packaging to a similar low level (Figs 1 and 2
), while deletion of both elements together reduces packaging drastically (Fig. 3
). We suspect that these two regions may form a higher-order structure(s). Our preliminary computer analysis of the 5' end of the FIV genome has revealed a complex secondary RNA structure. Whether this structure will be important for FIV RNA packaging remains to be determined.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Harrich, D., Hooker, C. W. & Parry, E. (2000). The human immunodeficiency virus type 1 TAR RNA upper stemloop plays distinct roles in reverse transcription and RNA packaging. J Virol 74, 56395646.
Johnston, J. C., Gasmi, M., Lim, L. E., Elder, J. H., Yee, J.-K., Jolly, D. J., Campbell, K. P., Davidson, B. L. & Sauter, S. (1999). Minimum requirement for efficient transduction of dividing and nondividing cells by feline immunodeficiency virus vectors. J Virol 73, 49915000.
Kim, H.-K., Lee, K. & O'Rear, J. J. (1994). A short sequence upstream of the 5' major splice site is important for encapsidation of HIV-1 genomic RNA. Virology 198, 336340.[CrossRef][Medline]
McBride, M. S. & Panganiban, A. T. (1996). The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol 70, 29632973.[Abstract]
McBride, M. S. & Panganiban, A. T. (1997). Position dependence of functional hairpins important for human immunodeficiency virus type 1 encapsidation in vivo. J Virol 71, 20502058.[Abstract]
McBride, M. S., Schwartz, M. D. & Panganiban, A. T. (1997). Efficient encapsidation of human immunodeficiency virus type 1 vectors and further characterization of cis elements required for encapsidation. J Virol 71, 45444554.[Abstract]
Poeschla, E. M., Wong-Staal, F. & Looney, D. J. (1998). Efficient transduction of nondividing cells by feline immunodeficiency virus lentiviral vectors. Nat Med 4, 354357.[Medline]
Rizvi, T. A. & Panganiban, A. T. (1993). Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles. J Virol 67, 26812688.[Abstract]
Schmidt, R. S., Mustafa, F., Lew, K. A., Browning, M. T. & Rizvi, T. A. (2003). Sequences both within the 5' untranslated region and the gag gene are important for efficient encapsidation of MasonPfizer monkey virus RNA. Virology (in press).
Sorge, J., Ricci, W. & Hughes, S. H. (1983). Cis-acting RNA packaging locus in the 115-nucleotide direct repeat of Rous sarcoma virus. J Virol 48, 667675.[Medline]
Yu, S. S., Kim, J.-M. & Kim, S. (2000). The 17 nucleotides downstream from the env gene stop codon are important for murine leukemia virus packaging. J Virol 74, 87758780.
Received 9 October 2002;
accepted 13 November 2002.