Biochemical characterization of the mouse ABCF3 protein, a partner of the flavivirus-resistance protein OAS1B.

Mammalian ATP-binding cassette (ABC) subfamily F member 3 (ABCF3) is a class 2 ABC protein that has previously been identified as a partner of the mouse flavivirus resistance protein 2',5'-oligoadenylate synthetase 1B (OAS1B). The functions and natural substrates of ABCF3 are not known. In this study, analysis of purified ABCF3 showed that it is an active ATPase, and binding analyses with a fluorescent ATP analog suggested unequal contributions by the two nucleotide-binding domains. We further showed that ABCF3 activity is increased by lipids, including sphingosine, sphingomyelin, platelet-activating factor, and lysophosphatidylcholine. However, cholesterol inhibited ABCF3 activity, whereas alkyl ether lipids either inhibited or resulted in a biphasic response, suggesting small changes in lipid structure differentially affect ABCF3 activity. Point mutations in the two nucleotide-binding domains of ABCF3 affected sphingosine-stimulated ATPase activity differently, further supporting different roles for the two catalytic pockets. We propose a model in which pocket 1 is the site of basal catalysis, whereas pocket 2 engages in ligand-stimulated ATP hydrolysis. Co-localization of the ABCF3-OAS1B complex to the virus-remodeled endoplasmic reticulum membrane has been shown before. We also noted that co-expression of ABCF3 and OAS1B in bacteria alleviated growth inhibition caused by expression of OAS1B alone, and ABCF3 significantly enhanced OAS1B levels, indirectly showing interaction between these two proteins in bacterial cells. As viral RNA synthesis requires large amounts of ATP, we conclude that lipid-stimulated ATP hydrolysis may contribute to the reduction in viral RNA production characteristic of the flavivirus resistance phenotype.

Insertion position as well as the inserted TRS and gene sequences differentially affect the retention of foreign gene expression by simian hemorrhagic fever virus (SHFV).

Recombinant SHFV infectious cDNA clones expressing a foreign gene from an additional sg mRNA were constructed. Two 3' genomic region sites, between ORF4' and ORF2b and between ORF4 and ORF5, were utilized for insertion of the myxoma M013 gene with a C-terminal V5 tag followed by one of the three inserted transcription regulatory sequences (TRS), TRS2', TRS4' or TRS7. M013 insertion at the ORF4'/ORF2b site but not the ORF4/ORF5 site generated progeny virus but only the recombinant virus with an inserted TRS2' retained the entire M013 gene through passage four. Insertion of an auto-fluorescent protein gene, iLOV, with an inserted TRS2' at the ORF4'/ORF2b site, generated viable progeny virus. iLOV expression was maintained through passage eight. Although regulation of SHFV subgenomic RNA synthesis is complex, the ORF4'/ORF2b site, which is located between the two sets of minor structural proteins, is able to tolerate foreign gene insertion.

New insights about the regulation of Nidovirus subgenomic mRNA synthesis.

The members of the Order Nidovirales share a similar genome organization with two overlapping nonstructural polyproteins encoded in the 5' two-thirds and the structural proteins encoded in the 3' third. They also express their 3' region proteins from a nested set of 3' co-terminal subgenomic messenger RNAs (sg mRNAs). Some but not all of the Nidovirus sg mRNAs also have a common 5' leader sequence that is acquired by a discontinuous RNA synthesis mechanism regulated by multiple 3' body transcription regulating sequences (TRSs) and the 5' leader TRS. Initial studies detected a single major body TRS for each 3' sg mRNA with a few alternative functional TRSs reported. The recent application of advanced techniques, such as next generation sequencing and ribosomal profiling, in studies of arteriviruses and coronaviruses has revealed an expanded sg mRNA transcriptome and coding capacity.

Antiviral activity of K22 against members of the order Nidovirales.

Recently, a novel antiviral compound (K22) that inhibits replication of a broad range of animal and human coronaviruses was reported to interfere with viral RNA synthesis by impairing double-membrane vesicle (DMV) formation (Lundin et al., 2014). Here we assessed potential antiviral activities of K22 against a range of viruses representing two (sub)families of the order Nidovirales, the Arteriviridae (porcine reproductive and respiratory syndrome virus [PRRSV], equine arteritis virus [EAV] and simian hemorrhagic fever virus [SHFV]), and the Torovirinae (equine torovirus [EToV] and White Bream virus [WBV]). Possible effects of K22 on nidovirus replication were studied in suitable cell lines. K22 concentrations significantly decreasing infectious titres of the viruses included in this study ranged from 25 to 50 µM. Reduction of double-stranded RNA intermediates of viral replication in nidovirus-infected cells treated with K22 confirmed the anti-viral potential of K22. Collectively, the data show that K22 has antiviral activity against diverse lineages of nidoviruses, suggesting that the inhibitor targets a critical and conserved step during nidovirus replication.

Expanded subgenomic mRNA transcriptome and coding capacity of a nidovirus.

Members of the order Nidovirales express their structural protein ORFs from a nested set of 3' subgenomic mRNAs (sg mRNAs), and for most of these ORFs, a single genomic transcription regulatory sequence (TRS) was identified. Nine TRSs were previously reported for the arterivirus Simian hemorrhagic fever virus (SHFV). In the present study, which was facilitated by next-generation sequencing, 96 SHFV body TRSs were identified that were functional in both infected MA104 cells and macaque macrophages. The abundance of sg mRNAs produced from individual TRSs was consistent over time in the two different cell types. Most of the TRSs are located in the genomic 3' region, but some are in the 5' ORF1a/1b region and provide alternative sources of nonstructural proteins. Multiple functional TRSs were identified for the majority of the SHFV 3' ORFs, and four previously identified TRSs were found not to be the predominant ones used. A third of the TRSs generated sg mRNAs with variant leader-body junction sequences. Sg mRNAs encoding E', GP2, or ORF5a as their 5' ORF as well as sg mRNAs encoding six previously unreported alternative frame ORFs or 14 previously unreported C-terminal ORFs of known proteins were also identified. Mutation of the start codon of two C-terminal ORFs in an infectious clone reduced virus yield. Mass spectrometry detected one previously unreported protein and suggested translation of some of the C-terminal ORFs. The results reveal the complexity of the transcriptional regulatory mechanism and expanded coding capacity for SHFV, which may also be characteristic of other nidoviruses.

Arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in West Nile virus-infected cells.

Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits for the virus during its lifecycle.

Simian hemorrhagic fever virus: Recent advances.

The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arteriviridae in encoding three papain-like one proteases (PLP1α, PLP1ß and PLP1γ) at the 5' end and two adjacent sets of four minor structural proteins at the 3' end. The catalytic Cys and His residues and cleavage sites for each of the SHFV PLP1s were predicted and their functionality was tested in in vitro transcription/translation reactions done with wildtype or mutant polyprotein constructs. Mass spectrometry analyses of selected autoproteolytic products confirmed cleavage site locations. The catalytic Cys of PLP1α is unusual in being adjacent to an Ala instead of a Typ. PLP1γ cleaves at both downstream and upstream sites. Intermediate precursor and alternative cleavage products were detected in the in vitro transcription/translation reactions but only the three mature nsp1 proteins were detected in SHFV-infected MA104 cell lysates with SHFV nsp1 protein-specific antibodies. The duplicated sets of SHFV minor structural proteins were predicted to be functionally redundant. A stable, full-length, infectious SHFV-LVR cDNA clone was constructed and a set of mutant infectious clones was generated each with the start codon of one of the minor structural proteins mutated. All eight of the minor structural proteins were found to be required for production of infectious extracellular virus. SHFV causes a fatal hemorrhagic fever in macaques but asymptomatic, persistent infections in natural hosts such as baboons. SHFV infections were compared in macrophages and myeloid dendritic cells from baboons and macaques. Virus yields were higher from macaque cells than from baboon cells. Macrophage cultures from the two types of animals differed dramatically in the percentage of cells infected. In contrast, similar percentages of myeloid dendritic cells were infected but virus replication was efficient in the macaque cells but inefficient in the baboon cells. SHFV infection induced the production of pro-inflammatory cytokines, including IL-1ß, IL-6, IL-12/23(p40), TNF-α and MIP-1α, in macaque cells but not baboon cells.

Each of the eight simian hemorrhagic fever virus minor structural proteins is functionally important.

The simian hemorrhagic fever virus (SHFV) genome differs from those of other members of the family Arterivirus in encoding two adjacent sets of four minor structural protein open reading frames (ORFs). A stable, full-length, infectious SHFV-LVR cDNA clone was constructed. Virus produced from this clone had replication characteristics similar to those of the parental virus. A subgenomic mRNA was identified for the SHFV ORF previously identified as 2b. As an initial means of analyzing the functional relevance of each of the SHFV minor structural proteins, a set of mutant infectious clones was generated, each with the start codon of one minor structural protein ORF mutated. Different phenotypes were observed for each ortholog of the pairs of minor glycoproteins and all of the eight minor structural proteins were required for the production of infectious extracellular virus indicating that the duplicated sets of SHFV minor structural proteins are not functionally redundant.

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

UNLABELLED: The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1ß, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1ß were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. IMPORTANCE: SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1α catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1γ can cleave at both the downstream nsp1γ-nsp2 and upstream nsp1ß-nsp1γ junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells.

Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity.

RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.

Differential responses of disease-resistant and disease-susceptible primate macrophages and myeloid dendritic cells to simian hemorrhagic fever virus infection.

Simian hemorrhagic fever virus (SHFV) causes a fatal hemorrhagic fever in macaques but an asymptomatic, persistent infection in baboons. To investigate factors contributing to this differential infection outcome, the targets of SHFV infection, macrophages (MΦs) and myeloid dendritic cells (mDCs), were differentiated from macaque and baboon peripheral blood monocytes and used to compare viral replication and cell responses. SHFV replicated in >90% of macaque MΦs but in only ∼10% of baboon MΦs. Although SHFV infected ∼50% of macaque and baboon mDCs, virus replication was efficient in macaque but not in baboon mDCs. Both types of macaque cultures produced higher virus yields than baboon cultures. A more efficient type I interferon response and the production of proinflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, IL-12/23(p40), tumor necrosis factor alpha (TNF-α), and macrophage inflammatory protein 1α (MIP-1α), in response to SHFV infection were observed in macaque but not baboon cultures, suggesting less efficient counteraction of these responses by viral proteins in macaque cells. Baboon cultures produced higher levels of IL-10 than macaque cultures both prior to and after SHFV infection. In baboon but not macaque cell cultures, SHFV infection upregulated IL-10R1, a subunit of the IL-10 receptor (IL-10R), and also SOCS3, a negative regulator of proinflammatory cytokine production. Incubation of macaque cultures with human IL-10 before and/or after SHFV infection decreased production of IL-6, IL-1ß, and MIP-1α but not TNF-α, suggesting a role for IL-10 in suppressing SHFV-induced proinflammatory cytokine production in macaques.

Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

RNase L plays a role in the antiviral response to West Nile virus.

Alleles at the Flv locus determine disease outcome after a flavivirus infection in mice. Although comparable numbers of congenic resistant and susceptible mouse embryo fibroblasts (MEFs) are infected by the flavivirus West Nile virus (WNV), resistant MEFs produce approximately 100- to 150-fold lower titers than susceptible ones and flavivirus titers in the brains of resistant and susceptible animals can differ by >10,000-fold. The Flv locus was previously identified as the 2'-5' oligoadenylate synthetase 1b (Oas1b) gene. Oas gene expression is up-regulated by interferon (IFN), and after activation by double-stranded RNA, some mouse synthetases produce 2-5A, which activates latent RNase L to degrade viral and cellular RNAs. To determine whether the lower levels of intracellular flavivirus genomic RNA from resistant mice detected in cells at all times after infection were mediated by RNase L, RNase L activity levels in congenic resistant and susceptible cells were compared. Similar moderate levels of RNase L activation by transfected 2-5A were observed in both types of uninfected cells. After WNV infection, the mRNAs of IFN-beta and three Oas genes were up-regulated to similar levels in both types of cells. However, significant levels of RNase L activity were not detected until 72 h after WNV infection and the patterns of viral RNA cleavage products generated were similar in both types of cells. When RNase L activity was down-regulated in resistant cells via stable expression of a dominant negative RNase L mutant, approximately 5- to 10-times-higher yields of WNV were produced. Similarly, about approximately 5- to 10-times-higher virus yields were produced by susceptible C57BL/6 RNase L-/- cells compared to RNase L+/+ cells that were either left untreated or pretreated with IFN and/or poly(I) . poly(C). The data indicate that WNV genomic RNA is susceptible to RNase L cleavage and that RNase L plays a role in the cellular antiviral response to flaviviruses. The results suggest that RNase L activation is not a major component of the Oas1b-mediated flavivirus resistance phenotype.

Two cellular proteins that interact with a stem loop in the simian hemorrhagic fever virus 3'(+)NCR RNA.

Both full-length and subgenomic negative-strand RNAs are initiated at the 3' terminus of the positive-strand genomic RNA of the arterivirus, simian hemorrhagic fever virus (SHFV). The SHFV 3'(+) non-coding region (NCR) is 76 nts in length and forms a stem loop (SL) structure that was confirmed by ribonuclease structure probing. Two cell proteins, p56 and p42, bound specifically to a probe consisting of the SHFV 3'(+)NCR RNA. The 3'(+)NCR RNAs of two additional members of the arterivirus genus specifically interacted with two cell proteins of the same size. p56 was identified as polypyrimidine tract-binding protein (PTB) and p42 was identified as fructose bisphosphate aldolase A. PTB binding sites were mapped to a terminal loop and to a bulged region of the SHFV 3'SL structure. Deletion of either of the PTB binding sites in the viral RNA significantly reduced PTB binding activity, suggesting that both sites are required for efficient binding of this protein. Changes in the top portion of the SHFV 3'SL structure eliminated aldolase binding, suggesting that the binding site for this protein is located near the top of the SL. These cell proteins may play roles in regulating the functions of the genomic 3' NCR.

The molecular biology of West Nile Virus: a new invader of the western hemisphere.

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. In recent years, the frequency of WNV outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. In 1999, the geographical distribution of WNV expanded to the Western hemisphere. WNV has a positive strand RNA genome of about 11 kb that encodes a single polyprotein. WNV replicates in the cytoplasm of infected cells. Although there are still many questions to be answered, a large body of data on the molecular biology of WNV and other flaviviruses has already been obtained. Aspects of virion structure, the viral replication cycle, viral protein function, genome structure, conserved viral elements, host factors, virus-host interactions, and vaccines are discussed in this review.