The bipartite promoter of Orthonairovirus hazaraense large segment.

IMPORTANCE: The realization that segmented negative-strand RNA virus genome ribonucleoproteins are never free as their RNA ends are always bound to the viral polymerase has highlighted the problem of how these genome segments are replicated and express their mRNAs while their RNA ends remain associated with the polymerase throughout the cycles of RNA synthesis. This study of the length and nucleotide composition of the Orthonairovirus hazaraense L segment-specific double-stranded RNA (dsRNA) promoter element (the promoter duplex) provides insight into how its mRNA might be initiated and suggests that this promoter element acts via its separated single strands as well as via dsRNA.

A Point Mutation in the Human Parainfluenza Virus Type 2 Nucleoprotein Leads to Two Separate Effects on Virus Replication.

Paramyxovirus genomes, like that of human parainfluenza virus type 2 (hPIV2), have lengths of precisely multiples-of-six nucleotides ("rule of six"), where each nucleoprotein subunit (NP) binds exactly six nucleotides. Ten residues of its RNA binding groove contact the genome RNA; but only one, Q202, directly contacts a nucleotide base. The mutation of NPQ202 leads to two phenotypes: the ability of the viral polymerase to replicate minigenomes with defective bipartite promoters where NPwt is inactive, and the inability to rescue rPIV2 carrying this point mutation by standard means. The absence of an rPIV2 NPQ202A prevented further study of the latter phenotype. By extensive and repeated cocultivation of transfected cells, an rPIV2 carrying this mutation was finally recovered, and this virus was apparently viable due to the presence of an additional NP mutation (I35L). Our results suggest that these two phenotypes are due to separate effects of the Q202 mutation, and that the problematic rescue phenotype may be due to the inability of the transfected cell to incorporate viral nucleocapsids during virus budding. IMPORTANCE Paramyxovirus genomes are contained within a noncovalent homopolymer of its nucleoprotein (NP) and form helical nucleocapsids (NC) whose 3' ends contain the promoters for the initiation of viral RNA synthesis. This work suggests that these NC 3' ends may play another role in the virus life cycle via their specific interaction with virus-modified cell membranes needed for the incorporation of viral NCs into budding virions.

γHV68 vGAT: a viral pseudoenzyme pimping for PAMPs.

In this issue, He et al. (2015) show how herpes virus usurps a cellular metabolic enzyme to induce RIG-I deamidation and RNA-independent activation, likely to better prevent further innate immune responses.

A paramyxovirus-like model for Ebola virus bipartite promoters.

Paramyxo- and filovirus nucleocapsids (NCs) have bipartite promoters at their 3' ends to initiate RNA synthesis. The 2 elements, promoter element 1 (PE1) and promoter element 2 (PE2), are separated by a spacer region that must be exactly a multiple of 6 nucleotides (nt) long. Paramyxovirus NCs have 13 nucleoprotein (NP) subunits/turn, such that PE1 and PE2 are juxtaposed on the same face of the NC helix, for concerted recognition by the viral polymerase. Ebola virus (EBOV) NCs, in contrast, have 25 to 28 subunits/turn, meaning that PE1 and PE2 cannot be juxtaposed. However, there is evidence that the number of subunits/turn at the 3' end of the EBOV NC is variable. We propose a paramyxovirus-like model for EBOV explaining why there are 8 contiguous copies of the PE2 repeat when 3 are sufficient, why expanding this run to 13 further improves minigenome performance, and why there is a limit to the number of hexa-nt that can be inserted in the spacer region.

Bipartite promoters and RNA editing of paramyxoviruses and filoviruses.

A primary property of paramyxovirus bipartite promoters is to ensure that their RNA genomes are imprinted with a hexamer phase via their association with nucleoproteins, in part because this phase as well the editing sequence itself controls mRNA editing. The question then arises whether a similar mechanism operates for filoviruses that also contain bipartite promoters that are governed by the "rule of six," even though these genomes need not, and given Ebola virus biology, cannot always be of hexamer genome length. This review suggests that this is possible and describes how it might operate, and that RNA editing may play a role in Ebola virus genome interconversion that helps the virus adapt to different host environments.

The control of paramyxovirus genome hexamer length and mRNA editing.

The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NPQ202A) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NPwt is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln202 of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the cis-acting mRNA editing sequence is maintained.

A Minigenome Study of Hazara Nairovirus Genomic Promoters.

Hazara nairovirus (HAZV) is a trisegmented RNA virus most closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) in the order Bunyavirales The terminal roughly 20 nucleotides (nt) of its genome ends are highly complementary, similar to those of other segmented negative-strand RNA viruses (sNSV), and act as promoters for RNA synthesis. These promoters contain two elements: the extreme termini of both strands (promoter element 1 [PE1]) are conserved and virus specific and are found bound to separate sites on the polymerase surface in crystal structures of promoter-polymerase complexes. The following sequences (PE2) are segment specific, with the potential to form double-stranded RNA (dsRNA), and the latter aspect is also important for promoter activity. Nairovirus genome promoters differ from those of peribunyaviruses and arenaviruses in that they contain a short single-stranded region between the two regions of complementarity. Using a HAZV minigenome system, we found the single-stranded nature of this region, as well as the potential of the following sequence to form dsRNA, is essential for reporter gene expression. Most unexpectedly, the sequence of the PE2 dsRNA appears to be equally important for promoter activity. These differences in sNSV PE2 promoter elements are discussed in light of our current understanding of the initiation of RNA synthesis.IMPORTANCE A minigenome system for HAZV, closely related to CCHFV, was used to study its genome replication. HAZV genome ends, like those of other sNSV, such as peribunyaviruses and arenaviruses, are highly complementary and serve as promoters for genome synthesis. These promoters are composed of two elements: the extreme termini of both 3' and 5' strands that are initially bound to separate sites on the polymerase surface in a sequence-specific fashion and the following sequences with the potential to anneal but whose sequence is not important. Nairovirus promoters differ from the other sNSV cited in that they contain a short single-stranded RNA (ssRNA) region between the two elements. The single-stranded nature of this region is an essential element of the promoter, whereas its sequence is unimportant. The sequence of the following complementary region is unexpectedly also important, a possible rare example of sequence-specific dsRNA recognition.

Complete genome sequence of Tacaribe virus.

Tacaribe virus (TCRV) is the prototype of the New World arenaviruses (also known as TCRV serocomplex viruses). While TCRV is not itself a human pathogen, many closely related members of this group cause hemorrhagic fever, and thus TCRV has long served as an important BSL2 system for research into diverse areas of arenavirus biology. Due to its widespread use, a coding-complete sequence for both the S and L segments of the bipartite genome has been publically available for almost 30 years. However, more recently, this sequence has been found to contain significant discrepancies compared to other samples of the same original strain (i.e., TRVL-11573). Further, it is incomplete with respect to the genome ends, which contain critical regulatory elements for RNA synthesis. In order to rectify these issues we now present the first complete genome sequence for this important prototype arenavirus. In addition to completing the S segment 5' end, we identified an apparent error in the L segment 3' end as well as substantial discrepancies in the S segment intergenic region likely to affect folding. Comparison of this sequence with existing partial sequences confirmed a 12-amino-acid deletion in GP, including putative glycosylation sites, and a 4-amino-acid exchange flanking the exonuclease domain of NP. Accounting for these corrections, the TRVL-11573 strain appears to be nearly identical to that isolated in Florida in 2012. The availability of this information provides a solid basis for future molecular and genetic work on this important prototype arenavirus.

A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity.

The genome RNA of human parainfluenza virus type 2 (hPIV2) that acts as the template for the polymerase complex is entirely encapsidated by the nucleoprotein (NP). Recently, the crystal structure of NP of PIV5, a virus closely related to hPIV2, was resolved in association with RNA. Ten amino acids that contact the bound RNA were identified and are strictly conserved between PIV5 and hPIV2 NP. Mutation of hPIV2 NP Q202 (which contacts a base rather than the RNA backbone) to various amino acids resulted in an over 30-fold increase of polymerase activity as evidenced by a minireplicon assay, even though the RNA-binding affinity was unaltered. Using various modified minireplicons, we found that the enhanced reporter gene expression could be accounted for by increased minigenome replication, whereas mRNA synthesis itself was not affected by Q202 mutation. Moreover, the enhanced activities were still observed in minigenomes partially lacking the leader sequence and which were not of hexamer genome length. Unexpectedly, recombinant hPIV2 possessing the NP Q202A mutation could not be recovered from cDNA.IMPORTANCE We examined the importance of amino acids in the putative RNA-binding domain of hPIV2 NP for polymerase activity using minireplicons. Abnormally enhanced genome replication was observed upon substitution mutation of the NP Q202 position to various amino acids. Surprisingly, this mutation enabled polymerase to use minigenomes that were partially lacking the leader sequence and not of hexamer genome length. This mutation does not affect fundamental properties of NP, e.g., recognition of gene junctional and editing signals. However, the strongly enhanced polymerase activity may not be viable for the infectious life cycle. This report highlights the potential of the polymerase complex with point mutations in NP and helps our detailed understanding of the molecular basis of gene expression.

Loss of Sendai virus C protein leads to accumulation of RIG-I immunostimulatory defective interfering RNA.

Retinoic acid inducible gene (RIG-I)-mediated innate immunity plays a pivotal role in defence against virus infections. Previously we have shown that Sendai virus (SeV) defective interfering (DI) RNA functions as an exclusive and potent RIG-I ligand in DI-RNA-rich SeV-Cantell infected cells. To further understand how RIG-I is activated during SeV infection, we used a different interferon (IFN)-inducing SeV strain, recombinant SeVΔC, which, in contrast to SeV-Cantell is believed to stimulate IFN production due to the lack of the SeV IFN antagonist protein C. Surprisingly, we found that in SevΔC-infected cells, DI RNAs also functioned as an exclusive RIG-I ligand. Infections with wild-type SeV failed to generate any RIG-I-associated immunostimulatory RNA and this correlated with the lack of DI genomes in infected cells, as well as with the absence of cellular innate immune responses. Supplementation of the C protein in the context of SeVΔC infection led to a reduction in the number of DI RNAs, further supporting the potential role of the C protein as a negative regulator of DI generation and/or accumulation. Our findings indicate that limiting DI genome production is an important function of viral IFN antagonist proteins.

A structure-based model of RIG-I activation.

A series of high-resolution crystal structures of RIG-I and RIG-I:dsRNA cocrystals has recently been reported. Comparison of these structures provides considerable insight into how this innate immune pattern recognition receptor is activated upon detecting and binding a certain class of viral RNAs.

Short double-stranded RNAs with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as RIG-I decoys.

Arenavirus RNA genomes are initiated by a "prime and realign" mechanism, such that the initiating GTP is found as a single unpaired (overhanging) nucleotide when the complementary genome ends anneal to form double-stranded (ds) RNA panhandle structures. dsRNAs modeled on these structures do not induce interferon (IFN), as opposed to blunt-ended (5' ppp)dsRNA. This study examines whether these viral structures can also act as decoys, by trapping RIG-I in inactive dsRNA complexes. We examined the ability of various dsRNAs to activate the RIG-I ATPase (presumably a measure of helicase translocation on dsRNA) relative to their ability to induce IFN. We found that there is no simple relationship between these two properties, as if RIG-I can translocate on short dsRNAs without inducing IFN. Moreover, we found that (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide can in fact competitively inhibit the ability of blunt-ended (5' ppp)dsRNAs to induce IFN when co-transfected into cells and that this inhibition is strongly dependent on the presence of the 5' ppp. In contrast, (5' ppp)dsRNAs with a single unpaired 5' ppp-nucleotide does not inhibit poly(I-C)-induced IFN activation, which is independent of the presence of a 5' ppp group.

Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus.

Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.

Human parainfluenza virus type 2 L protein regions required for interaction with other viral proteins and mRNA capping.

The large RNA polymerase (L) protein of human parainfluenza virus type 2 (hPIV2) binds the nucleocapsid, phosphoprotein, and V protein, as well as itself, and these interactions are essential for transcription and replication of the viral RNA genome. Although all of these interactions were found to be mediated through the domains within the N terminus of L, the C terminus of the L protein was also required for minigenome reporter gene expression. We have identified a highly conserved rubulavirus domain near the C terminus of the L protein that is required for mRNA synthesis but not for genome replication. Remarkably, this region of L shares homology with a conserved region of cellular capping enzymes that binds GTP and forms a lysyl-GMP enzyme intermediate, the first step in the cellular capping reaction. We propose that this conserved region of L also binds GTP (or GDP) to carry out the second step of the unconventional nonsegmented negative-strand virus capping reaction.

Unpaired 5' ppp-nucleotides, as found in arenavirus double-stranded RNA panhandles, are not recognized by RIG-I.

Arenavirus and bunyavirus RNA genomes are unusual in that they are found in circular nucleocapsids, presumably due to the annealing of their complementary terminal sequences. Moreover, arenavirus genome synthesis initiates with GTP at position +2 of the template rather than at the precise 3' end (position +1). After formation of a dinucleotide, 5' pppGpC(OH) is then realigned on the template before this primer is extended. The net result of this "prime and realign" mechanism of genome initiation is that 5' pppG is found as an unpaired 5' nucleotide when the complementary genome ends anneal to form a double-stranded (dsRNA) panhandle. Using 5' pppRNA made in vitro and purified so that all dsRNA side products are absent, we have determined that both this 5' nucleotide overhang, as well as mismatches within the dsRNA (as found in some arenavirus genomes), clearly reduce the ability of these model dsRNAs to induce interferon upon transfection into cells. The presence of this unpaired 5' ppp-nucleotide is thus another way that some viruses appear to use to avoid detection by cytoplasmic pattern recognition receptors.

Sendai Virus and a Unified Model of Mononegavirus RNA Synthesis.

Vesicular stomatitis virus (VSV), the founding member of the mononegavirus order (Mononegavirales), was found to be a negative strand RNA virus in the 1960s, and since then the number of such viruses has continually increased with no end in sight. Sendai virus (SeV) was noted soon afterwards due to an outbreak of newborn pneumonitis in Japan whose putative agent was passed in mice, and nowadays this mouse virus is mainly the bane of animal houses and immunologists. However, SeV was important in the study of this class of viruses because, like flu, it grows to high titers in embryonated chicken eggs, facilitating the biochemical characterization of its infection and that of its nucleocapsid, which is very close to that of measles virus (MeV). This review and opinion piece follow SeV as more is known about how various mononegaviruses express their genetic information and carry out their RNA synthesis, and proposes a unified model based on what all MNV have in common.

The double-stranded RNA binding domain of the vaccinia virus E3L protein inhibits both RNA- and DNA-induced activation of interferon beta.

Vaccinia virus, a large DNA virus that replicates in the cytoplasm, expresses its E3L protein to inhibit the cellular innate immune response and apoptosis. E3L is a bifunctional protein that contains an N-terminal DNA binding domain (BD) and a C-terminal double-stranded RNA (dsRNA)-BD (residues 100-190), both of which contribute to viral pathogenesis by blocking the activation of cellular genes that respond to the viral infection. We report that expression of the dsRNA-BD alone inhibits not only the dsRNA-induced activation of interferon beta (IFNbeta) but also that of 5'-triphosphate single-stranded RNA and DNA-induced IFNbeta activation even though E3L(100-190) does not bind the latter two pathogen-associated molecular patterns. This inhibition occurs in both human HeLa and A549 cells, where RIG-I appears to be required for dsDNA-induced IFNbeta activation. Unexpectedly, the two residues most important for dsRNA binding are also critical for this domain's ability to inhibit all three nucleic acid-induced cellular responses.

Human parainfluenza virus type 2 V protein inhibits genome replication by binding to the L protein: possible role in promoting viral fitness.

The human parainfluenza virus type 2 (hPIV2) V protein plays important roles in inhibiting the host interferon response and promoting virus growth, but its role in hPIV2 replication and transcription is not clear. A green fluorescent protein (GFP)-expressing a negative-sense minigenomic construct of hPIV2 has been established by standard technology, with helper plasmids expressing the nucleocapsid protein (NP), phosphoprotein (P), and large RNA polymerase (L) protein, to examine the role of V protein. We found that the simultaneous expression of wild-type V protein in the minigenome system inhibited GFP expression, at least in part, by inhibiting minigenome replication. In contrast, expression of C terminally truncated or mutant hPIV2 V proteins had no effect. Moreover, the V protein of simian virus 41, the rubulavirus most closely related virus to hPIV2, also inhibited GFP expression, whereas that of PIV5, a more distantly related rubulavirus, did not. Using these other rubulavirus V proteins, as well as various mutant hPIV2 V proteins, we found that the ability of V protein to inhibit GFP expression correlated with its ability to bind to L protein via its C-terminal V protein-specific region, but there was no correlation with NP binding. A possible role for this inhibition of genome replication in promoting viral fitness is discussed.