Moloney murine leukemia virus genomic RNA packaged in the absence of a full complement of wild type nucleocapsid protein

The current model for MLV genomic RNA (gRNA) packaging predicts that of the thousands of Gag proteins in a budding virion, only a small number (≤1%) may be necessary to recruit gRNA. Here, we examined the threshold limits of functional Gag required to package gRNA using wild-type (WT) and packaging deficient mutant nucleocapsid (NC) phenotypically mixed virions. Although gRNA packaging was severely diminished for the NC mutant, the residual encapsidated RNA dimer displayed motility on gels, thermostability, and integrity that was indistinguishable from that of WT. In phenotypically mixed virions, gRNA encapsidation recovered to within approximately two-fold of WT levels when the amount of WT NC was 5-10% of the total. Our results demonstrate that NC's roles in gRNA dimerization and packaging are genetically separable. Additionally, MLV gRNA packaging does not require 100% WT NC, but the amount of functional NC required is greater than the predicted minimum.     

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.     

Dispensability of 3' tRNA-like sequence for packaging cowpea chlorotic mottle virus genomic RNAs

The 3' ends of three genomic RNAs (gRNAs) of cowpea chlorotic mottle virus (CCMV) terminate in a highly conserved tRNA-like structure (3'TLS). To examine the intrinsic role played the 3'TLS in packaging, the competence of each gRNA lacking the 3' TLS (DeltaTLS-gRNA) to interact with dissociated coat protein (CP) subunits and form virions was assayed in vitro. In contrast to the well established requirement for the participation of either viral 3'TLS or host-tRNAs in the assembly of RNA-containing virions in brome mosaic virus (BMV; Choi, Y, G., Dreher, T. W., Rao, A. L. N. 2002. tRNA elements mediate the assembly of an icosahedral RNA virus. Proc. Natl. Acad. Sci. 99, 655-660), CCMV CP does not require the presence of viral TLS in cis or in trans. Similar in vitro assembly assays showed that CCMV CP subunits also packaged BMV RNAs lacking 3' TLS as well as two other non-bromoviral RNAs although with lesser efficiency. To characterize sequences of CCMV RNA3 (C3) required for packaging, a series deletions was engineered into C3 and their effect on virus assembly was examined. It was observed that, unlike BMV RNA3 whose packaging requires a bipartite signal (Choi, Y. G., Rao, A. L. N. 2003. Packaging of brome mosaic virus RNA3 is mediated through a bipartite signal. J. Virol. 77, 9750-9757), packaging of C3 is independent of either movement protein (MP) ORF or CP ORF or 3' non-coding regions. Based on the differential prerequisites identified in this study for the assembly of BMV and CCMV, we hypothesize that the adaptive condition for movement in monocotyledonous host has made packaging a necessary co-requirement for BMV.     

Human immunodeficiency virus type 1 preferentially encapsidates genomic RNAs that encode Pr55(Gag): functional linkage between translation and RNA packaging

Full-length retroviral RNA serves as both messenger and genomic RNA. Therefore, an unspliced RNA could play both roles: viral mRNA could be bound in cis by the same Gag polyprotein that it produced, becoming a packaged genomic RNA. To test this possibility, we used in vivo packaging experiments which coexpressed wild-type NL4-3 RNA and NL4-3-based mutant RNA that, ideally, could not translate Gag. However, mutating the gag initiator produced a mutant (pNLX) that expressed a truncated Gag, Gag*, initiated at methionine 10 in the CA region (142 of Pr55(Gag)). Gag* can be rescued into virions by Gag and, as it contains the NC domain, could package RNA in cis. To eliminate NC and the CA dimerization domain, a nonsense mutation in CA at residue 99 was introduced into pNLX to produce pNLXX, which expresses an RNA that should only be packaged in trans. Cotransfection packaging experiments revealed that wild-type genomic RNA was packaged at an 8-fold greater level than NLXX RNA given equal expression of both RNAs. Experiments that varied the relative amounts of these RNAs in the cell found that the wild-type RNA was encapsidated with a packaging preference (i.e., the relative amount of this RNA in virions versus cells) of 6- to 13-fold over the NLXX RNA, showing that the NLXX RNA did not efficiently compete with NL4-3 RNA. These data suggest that the wild-type RNA's ability to express Pr55(Gag) and, by inference, actively translate Gag confers an advantage in packaging over the nearly identical NLXX RNA. In contrast, the NLX RNA competed with wild-type RNA at a 1-to-3 preference. This ratio is similar to the amounts of Gag* rescued by Gag, suggesting that the presence of Gag* assists in the encapsidation of NLX RNA. Together, our data link translation and particle formation to the packaging of viral RNA and support a model of cis packaging where nascent Gag proteins encapsidate their cognate RNA.     

Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging

To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 or RNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.     

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.     

Functional analysis of dengue virus cyclization sequences located at the 5' and 3'UTRs

Flavivirus RNA replication involves cyclization of the viral genome. A model for this process includes a promoter element at the 5' end of the genome and long-range RNA-RNA interactions. Two pairs of complementary sequences present at the ends of the viral RNA, known as 5'-3'CS and 5'-3'UAR, have been proposed to be involved in dengue virus genome cyclization. The requirement of 5'-3'CS complementarity for viral replication has been experimentally demonstrated for dengue and other mosquito borne flaviviruses. Here, we performed a functional analysis to study the role of 5'-3'UAR sequences using genomic and subgenomic dengue virus RNAs. We found that single mutations disrupting 5'-3' complementarity greatly compromised viral RNA synthesis. Although in most of the cases incorporation of compensatory mutations re-established viral RNA replication, certain nucleotides were found to be involved in alternative secondary structures also important for viral replication. In addition, mutations within 5' or 3'UAR in the context of an infectious dengue virus RNA resulted in spontaneous mutations that restored UAR base pairings. Together, we propose that specific UAR nucleotides as well as 5'-3'UAR complementarity constitute cis-acting signals involved in amplification of the dengue virus genome.     

Molecular dissection of Flock house virus protein B2 reveals that electrostatic interactions between N-terminal domains of B2 monomers are critical for dimerization

Flock house virus (FHV) encodes a suppressor protein B2 to overcome antiviral RNA silencing during infection. Biochemical analyses have shown that a homodimer of B2 binds to double-stranded RNA to inhibit dicer-mediated cleavage of dsRNA and incorporation of small interfering RNAs into the RNA-induced silencing complex. In this study, using FHV-Nicotiana benthamiana system, we identified that the charged amino acids at the N-terminus of B2 are critical for dimerization. Interestingly, B2 mutants defective in dimerization exhibited enhanced silencing suppressor activity, Furthermore, we found that the C-terminal charged amino acids are dispensable for B2 dimerization and viral RNA silencing suppression but are critical for transgene silencing suppression. Additional yeast two hybrid assays revealed that dimerization of B2 is not essential for interacting with the RNA silencing machinery. Taken together, our data provide evidence that both monomeric and dimeric B2 proteins function in different modes to suppress RNA silencing.     

The Red clover necrotic mosaic virus origin of assembly is delimited to the RNA-2 trans-activator

The bipartite RNA genome of Red clover necrotic mosaic virus (RCNMV) is encapsidated into icosahedral virions that exist as two populations: i) virions that co-package both genomic RNAs and ii) virions packaging multiple copies of RNA-2. To elucidate the packaging mechanism, we sought to identify the RCNMV origin of assembly sequence (OAS). RCNMV RNA-1 cannot package in the absence of RNA-2 suggesting that it does not contain an independent packaging signal. A 209 nt RNA-2 element expressed from the Tomato bushy stunt virus CP subgenomic promoter is co-assembled with genomic RNA-1 into virions. Deletion mutagenesis delimited the previously characterized 34 nt trans-activator (TA) as the minimal RCNMV OAS. From this study we hypothesize that RNA-1 must be base-paired with RNA-2 at the TA to initiate co-packaging. The addition of viral assembly illustrates the critical importance of the multifunctional TA element as a key regulatory switch in the RCNMV life cycle.     

Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control

Humans express four distinct non-protein-coding Y RNAs (ncRNAs). To investigate Y RNA functional diversification, we exploited an RNA-based affinity purification method to isolate ribonucleoproteins (RNPs) assembled on individual human Y RNAs. Silver staining and mass spectrometry revealed that the Ro and La proteins assemble with all Y RNAs, while additional proteins associate with specific Y RNAs. Unexpectedly, Y5 RNA uniquely copurified ribosomal protein L5 and its binding partner 5S RNA. These findings reveal a contribution of Y5 to 5S surveillance and suggest that interactions between Ro-Y5 and L5-5S RNPs establish 5S RNA as a target of quality control.     

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.     

Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV) was shown to produce atypical subgenomic RNAs that contain open reading frame la nucleotides and are present under a wide variety of culture conditions, including high and low multiplicities of infection, in simian and porcine host cells, and during infection with cell-adapted and wild-type PRIRSV strains. Sequence analysis demonstrated that they are heterogeneous in 5-3' junction sequence and size and may code for different predicted fusion proteins. This is the first report of these novel RNA5 in arteriviruses and we have termed them heteroclite (meaning 'deviating from common forms or rules") subgenomic RNAs. The unique properties of these subgenomic RNAs include (a) apparent association with normal virus infection and stability during serial passage, (b) packaging of heteroclite RNAs into virus-like particles, (c) short, heterogeneous sequences which may mediate the generation of these RNAs, (d) a primary structure which consists of the two genomic termini with one large internal deletion, and (eJ little apparent interference with parental virus replication. These subgenomic RNA5 may be critical to, or a necessary side product of, viral replication. The expression of these novel RNA species support the template-switching model of similarity-assisted RNA recombination. In summary, PRRSV readily undergoes nonhomologous RNA recombination to generate heteroclite sub-genomic RNA5.