End Of Genomic RNA Research Articles

The highly conserved RNA-binding specificity of nucleocapsid protein facilitates the identification of drugs with broad anti-coronavirus activity

The binding of SARS-CoV-2 nucleocapsid (N) protein to both the 5′- and 3′-ends of genomic RNA has different implications arising from its binding to the central region during virion assembly. However, the mechanism underlying selective binding remains unknown. Herein, we performed the high-throughput RNA-SELEX (HTR-SELEX) to determine the RNA-binding specificity of the N proteins of various SARS-CoV-2 variants as well as other β-coronaviruses and showed that N proteins could bind two unrelated sequences, both of which were highly conserved across all variants and species. Interestingly, both sequences are virtually absent from the human transcriptome; however, they exhibit a highly enriched, mutually complementary distribution in the coronavirus genome, highlighting their varied functions in genome packaging. Our results provide mechanistic insights into viral genome packaging, thereby increasing the feasibility of developing drugs with broad-spectrum anti-coronavirus activity by targeting RNA binding by N proteins.

VPg unlinkase/TDP2 in cardiovirus infected cells: Re-localization and proteolytic cleavage

Cardioviruses cause diseases in many animals including, in rare cases, humans. Although they share common features with all picornaviruses, cardioviruses have unique properties that distinguish them from other family members, including enteroviruses. One feature shared by all picornaviruses is the covalent attachment of VPg to the 5′ end of genomic RNA via a phosphotyrosyl linkage. For enteroviruses, this linkage is cleaved by a host cell protein, TDP2. Since TDP2 is divergently required during enterovirus infections, we determined if TDP2 is necessary during infection by the prototype cardiovirus, EMCV. We found that EMCV yields are reduced in the absence of TDP2. We observed a decrease in viral protein accumulation and viral RNA replication in the absence of TDP2. In contrast to enterovirus infections, we found that TDP2 is modified at peak times of EMCV infection. This finding suggests a unique mechanism for cardioviruses to regulate TDP2 activity during infection.

Secondary Structure of the HIV Reverse Transcription Initiation Complex by NMR

Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus (HIV) upon infection of a host cell. Viral reverse transcriptase initiates from a specific RNA–RNA complex formed between a host transfer RNA (tRNA Lys 3) and a region at the 5′ end of genomic RNA; the 3′ end of the tRNA acts as a primer for reverse transcription of genomic RNA. We report here the secondary structure of the HIV genomic RNA–human tRNA Lys 3 initiation complex using heteronuclear nuclear magnetic resonance methods. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of the 18-bp primer helix with the 3′ end of tRNA Lys 3 drives large conformational rearrangements of the tRNA at the 5′ end while maintaining the anticodon loop for potential loop–loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse transcription.

A unique, thermostable dimer linkage structure of RD114 retroviral RNA

Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem-loop (psl) sequences at the 5' end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55 degrees C-70 degrees C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87 degrees C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 5' region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 5' R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 3' R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 3' end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.

Detection of small RNAs containing the 5′- and the 3′-end sequences of viral genome during West Nile virus replication

In the first step of flavivirus replication, the 5′-end of viral genomic RNA is thought to interact with the 3′-end of the genomic RNA at the complimentary sequences (CSs) located at both ends of the genomic RNA. However, there is little evidence of direct interaction between the two ends of the viral genomic RNA in virus-replicating cells. Herein, we show that viral small negative-strand RNA species, composed of two ends corresponding to the upstream of the 5′-end CS and the downstream of the 3′-end CS of viral genomic RNA, were synthesized during viral replication. We hypothesized that the viral small negative-sense RNAs were synthesized during viral negative-sense RNA synthesis through the template-jumping of viral RNA-dependent RNA polymerase from the 3′-end to the 5′-end of viral genomic RNA used as a template. Our present results strongly indicate that the two ends of viral genomic RNA associate with each other during viral replication.

Complete nucleotide sequence of the hirame rhabdovirus, a pathogen of marine fish

Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed for the hirame rhabdovirus (HIRRV) CA-9703 strain from Korea, and the DNA was sequenced. The 3′-end of genomic RNA was cloned by poly(A)-tailing of the genomic RNA before reverse transcription, and the 5′-end of the genome was cloned by poly(G)- or poly(C)-tailing of the first strand, followed by PCR. The remainder of the genomic DNA was cloned by reverse transcription-polymerase chain reaction using primers that were based on the published rhabdovirus sequences. The complete genome of HIRRV CA-9703 strain comprises 11,034 nucleotides and encodes six genes in the order of: 3′-leader, N, P, M, G, NV, L, and 5′-trailer. These genes are separated by conserved sequences or gene junctions, with one-nucleotide gene spacers. The first 16 of the 19 nucleotides at the ends of the HIRRV genome are complementary, and the first four nucleotides at the 3′-ends of the HIRRV, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and snakehead rhabdovirus (SHRV) genomes are identical. The HIRRV proteins share the highest amino acid sequence homology (ranging from 72% to 92%) with the proteins of IHNV, of all the known fish rhabdoviruses, and the highest sequence homology with respect to the L protein was shared among HIRRV, IHNV, VHSV, and SHRV. Although there were differences in the degrees of relatedness, phylogenetic trees that were derived from multiple sequence alignments of the rhabdovirus proteins showed similar patterns of relationship among these viruses, in which fish Novirhabdoviruses formed a separate clade from spring viremia of carp virus (SVCV), unassigned fish rhabdovirus that was closer to mammalian rhabdoviruses.

Leader RNA of Rinderpest virus binds specifically with cellular La protein: a possible role in virus replication

Rinderpest virus (RPV) is an important member of the Morbillivirus genus in the family Paramyxoviridae and employs a similar strategy for transcription and replication of its genome as that of other negative sense RNA viruses. Cellular proteins have earlier been shown to stimulate viral RNA synthesis by isolated nucleocapsids from purified virus or from virus-infected cells. In the present work, we show that plus sense leader RNA of RPV, transcribed from 3′ end of genomic RNA, specifically interacts with cellular La protein employing gel mobility shift assay as well as UV cross-linking of leader RNA with La protein. The leader RNA synthesized in virus-infected cells was shown to interact with La protein by immunoprecipitation of leader RNA bound to La protein and detecting the leader RNA in the immunoprecipitate by Northern hybridization with labeled antisense leader RNA. Employing a minireplicon system, we demonstrate that transiently expressed La protein enhances the replication/transcription of the RPV minigenome in cells. Sub-cellular immunolocalization shows that La protein is redistributed from nucleus to the cytoplasm upon infection. Our results strongly suggest that La protein may be involved in regulation of Rinderpest virus replication.

Maize Chlorotic Mottle Machlomovirus Expresses Its Coat Protein from a 1.47-kb Subgenomic RNA and Makes a 0.34-kb Subgenomic RNA

Analysis of double-stranded RNAs produced in maize plants infected with maize chlorotic mottle machlomovirus (MCMV) and Northern blots of total RNA from infected plants or protoplasts showed two subgenomic RNAs (sgRNAs). Primer extension was used to map these sgRNAs, which are 1.47 and 0.34 kb long. The transcription start sites are nucleotide (nt) 2970 or 2971 for sgRNA1 and nt 4101 for sgRNA2. The 5′ ends of the sgRNAs are similar to one another and to the 5′ end of genomic RNA, and 11 nt sequences immediately upstream of their transcription start sites are similar. The location of the sgRNA1 transcription start site indicates that MCMV expresses a 7-kDa open reading frame (ORF) from nt 2995 to 3202 instead of the predicted 9-kDa ORF from nt 2959 to 3202. In protoplast inoculation experiments, a silent mutation at nt 2965 and a 4-nt change at nt 2959–2962 stopped the synthesis of sgRNA1 and expression of the coat protein ORF, which begins more than 400 nt downstream. Replication of MCMV does not require the expression of any of the ORFs encoded on sgRNA1. SgRNA2 has the potential to encode 2.3-, 2.7-, and 4.6-kDa peptides, but the function, if any, of sgRNA2 is unknown.

VPg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement.

The V20 cultivar of Nicotiana tabacum was shown previously to exhibit a strain-specific restriction of long-distance movement of tobacco etch potyvirus (TEV). In V20, both TEV-HAT and TEV-Oxnard strains are capable of genome amplification and cell-to-cell movement, but only TEV-Oxnard is capable of systemic infection by vasculature-dependent long-distance movement. To investigate the basis for host-specific movement of TEV, chimeric virus genomes were assembled from TEV-HAT and TEV-Oxnard. Viruses containing the TEV-Oxnard coding regions for HC-Pro and/or capsid protein (CP), two proteins that are known to be essential for TEV long-distance movement, failed to infect V20 systemically. In contrast, chimeric viruses encoding the TEV-Oxnard VPg domain of NIa were able to infect V20 systemically. The critical region controlling the infection phenotype in V20 was mapped to a 67-nucleotide segment containing 10-nucleotide differences, but only five amino acid differences, between TEV-HAT and TEV-Oxnard. In V20 coinfection experiments, a restricted strain had no effect on systemic infection by a long-distance movement-competent chimeric strain, suggesting that the restricted strain was not inducing a generalized systemic resistance response. These data suggest that the VPg domain, which is covalently attached to the 5' end of genomic RNA, interacts either directly or indirectly with host components to facilitate long-distance movement.

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

Reverse transcriptase must perform two specialized template switches during retroviral DNA synthesis. Here, we used Moloney murine leukemia virus-based vectors to examine the site of one of these switches during intracellular reverse transcription. Consistent with original models for reverse transcription, but in contrast to previous experimental data, we observed that this first strand transfer nearly always occurred precisely at the 5' end of genomic RNA. This finding allowed us to use first strand transfer to study the classes of errors that reverse transcriptase can and/or does make when it switches templates at a defined position during viral DNA synthesis. We found that errors occurred at the site of first strand transfer approximately 1000-fold more frequently than reported average reverse transcriptase error rates for template-internal positions. We then analyzed replication products of specialized vectors that were designed to test possible origins for the switch-associated errors. Our results suggest that at least some errors arose via non-templated nucleotide addition followed by mismatch extension at the point of strand transfer. We discuss the significance of our findings as they relate to the possible contribution that template switch-associated errors may make to retroviral mutation rates.

Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands.

We have developed a new model for coronavirus transcription, which we call discontinuous extension, to explain how subgenome-length negatives stands are derived directly from the genome. The current model called leader-primed transcription, which states that subgenomic mRNA is transcribed directly from genome-length negative-strands, cannot explain many of the recent experimental findings. For instance, subgenomic mRNAs are transcribed directly via transcription intermediates that contain subgenome-length negative-strand templates; however subgenomic mRNA does not appear to be copied directly into negative strands. In our model the subgenome-length negative strands would be derived using the genome as a template. After the polymerase had copied the 3'-end of the genome, it would detach at any one of the several intergenic sequences and reattach to the sequence immediately downstream of the leader sequence at the 5'-end of genome RNA. Base pairing between the 3'-end of the nascent subgenome-length negative strands, which would be complementary to the intergenic sequence at the end of the leader sequence at the 5'-end of genome, would serve to align the nascent negative strand to the genome and permit the completion of synthesis, i.e., discontinuous extension of the 3'-end of the negative strand. Thus, subgenome-length negative strands would arise by discontinuous synthesis, but of negative strands, not of positive strands as proposed originally by the leader-primed transcription model.

Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription.

We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The identification of an upstream cis-acting element for MHV subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.

Genomic Organization of Lettuce Necrotic Yellows Rhabdovirus

We have mapped the genome of lettuce necrotic yellows virus (LNYV), the type member of the genus cytorhabdo-virus of the family Rhabdoviridae. We have cloned and sequenced all intergenic regions and the 3′ leader and 5′ trailer of the negative-sense, single-stranded RNA genome of LNYV. The LNYV genome appears to contain six genes, the five expected genes coding for the virion proteins, and a sixth gene of unknown function, as for sonchus yellow net virus (SYNV), a member of the genus nucleorhabdovirus. The proposed LNYV genomic map is 3′-N-4a-4b-M-G-L-5′, where N is the nucleocapsid protein gene; 4a and 4b are two genes, one of which codes for the proposed phosphoprotein P and the other for a putative protein of unknown function; M is the proposed matrix protein gene; G is the proposed glycoprotein gene; and L is the proposed transcriptase gene. The different LNYV intergenic regions have highly conserved consensus sequences, which could be divided into three components: the sequences corresponding to the 3′ end of the mRNAs, intergenic sequences of variable length, and the sequences corresponding to the 5′ end of the mRNAs. A leader sequence of 84 nucleotides (nt) at the 3′ end of the LNYV genomic RNA preceeded the N gene. A trailer sequence of 187 nt at the 5′ end of the genomic RNA followed the L gene. A comparison between LNYV leader and trailer sequences revealed complementary 3′ and 5′ ends, which could give rise to a putative "panhandle" structure with a two bases overhang in the leader sequence. We have compared these sequences to the corresponding sequences of SYNV as well as to vesicular stornatitis virus (VSV) and rabies virus (RV), the type members of the vesiculovirus and lyssavirus genera, respectively, of animal rhabdoviruses. Homologies were found in the intergenic regions between LNYV, SYNV, VSV, and RV, at the 3′ ends of the mRNAs. LNYV intergenic sequences were of variable lengths, as were those found in RV. The consensus sequences found at the 5′ ends of LNYV mRNAs differed from the highly conserved consensus transcription start sequence UUGU/A found in SYNV, VSV, and RV. Conserved sequences were also found in the first 30 nt of the leader and the last 30 nt of the trailer, between LNYV, SYNV, VSV, and RV.

Evidence for coronavirus discontinuous transcription.

Coronavirus subgenomic mRNA possesses a 5'-end leader sequence which is derived from the 5' end of genomic RNA and is linked to the mRNA body sequence. This study examined whether coronavirus transcription involves a discontinuous transcription step; the possibility that a leader sequence from mouse hepatitis virus (MHV) genomic RNA could be used for MHV subgenomic defective interfering (DI) RNA transcription was examined. This was tested by using helper viruses and DI RNAs that were easily distinguishable. MHV JHM variant JHM(2), which synthesizes a subgenomic mRNA encoding the HE gene, and variant JHM(3-9), which does not synthesize this mRNA, were used. An MHV DI RNA, DI(J3-9), was constructed to contain a JHM(3-9)-derived leader sequence and an inserted intergenic region derived from the region preceding the MHV JHM HE gene. DI(J3-9) replicated efficiently in JHM(2)- or JHM(3-9)-infected cells, whereas synthesis of subgenomic DI RNAs was observed only in JHM(2)-infected cells. Sequence analyses demonstrated that the 5' regions of both helper virus genomic RNAs and genomic DI RNAs maintained their original sequences in DI RNA-replicating cells, indicating that the genomic leader sequences derived from JHM(2) functioned for subgenomic DI RNA transcription. Replication and transcription of DI(J3-9) were observed in cells infected with an MHV A59 strain whose leader sequence was similar to that of JHM(2), except for one nucleotide substitution within the leader sequence. The 5' region of the helper virus genomic RNA and that of the DI RNA were the same as their original structures in virus-infected cells, and the leader sequence of DI(J3-9) subgenomic DI RNA contained the MHV A59-derived leader sequence. The leader sequence of subgenomic DI RNA was derived from that of helper virus; therefore, the genomic leader sequence had a trans-acting property indicative of a discontinuous step in coronavirus transcription.

An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation

Selective encapsidation of hepatitis B virus (HBV) genomic RNA within cytoplasmic core particles requires recognition of the cis-encapsidation signal, (termed epsilon) located at the 5' end of genomic RNA. By transfecting plasmids expressing chimeric RNAs bearing HBV sequences fused to lacZ, we have mapped the minimal region of epsilon to the 5' 94 nucleotides (nt) of genomic RNA. Enzymatic probing of the RNA secondary structure in this region (by using either in vitro transcripts or RNA extracted from HBV core particles) reveals a stem-loop structure containing a lower stem, a 6-nt bulge, an upper stem with a single unpaired U residue, and a 6-nt loop. The functional role of this structure in encapsidation was explored by examining the effects of mutations in epsilon on encapsidation of RNA in vivo. These studies reveal that (i) in the lower stem, base pairing but not specific primary sequence is required for function; (ii) there is no requirement for base pairing in the lower portion of the upper stem, but base pairing elsewhere in this stem contributes to packaging efficiency; (iii) the presence of the 6-nt bulge, but not its primary sequence, is important for function; and (iv) specific nucleotide sequences in the loop and in regions of the upper stem are critical for RNA encapsidation.

Regulation of polyadenylation in hepatitis B viruses: stimulation by the upstream activating signal PS1 is orientation-dependent, distance-independent, and additive

Hepatitis B viruses replicate by reverse transcription of a genomic RNA which harbors terminal redundancies. The synthesis of this RNA requires that transcription proceed twice through the polyadenylation (pA) site which, in mammalian strains, is flanked by the variant hexanucleotide UAUAAA and a T-rich downstream domain. These core elements are by themselves virtually defective in 3' end processing and require multiple upstream accessory elements which regulate pA site use. In ground squirrel hepatitis B virus (GSHV), one of these signals (PS1; -215 to -107 relative to UAUAAA) is transcribed only at the 3' end of genomic RNA and as such is analogous to retroviral U3 sequences. PS1 cooperates with other signals to enhance pA site use to very high levels and can be further sub-divided into two regions (A and B) which contribute equally to 3' end processing. Critical residues within PS1B have been localized to a 15 bp A/T-rich stretch which displays homology to other known upstream activating signals. A 15 bp segment within PS1A which has the identical A/T content but a divergent primary sequence plays a diminished role in processing. Furthermore, PS1 can activate GSHV core element usage autonomously. This stimulation has been shown to be additive since multiple copies of PS1 progressively increase polyadenylation, a phenomenon which also demands that PS1 exert its influence from a variety of distances from the hexanucleotide signal.

Detection of avian infectious bronchitis viral infection using in situ hybridization and recombinant DNA

A recombinant DNA probe with specificity for the 3′ end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2, 4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation.

Promoter analysis of influenza virus RNA polymerase

Influenza virus polymerase, which was prepared depleted of viral RNA, was used to copy small RNA templates prepared from plasmid-encoded sequences. Template constructions containing only the 3' end of genomic RNA were shown to be efficiently copied, indicating that the promoter lay solely within the 15-nucleotide 3' terminus. Sequences not specific for the influenza virus termini were not copied, and, surprisingly, RNAs containing termini identical to those from plus-sense cRNA were copied at low levels. The specificity for recognition of the virus sense promoter was further defined by site-specific mutagenesis. It was also found that increased levels of viral protein were required in order to catalyze both the cap endonuclease-primed and primer-free RNA synthesis from these model templates, as well as from genomic-length RNAs. This finding indicates that the reconstituted system has catalytic properties very similar to those of native viral ribonucleoprotein complexes.

Evolution of the 5′-end of genomic rna of murine coronaviruses during passages in vitro

The 5′-ends of the genomic RNA and subgenomic mRNAs of murine coronavirus (MHV) have a stretch of approximately 70 nucleotides of leader sequences. The 3'-region of this leader sequence contains several repeats of a pentanucleotide (UCUAA), whose number varies among different MHV strains. It has been demonstrated that this UCUAA repeat plays crucial roles in the discontinuous transcription of MHV mRNAs. In the present study, we demonstrate that the number of UCUAA repeats in the leader sequence of MHV genome rapidly decreases during serial passages of viruses on susceptible cells. The downward evolution of the number of UCUAA repeats was not due to a higher growth rate of the viruses with fewer repeats, but seemed to be due to homologous interference between viruses with different numbers of UCUAA repeat. The ease with which these variant viruses arose suggests the high frequency of the occurrence of this deletion during RNA replication. This finding is in agreement with the proposed discontinuous and nonprocessive mode of coronavirus RNA synthesis. Analysis of the intracellular subgenomic mRNA species of viruses with different numbers of UCUAA repeats and of MHV recombinant viruses suggests that the number of this pentanucleotide repeat at the 3′-end of the leader sequence may regulate the synthesis of certain mRNA species, in agreement with the leader-primed transcription mechanism.

Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription.

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.