Cis-acting Packaging Signal Research Articles

Structural phylogenetic analysis reveals lineage-specific RNA repetitive structural motifs in all coronaviruses and associated variations in SARS-CoV-2.

In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA–protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5′-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5′-gUUYCGUc-3′ RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5′-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5′-agc-3′/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5′-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5′-gUUUCGUc-3′ motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2Min, a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2Min, 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either (a) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or (b) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation.

On the Selective Packaging of Genomic RNA by HIV-1.

Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag–gRNA complex will nucleate particle assembly more efficiently than other Gag–RNA complexes. New data shows that among cellular mRNAs, those with long 3′-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3′-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

Experimental Approaches to Study Genome Packaging of Influenza A Viruses

The genome of influenza A viruses (IAV) consists of eight single-stranded negative sense viral RNAs (vRNAs) encapsidated into viral ribonucleoproteins (vRNPs). It is now well established that genome packaging (i.e., the incorporation of a set of eight distinct vRNPs into budding viral particles), follows a specific pathway guided by segment-specific cis-acting packaging signals on each vRNA. However, the precise nature and function of the packaging signals, and the mechanisms underlying the assembly of vRNPs into sub-bundles in the cytoplasm and their selective packaging at the viral budding site, remain largely unknown. Here, we review the diverse and complementary methods currently being used to elucidate these aspects of the viral cycle. They range from conventional and competitive reverse genetics, single molecule imaging of vRNPs by fluorescence in situ hybridization (FISH) and high-resolution electron microscopy and tomography of budding viral particles, to solely in vitro approaches to investigate vRNA-vRNA interactions at the molecular level.

Identification of cis-acting packaging signals in the coding regions of the influenza B virus HA gene segment.

For influenza A and B viruses to be infectious, they require eight viral RNA (vRNA) genome segments to be packaged into virions. For efficient packaging, influenza A viruses utilize cis-acting vRNA sequences, containing both non-coding and protein coding regions of each segment. Whether influenza B viruses have similar packaging signals is unknown. Here we show that coding regions at the 3' and 5' ends of the influenza B virus vRNA segment 4 are required for genome packaging, with the first 30 nt at each end essential for this process. Synonymous mutation of these regions led to virus attenuation, an increase in defective particle production and a reduction in packaging of multiple vRNAs. Overall, our data suggest that the influenza B virus vRNA gene segments likely interact with each other during the packaging process, which is driven by cis-acting packaging signals that extend into protein coding regions of the vRNA.

Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP–packaging signal RNA interaction in living cells.

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs--only four nucleotides per genomic RNA--reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

RNA packaging in lentiviruses

The selection by a lentivirus of its genomic RNA from the background of a vast excess of cellular messenger RNAs and sub-genomic viral RNAs occurs with high specificity. The nature and site of the specific interaction between the unspliced RNA genome and the gag polyprotein, which is responsible for its capture, is still poorly understood and the routes by which the two partners arrive at the initial site of interaction and what happens thereafter are similarly obscure. We have been studying this process in a number of different lentiviruses including HIV-1, HIV-2, SIV and FIV. A number of common themes emerge. Specificity of the interaction depends on RNA motifs, commonly short runs of purines in the context of specific structures. The cis-acting packaging signal structures are at the 5' end of the genome but may be upstream or downstream of the major splice donor, hence they may require additional mechanisms for specificity in some cases. The 5' structured untranslated region, together with sequences stretching into the gag open reading frame appear to be able to form more than one structure commonly involving long-range interactions and the different structures may contribute to the different functions of spliced and unspliced RNA. Different structures also very likely form at different stages of the passage of the genomic RNA from transcription through to encapsidation, reflecting interactions with different cellular chaperone proteins as well as trans acting viral proteins involved in trafficking of the RNA through the cell. There is good evidence for significant structural change occurring when the RNA interacts with protein ligands such as the Gag and the degree of conformational change may be quite profound. There is a close link between RNA dimerisation and packaging and it seems likely that the RNA is initially packaged as a dimer, however, whether RNA dimerisation is a necessary step for packaging is less clear and evidence from some viral systems, including HIV-2, suggests that the link with packaging is more critical to ensure a dimeric genome is present for maximum infectivity of the virus when it enters the next host cell. Newer structural analytical techniques are teaching us more about the nature of the RNA genome and its structural changes during the packaging process.

Production of viral vectors using recombinase-mediated cassette exchange

DNA viruses are often used as vectors for foreign gene expression, but large DNA region from cloned or authentic viral genomes must usually be handled to generate viral vectors. Here, we present a unique system for generating adenoviral vectors by directly substituting a gene of interest in a small transfected plasmid with a replaced gene in a replicating viral genome in Cre-expressing 293 cells using the recombinase-mediated cassette exchange (RMCE) reaction. In combination with a positive selection of the viral cis-acting packaging signal connected with the gene of interest, the purpose vector was enriched to 97.5 and 99.8% after three and four cycles of infection, respectively. Our results also showed that the mutant loxP V (previously called loxP 2272), a variant target of Cre used in the RMCE reaction, was useful as a non-compatible mutant to wild-type loxP. This method could be useful for generating not only a large number of adenovirus vectors simultaneously, but also other DNA virus vectors including helper-dependent adenovirus vector.

Inhibition of HIV-1 replication by 5'LTR decoy RNA

Long terminal repeats (5'-LTR) contain essential enhancer and promoter elements that act as targets for cellular and viral regulatory proteins. The interplay of these cis-acting sequences with specific transcription factors (NF kappa B, Ap1, and Sp1) present in infected cells determines the rate of transcription initiation from the 5'-LTR promoter element, and thus strongly influences the level of viral replication. Furthermore, Tat functions to enhance transcriptional elongation by binding to the trans-activation response element (TAR). These transcriptional factors enhance the transcriptional elongation from the HIV-1 promoter. HIV-1 genomic RNA packaging is a highly specific process involving interactions between the viral Gag precursor and cis acting packaging signals in the 5' leader sequence of HIV-1 genomic RNA. We examined the suppression effect of HIV-1 expression by the decoy RNAs targeted to these HIV-1 transcriptional and packaging regulatory regions. HIV-1 expression was inhibited by the 5'-LTR decoy RNA, and measured by the amount of HIV-1 gag p24 protein, but no inhibition of HIV-1 packaging could be detected.

Functional analysis of the core human immunodeficiency virus type 1 packaging signal in a permissive cell line.

Packaging of type C retrovirus genomic RNAs into budding virions requires a highly specific interaction between the viral Gag precursor and unique cis-acting packaging signals on the full-length RNA genome, allowing the selection of this RNA species from among a pool of spliced viral RNAs and similar cellular RNAs. This process is thought to involve RNA secondary and tertiary structural motifs since there is little conservation of the primary sequence of this region between retroviruses. To confirm RNA secondary structures, which we and others have predicted for this region, disruptive, compensatory, and deletion mutations were introduced into proviral constructs, which were then assayed in a permissive cell line. Disruption of either of two predicted stem-loops was found to greatly reduce RNA encapsidation and replication, whereas compensatory mutations restoring base pairing to these stem-loops had a wild-type phenotype. A GGNGR motif was identified in the loops of three hairpins in this region. Results were consistent with the hypothesis that the process of efficient RNA encapsidation is linked to dimerization. Replication and encapsidation were shown to occur at a reduced rate in the absence of the previously described kissing hairpin motif.

Location of cis-acting signals important for RNA encapsidation in the leader sequence of human immunodeficiency virus type 2.

We used a series of deletion mutations in the untranslated leader region of human immunodeficiency virus type 2 (HIV-2) to seek cis-acting packaging signals. Sequences between the 5' major splice donor and the gag initiation codon, where such signals have been identified in HIV-1, appear to make a measurable but very minor contribution to genomic RNA packaging, and deletions here had little effect on viral replication in vitro. Immediately 5' to the splice donor, two regions were identified which, when deleted, caused a significant replication defect. The most proximal of these to the splice donor demonstrated a phenotype consistent with its being a major cis-acting packaging signal in HIV-2.

Mutational analysis of cis-acting packaging signals in human immunodeficiency virus type 1 RNA.

We previously identified blocks of sequence near the 5' end of the human immunodeficiency virus (HIV-1) genome which conferred on RNA the ability to bind specifically to the HIV-1 Gag polyprotein, Pr55gag (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luban, and S. P. Goff, J. Virol. 67:7190-7200, 1993). Here we report the use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstrated with wild-type HIV-1 sequences that in comparison with spliced viral RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinger, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrupted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletion mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging sequences. Using this system, we were able to demonstrate significant effects on packaging of RNAs with mutations immediately preceding the first codon of gag. The greatest reduction in packaging was seen with RNAs lacking the first 40 nucleotides of gag coding sequence, although sequences more 3' had slight additional effects.

A conserved cis-acting sequence in the 5' leader of avian sarcoma virus RNA is required for packaging.

The deletion of a conserved sequence of ca. 30 nucleotides in the 5' noncoding leader region of an avian sarcoma virus DNA clone resulted in a loss of infectivity after transfection of chicken embryo fibroblasts. Genetic and biochemical analysis of a representative mutant demonstrated that the env gene was expressed normally. Thus, viral RNA transcription, splicing, and translation were not impaired. The amount of mutant viral RNA encapsidated into virions, however, was severely reduced despite the presence of helper-virus. We conclude that the deleted sequence is an essential cis-acting packaging signal.