Specific high-affinity binding of host cell proteins to the 3' region of rubella virus RNA

Replication of rubella virus is initiated at the 3' end of the genomic RNA. An inverted repeat sequence of 12 nucleotides that is capable of forming a stem-loop structure is located at the 3' end of the RNA, 59 nucleotides upstream from the poly (A) tail. We screened the 158-bp region of the 3' end of the virus, including the stem-loop structure, for its ability to bind to host-cell proteins. Specific high-affinity binding of three cytosolic proteins with relative molecular masses (Mr) of 61, 63 and 68 kD to the stem-loop structure was observed by UV-induced covalent crosslinking. Altering the stem structure by removal of specific bases abolished the binding interactions. The binding of the host proteins is greatly increased after infection and coincides with the appearance of negative strand RNA synthesis. The increase in binding is dependent on new protein synthesis. The amount of the 61-kD protein that binds varies in uninfected cells and is maximal in cells that are in the stationary phase of growth. All binding activity could be abrogated by alkaline phosphatase treatment of cell lysates. A possible role of these host proteins in the replication of rubella virus is discussed.     

Multiple binding sites for cellular proteins in the 3' end of Sindbis alphavirus minus-sense RNA.

The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The first binding domain was found in the first 60 nucleotides, which represents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and the fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, Kr, of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Deletion mapping of the first domain showed that the first 10 nucleotides were critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides was deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40, or 41 to 55 had little effect on the binding, and deletion of nucleotides 15 to to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross River virus and Semliki Forest virus, and from rubella virus were also able to interact with the 50- and 52-kDa proteins. The Kr value for the Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times greater. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replication. We suggest that the binding of the 50- and 52-kDa proteins to the 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.     

Interaction of cellular proteins with the 5' end of Norwalk virus genomic RNA

The lack of a susceptible cell line and an animal model for Norwalk virus (NV) infection has prompted the development of alternative strategies to generate in vitro RNAs that approximate the authentic viral genome. This approach has allowed the study of viral RNA replication and gene expression. In this study, using mobility shift and cross-linking assays, we detected several cellular proteins from HeLa and CaCo-2 cell extracts that bind to, and form stable complexes with, the first 110 nucleotides of the 5' end of NV genomic RNA, a region previously predicted to form a double stem-loop structure. These proteins had molecular weights similar to those of the HeLa cellular proteins that bind to the internal ribosomal entry site of poliovirus RNA. HeLa proteins La, PCBP-2, and PTB, which are important for poliovirus translation, and hnRNP L, which is possibly implicated in hepatitis C virus translation, interact with NV RNA. These protein-RNA interactions are likely to play a role in NV translation and/or replication.     

Specific binding of host cell proteins to the 3''-terminal stem-loop structure of rubella virus negative-strand RNA.

At the 5' end of the rubella virus genomic RNA, there are sequences that can form a potentially stable stem-loop (SL) structure. The complementary negative-strand equivalent of the 5'-end SL structure of positive-strand rubella virus RNA [5' (+) SL structure] is thought to serve as a promoter for the initiation of positive-strand synthesis. We screened the negative-strand equivalent of the 5' (+) SL structure (64 nucleotides) and the adjacent region of the negative-strand RNA for their ability to bind to host cell proteins. Specific binding to the 64-nucleotide-long potential SL structure of three cytosolic proteins with relative molecular masses of 97, 79, and 56 kDa was observed by UV-induced covalent cross-linking. There was a significant increase in the binding of the 97-kDa protein from cells upon infection with rubella virus. Altering the SL structure by deleting sequences in either one of the two potential loops abolished the binding interaction. The 56-kDa protein also appeared to bind specifically to an SL derived from the 3' end of positive-strand RNA. The 3'-terminal structure of rubella virus negative-strand RNA shared the same protein-binding activity with similar structures in alphaviruses, such as Sindbis virus and eastern equine encephalitis virus. A possible role for the host proteins in the replication of rubella virus and alphaviruses is discussed.     

Three different cellular proteins bind to complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA.

The termini of viral genomic RNA and its complementary strand are important in the initiation of viral RNA replication, which probably involves both viral and cellular proteins. To detect the possible cellular proteins involved in the replication of mouse hepatitis virus RNA, we performed RNA-protein binding studies with RNAs representing both the 5' and 3' ends of the viral genomic RNA and the 3' end of the negative-strand complementary RNA. Gel-retardation assays showed that both the 5'-end-positive- and 3'-end-negative-strand RNA formed an RNA-protein complex with cellular proteins from the uninfected cells. UV cross-linking experiments further identified a 55-kDa protein bound to the 5' end of the positive-strand viral genomic RNA and two proteins 35 and 38 kDa in size bound to the 3' end of the negative-strand cRNA. The results of the competition assay confirmed the specificity of this RNA-protein binding. No proteins were found to bind to the 3' end of the viral genomic RNA under the same conditions. The binding site of the 55-kDa protein was mapped within the 56-nucleotide region from nucleotides 56 to 112 from the 5' end of the positive-strand RNA, and the 35- and 38-kDa proteins bound to the complementary region on the negative-strand RNA. The 38-kDa protein was detected only in DBT cells but was not detected in HeLa or COS cells, while the 35-kDa protein was found in all three cell types. The juxtaposition of the different cellular proteins on the complementary sites near the ends of the positive- and negative-strand RNAs suggests that these proteins may interact with each other and play a role in mouse hepatitis virus RNA replication.     

Specific binding of human immunodeficiency virus type 1 (HIV-1) Gag-derived proteins to a 5' HIV-1 genomic RNA sequence.

We developed an in vitro binding assay to study the specific interaction between human immunodeficiency virus type 1 (HIV-1) RNA and the Gag polyprotein. Binding of the in vitro-expressed protein to in vitro-transcribed RNA was determined by altered migration of the protein in polyacrylamide gels. We found that a Gag precursor lacking the matrix domain bound specifically to HIV-1 RNA, while deletion of both matrix and capsid domains diminished the specificity of binding. Among several regions of HIV-1 RNA tested, strongest binding was seen with the 5'-most 261 nucleotides, while antisense RNA from the same region did not bind.     

RNA-protein interactions directed by the 3' end of human rhinovirus genomic RNA.

The replication of a picornavirus genomic RNA is a template-specific process involving the recognition of viral RNAs as target replication templates for the membrane-bound viral replication initiation complex. The virus-encoded RNA-dependent RNA polymerase, 3Dpol, is a major component of the replication complex; however, when supplied with a primed template, 3Dpol is capable of copying polyadenylated RNAs which are not of viral origin. Therefore, there must be some other molecular mechanism to direct the specific assembly of the replication initiation complex at the 3' end of viral genomic RNAs, presumably involving cis-acting binding determinants within the 3' noncoding region (3' NCR). This report describes the use of an in vitro UV cross-linking assay to identify proteins which interact with the 3' NCR of human rhinovirus 14 RNA. A cellular protein(s) was identified in cytoplasmic extracts from human rhinovirus 14-infected cells which had a marked binding preference for RNAs containing the rhinovirus 3' NCR sequence. This protein(s) showed reduced cross-linking efficiency for a 3' NCR with an engineered deletion. Virus recovered from RNA transfections with in vitro transcribed RNA containing the same 3' NCR deletion demonstrated a defective replication phenotype in vivo. Cross-linking experiments with RNAs containing the poliovirus 3' NCR and cytoplasmic extracts from poliovirus-infected cells produced an RNA-protein complex with indistinguishable electrophoretic properties, suggesting that the appearance of the cellular protein(s) may be a common phenomenon of picornavirus infection. We suggest that the observed cellular protein(s) is sequestered or modified as a result of rhinovirus or poliovirus infection and is utilized in viral RNA replication, perhaps by binding to the 3' NCR as a prerequisite for replication complex assembly at the 3' end of the viral genomic RNA.     

Kissing-loop interaction in the 3' end of the hepatitis C virus genome essential for RNA replication

The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3' NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3' NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3' end of the HCV genome that is essential for replication.     

RNA of mouse hepatitis virus.

The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.     

Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.     

A single element within the hepatitis B virus enhancer binds multiple proteins and responds to multiple stimuli.

The hepatitis B virus enhancer can be dissected into multiple functional elements, one of which is the E element. We show here that the E element binds multiple nuclear proteins that are essential for its enhancer activity. These findings, together with the ability of this element to respond to at least two different viral transactivators, suggest that the E element is an enhancer modulator capable of binding different factors and responding to multiple stimuli.     

Ribosomal proteins. XXX. Specific protein binding sites on 23S RNA of Escherichia coli

Summary Strains of A. nidulans with a chromosome segment in duplicate (one in normal position, one translocated to another chromosome) are unstable at mitosis. During vegetative growth they produce variants which result from deletions in either of the duplicate segments.Caffeine increased the frequency of deletions from the duplicate segments of an unbalanced haploid a) without changing the proportions of the different deletion types and b) under conditions in which there were few, if any, induced breaks in the same segments of a balanced diploid. One possible explanation is that caffeine stimulates the mechanism which, in unbalanced strains, produces replication errors leading to deletions; an alternative is that it exposes the intrinsic instability of duplication strains by preventing the repair of spontaneous replication errors.     

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.