Department of Molecular Virology, Immunology, and Medical Genetics, Center for Retrovirus Research, and Comprehensive Cancer Center, Ohio State University Medical Center, 2078 Graves Hall, 333 West 10th Ave, Columbus, OH 43210, USA1
Molecular, Cellular, and Developmental Biology Graduate Program, Ohio State University, USA2
Author for correspondence: Louis Mansky. Fax +1 614 292 9805. e-mail mansky.3{at}osu.edu
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Introduction |
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RNAprotein interactions involved in genome recognition |
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RNA signals for genome recognition |
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HIV-1 SL1, SL2 and SL3 are likely not the only cis-acting elements within the genomic RNA involved in RNA encapsidation. Recent data suggest that the region upstream of the primary HIV-1 encapsidation signal may contribute to optimal encapsidation of the viral genomic RNA. Within this region is the TAR stemloop, a stemloop distal to the TAR element termed the r-U5, and a structure designated u5-l that surrounds the pbs (McBride et al., 1997 ). Deletion of these three secondary structures produced encapsidation efficiencies five- to tenfold lower than wt, similar to those observed when the region of SL1SL4 was mutated (McBride et al., 1997
). HIV-1 TAR primarily functions to regulate virus transcription through interaction with the Tat protein. Mutations constructed at the bulge and loop regions of TAR, where Tat binds, had no effect on RNA encapsidation, indicating that the role of the bulgeloop region of TAR in encapsidation is independent of Tat (Helga-Maria et al., 1999
; McBride et al., 1997
). The lower portion of the TAR stem was mutated to disrupt the base-pairing, resulting in an encapsidation efficiency that was 10- to 25-fold lower than wt. When compensatory mutations were introduced that recreated the stemloop, the encapsidation efficiency was fully restored (Helga-Maria et al., 1999
). For HIV-1 encapsidation, it therefore appears that the formation of the TAR stem is the important factor and not the sequence of this region (Helga-Maria et al., 1999
). For the other two regions, it seems that the structures are important factors also. The secondary structure of r-U5 was found not to be essential for encapsidation, but conservation among primate lentiviruses suggests that r-U5 may contribute to optimal encapsidation (McBride et al., 1997
). Although the exact structure surrounding the pbs is not agreed upon, the top of the u51 stemloop contains a secondary structure that is conserved among different retroviruses (McBride et al., 1997
).
HIV-2 RNA encapsidation has been reported to be distinctly different to that of HIV-1 encapsidation. Four deletion mutations were introduced into the 5 UTR of HIV-2, where two of the deletions removed sequences upstream of the major splice donor site and the other two deletions removed sequences downstream (McCann & Lever, 1997 ). The downstream mutations reduced encapsidation efficiency by twofold, whereas analogous mutations in HIV-1 produced a 10- to 100-fold reduction in encapsidation efficiency (McCann & Lever, 1997
). Mutations upstream of the HIV-2 splice donor resulted in a significant deficiency, three- to sixfold, in encapsidation. The upstream deletions not only affected encapsidation, but replication as well. In HIV-2, the upstream deletion that removed the dimerization signal produced the most severe results (McCann & Lever, 1997
). (The potential role of RNA dimerization in genome recognition and RNA encapsidation will be addressed later in this review.) The inverse occurs in HIV-1, where mutations downstream of the splice donor affect both encapsidation and replication. These data suggest that the HIV-2 encapsidation signal is positioned on both spliced and unspliced viral RNAs, though no evidence of efficient encapsidation of spliced RNA has been shown. It has been proposed that HIV-2 employs a novel mechanism for the selection of unspliced viral RNA, in which unspliced RNA is translated, producing the Gag polyprotein. This polyprotein then binds to the encapsidation signal on that same viral RNA, thus directing that RNA for encapsidation (Kaye & Lever, 1999
). (The potential interplay of RNA encapsidation and translation is discussed later.) The initial binding of the Gag polyprotein to the viral RNA could lead to the attraction of other Gag polyproteins to the viral RNA. The Gag monomers could bind to each other via homology regions and the NC subdomains would bind the viral RNA through non-specific interactions (Kaye & Lever, 1999
).
HTLV-BLV
The encapsidation signal region of BLV was initially mapped by deletion analysis (Mansky et al., 1995 ). The efficiency of RNA encapsidation revealed two important regions. The first region includes sequences downstream of the pbs and near the gag gene start codon, and was found to be essential for RNA encapsidation. The second region was a 132 nucleotide base sequence within the gag gene that facilitates efficient RNA encapsidation in the presence of the first region. These results led to the conclusion that the encapsidation signal necessary for efficient RNA packaging and virus production is discontinuous. Structurefunction analysis (Mansky & Wisniewski, 1998
) has provided genetic evidence that the primary encapsidation signal region of BLV consists of two stable RNA stemloop structures located just downstream of the gag start codon in the MA domain that are required for RNA encapsidation and virus production (Fig. 2b
). A secondary encapsidation signal was characterized in the CA domain of Gag that consists of one stable stemloop structure. It was also found that the encapsidation signal region of either HTLV-1 or HTLV-2 can replace the BLV primary encapsidation signal region and lead to either efficient or a modest level of replication of the chimeric virus, respectively. HTLV-1 and HTLV-2 have similar SL1 and SL2 structures downstream of the Gag start codon (Fig. 2c
), and the HTLV-1 SL1 and SL2 sequence has been shown to specifically replace the BLV SL1 and SL2 in a BLV vector (unpublished results). In comparison, the Moloney murine leukaemia virus (MoMLV) encapsidation signal includes four motifs (Fig. 2d
) (Mougel et al., 1996
). Motifs C and D are necessary for efficient encapsidation, and the presence of either motif C or D is crucial for encapsidation and virus replication. These observations indicate that simple and complex retroviruses may not be that easily separated simply on the basis of RNA encapsidation signal architecture.
Spumavirus
The cis-acting sequences that are likely required for spumavirus RNA encapsidation have been mapped in the HFV genome. Although spumaviruses contain viral DNA in their particles, the genome recognition event involves the unspliced viral RNA (pregenome) (Linial, 1999 ; Yu et al., 1999
). Two regions in the HFV genome have been identified as being important for efficient replication of spumavirus-based vectors (Erlwein et al., 1998
; Heinkelein et al., 1998
; Wu et al., 1998
). One region spans from the r region to the 5 end of the gag gene, while the second region is located towards the 3 end of the pol gene. The location of encapsidation sequences in the pol gene would be a unique observation. There are three sites at the 5 end of the unspliced RNA that have been reported to represent the dimer linkage structure (DLS), and mutation of the upstream site diminished RNA dimerization and inhibited HFV vector transfer by wt helper HFV (Erlwein et al., 1997
, 1998
). Structurefunction analyses of the RNA structures involved in HFV RNA encapsidation have not been reported to date.
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Protein domains involved in specific RNA binding |
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Other experiments that altered the basic residues flanking the zinc finger motifs resulted in a general decrease in the ability of HIV-1 NC to bind RNA. Arginine and lysine residues of HIV-1 NC were mutated by alanine scanning mutagenesis to determine the role of these basic residues in HIV-1 encapsidation (Poon et al., 1996 ). Mutation of lysine residues 11 and 14, which are located at the amino terminus of Cys-His box 1, produced encapsidation efficiencies fourfold lower than wt (Poon et al., 1996
). Substitution of arginine 7 with alanine reduced encapsidation of viral RNA by twofold (Poon et al., 1996
). Additionally, altering arginine 7 to a neutral amino acid drastically reduces the binding affinity of NC for viral RNA, more than any other single amino acid mutation (Schmalzbauer et al., 1996
). Mutation of arginine 32 and lysine 33, located between the two Cys-His boxes, also leads to a decrease in binding ability (Schmalzbauer et al., 1996
). RNA-binding proteins contain a consensus sequence of RXXRR or RXXRK (where X is any amino acid); arginines 7 and 29 each correspond to the beginning of this motif in the NC protein of HIV-1. This RNA-binding motif is also found in HIV-2, simian immunodeficiency virus, Rous sarcoma virus and MoMLV (Schmalzbauer et al., 1996
).
Complex retroviruses, with the exception of the spumaviruses, contain two zinc-binding motifs in the NC domain that are required for replication. Ablation of one of the Cys-His motifs results in non-infectious viruses and insufficient encapsidation. As eluded to above, the two Cys-His motifs are not interchangeable or functionally equivalent. The first zinc-binding motif plays a prominent role in RNA selection and encapsidation, as evidenced by experiments in which the two motifs were either switched or duplicated. Mutant HIV-1 viruses that contained two copies of the second Cys-His motif, or with the positions of the first and second motifs reversed, encapsidated viral RNA at less than 15% of wt virus (Gorelick et al., 1993 ). This low level of encapsidation is similar to that observed in mutants with altered zinc-binding ability. In contrast, HIV-1 mutants that contained two copies of the first Cys-His motif encapsidated viral RNA at 70% of the wt level (Gorelick et al., 1993
). This suggests that the first zinc-binding motif needs to be in the primary position for effective encapsidation. Comparison of the amino acid sequence of the Cys-His motifs reveals that the first motif is more highly conserved among retroviruses, implying that these residues may assist with the recognition and encapsidation of viral RNA (Gorelick et al., 1993
).
While it is known that the NC domain is necessary for RNA encapsidation, it is not certain whether NC actually confers the selective recognition of the viral genomic RNA, as it is known that NC possesses a non-specific RNA-binding activity (Berkowitz et al., 1995 ). In order to determine the specificity of HIV-1 NC for viral RNA, a chimera was constructed in which the entire HIV-1 NC domain of Gag was substituted with the mouse mammary tumour virus (MMTV) NC domain (Fig. 3
). This HIV-1 chimera preferentially encapsidated HIV-1 genomic RNA when both HIV and MMTV genomes were present. In the reciprocal experiment, the MMTV NC domain of MMTV Gag was replaced with the HIV-1 NC domain and this MMTV chimeric Gag was found to encapsidate the MMTV genome when both the MMTV and HIV-1 genomes were present (Poon et al., 1998
). These observations indicate that the NC domain and zinc-binding motifs are not solely responsible for specific HIV-1 RNA packaging, and support the hypothesis that not all the protein domains required for specificity of genome recognition and RNA encapsidation lie within the NC domain. To test this, an MA domain deletion mutant revealed that the MA domain was not required for RNA packaging (Poon et al., 1998
). [It is worthy to note here that 90% of the MA domain in HIV-1 has been found to be dispensable for virus replication in a cell line in which the cytoplasmic domain of Env is not required (Reil et al., 1998
).] In total, these results suggest that the CA domain or other viral proteins may contribute to HIV-1 RNA packaging.
In contrast to the results described above, an earlier study created a chimeric HIV-1 Gag in which the MoMLV NC domain (Fig. 3) was substituted for the HIV-1 NC domain, as well as the creation of a chimeric MoMLV that contained the HIV-1 NC in place of the MoMLV NC domain. The MoMLV mutant, with HIV-1 NC, preferentially encapsidated unspliced HIV-1 viral RNA over spliced HIV-1, while the HIV-1 chimeric mutant, with MoMLV NC, encapsidated RNA that contained the MoMLV encapsidation signal (Berkowitz et al., 1995
). These results imply that the NC domain is solely responsible for genome recognition. The important consideration when comparing the results between these two reports is the number of Cys-His motifs in the NC domains. The MMTV has two zinc-binding domains (and is therefore similar to the HIV-1 NC), while the MoMLV has only one. As noted above, the number of motifs plays an important role in encapsidation. Therefore, taken together, the data from these two papers indicate that the two zinc-binding domains are necessary but not sufficient for specific HIV-1 RNA encapsidation (Poon et al., 1998
).
Selective encapsidation of viral RNA may occur through the non-specific binding ability of NC coupled to the specific recognition of the viral genome via another region in the Gag polyprotein (Berkowitz et al., 1995 ). There has been one suggestion that p2, the spacer peptide between CA and NC, may be that region (Kaye & Lever, 1998
). Through cross-packaging experiments, it was found that HIV-1 could encapsidate both HIV-1 and HIV-2 RNA, yet HIV-2 was unable to encapsidate HIV-1 RNA (Kaye & Lever, 1998
). However, HIV-2 chimeras that contained the HIV-1 NC and p2 domains were able to encapsidate HIV-1 RNA. HIV-2 chimeras with only the HIV-1 NC domain also exhibited the ability to encapsidate HIV-1 RNA, although at a lower level than when both NC and p2 were present (Kaye & Lever, 1998
). HIV-1 and HIV-2 NC proteins have a high level of amino acid sequence identity (60%), as well as many conservative substitutions elsewhere in the protein, while within the p2 domain they share only 35% amino acid identity, supporting the hypothesis that selective recognition may be found in other regions of the Gag precursor (Kaye & Lever, 1998
). The identity between the NC domains of HIV-1 and HIV-2 is likely due to the mechanistic binding of the NC domain to the encapsidation signal.
The NMR solution structure of HIV-1 NC protein with SL3 has been determined and provides structural insight into the genome recognition event. The basic residues, arginine and lysine, at positions 3 and 10 of NC, form a 310 helix that interacts with the RNA major groove (De Guzman et al., 1998 ). The two zinc-binding motifs of HIV-1 NC form hydrogen bonds to the exposed guanosines on the SL3 loop, providing the main interaction between the NC and encapsidation signal (Fig. 5
) (De Guzman et al., 1998
). Structurally, the SL3 NCRNA complex differs from other characterized proteinRNA complexes in which purine-purine base pairs open up the major groove to the insertion of alpha helices or beta sheets. In this complex, a kink in the RNA background is created, producing a widening of the major groove which allows penetration of the Lys-Arg helix (Fig. 5
) (De Guzman et al., 1998
). The association between the NC and SL3 is further stabilized by additional intra- and intermolecular interactions between the amino acids of the Cys-His motif and nucleic acids of the SL3 loop.
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Spumavirus
HFV encodes a 78 kDa Gag polyprotein that has MA, CA and NC domains (Linial, 1999 ; Pfrepper et al., 1999
). Unique to the spumaviruses is the fact that the major homology region in the CA domain and the Cys-His boxes in the NC domain are not present in Gag. In the NC domain, there are three glycine-arginine-rich domains located at the carboxyl-terminal end of the polyprotein (Fig. 3
) (Yu et al., 1996
). The Gag protein does not appear to be efficiently cleaved and assays of extracellular proteins show two predominant forms of Gag, 78 and 74 kDa (Enssle et al., 1997
; Linial, 1999
; Pfrepper et al., 1999
). This indicates that Gag is not cleaved by the viral protease into the MA, CA and NC polypeptides in mature virus particles. The cleavage of the 78 kDa protein to the 74 kDa protein requires the viral protease and is needed for virus infectivity (Enssle et al., 1997
; Linial, 1999
). The absence of a mature NC protein in foamy virus particles is notable, and may correlate with the observation of full-length viral DNA in particles. Thus, foamy viruses provide some clear evidence that full-length Gag is the molecule that recognizes the viral genomic RNA and initiates the assembly process. Once encapsidated, the RNA is reverse-transcribed to DNA and Gag is not processed into MA, CA and NC polypeptides. The NC domain of Gag is likely to be involved in foamy virus genome recognition, but this has not been studied in great detail. Alignment of the HFV NC domain with other complex retroviruses does not indicate significant homologies.
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Beyond genome recognition: complete encapsidation of the viral RNA and virus assembly |
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The basic residues of the HIV-1 MA have been shown to contribute to GagGag interaction in the presence of RNA and the absence of the NC domain (Burniston et al., 1999 ). This indicates that these residues have the potential to interact with RNA (Lochrie et al., 1997
) and that MARNA interaction may play an important role in complete RNA encapsidation and virus particle assembly. Specific to HTLV-BLV group viruses, the MA domain of BLV Gag has been implicated in specific binding with BLV RNA (Katoh et al., 1991
, 1993
), and could therefore also play a specific role in the genome recognition step, or at least in subsequent RNA encapsidation. The presence of genomic RNA appears to enhance the multimerization of HIV-1 Gag, and it is likely that Gag multimerization is important in the RNA encapsidation process (Morikawa et al., 1999
, 2000
).
HIV-1 MA has been reported to have a nuclear export signal (located in the N-terminal end of MA) which could target both Gag and the genomic RNA to the cell membrane (Fig. 1) (Dupont et al., 1999
). The cytoskeleton may play an important role in transporting the Gagviral RNA complex to the cell membrane. In particular, the actin cytoskeleton has been found to interact with HIV-1 Gag through the NC domain (Liu et al., 1999
; Wilk et al., 1999
).
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Other issues related to genome recognition, RNA encapsidation and virus assembly |
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The mechanisms behind how viral genomic RNA is packaged, preferentially to that of spliced viral RNAs among the complex retroviruses, is not entirely clear. While for most retroviruses the primary encapsidation signal is found only in the unspliced genomic RNA, many of the spliced viral mRNAs contain secondary encapsidation signals. For HIV-1, spliced mRNAs can be encapsidated to the order of 10% of the levels of genomic RNA (Katz et al., 1986 ; Luban & Goff, 1994
). Why these sequences are located in regions of the RNA that are present in the spliced RNA is not clear. The role of packaging the spliced viral RNAs in the retrovirus life-cycle is unknown.
Little is known regarding variables that determine whether newly synthesized viral unspliced RNA will be encapsidated or translated. It is possible that there is no regulation, and that all RNAs are competent for both translation and packaging. In this situation, it is likely that the viral RNAs would be initially translated until enough Gag polyprotein had been made to initiate the process of RNA encapsidation. However, it is plausible that there may be some discrimination between these functions of the unspliced viral RNA.
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Antiviral drugs |
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Summary |
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Acknowledgments |
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References |
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