The myeloproliferative disorder-associated JAK2 V617F mutant escapes negative regulation by suppressor of cytokine signaling 3

The somatic JAK2 valine-to-phenylalanine (V617F) mutation has been detected in up to 90% of patients with polycythemia and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis. Suppressor of cytokine signaling 3 (SOCS3) is known to be a strong negative regulator of erythropoietin (EPO) signaling through interaction with both the EPO receptor (EPOR) and JAK2. We report here that JAK2 V617F cannot be regulated and that its activation is actually potentiated in the presence of SOCS3. Instead of acting as a suppressor, SOCS3 enhanced the proliferation of cells expressing both JAK2 V617F and EPOR. Additionally, although SOCS1 and SOCS2 are degraded in the presence of JAK2 V617F, turnover of SOCS3 is inhibited by the JAK2 mutant kinase and this correlated with marked tyrosine phosphorylation of SOCS3 protein. We also observed constitutive tyrosine phosphorylation of SOCS3 in peripheral blood mononuclear cells (PBMCs) derived from patients homozygous for the JAK2 V617F mutant. These findings suggest that the JAK2 V617F has overcome normal SOCS regulation by hyperphosphorylating SOCS3, rendering it unable to inhibit the mutant kinase. Thus, JAK2 V617F may even exploit SOCS3 to potentiate its myeloproliferative capacity.

Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units.

Transcription from morbillivirus genomes commences at a single promoter in the 3' non-coding terminus, with the six genes being transcribed sequentially. The 3' and 5' untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5' UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5' UTRs. Insertions into the 5' UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5' UTR, govern this decreased expression from TU2.

Molecular mechanisms of measles virus persistence.

As measles virus causes subacute sclerosing panencephalitis and measles inclusion body encephalitis due to its ability to establish human persistent infection, without symptoms for the time between the acute infection and the onset of clinical symptoms, it has been the paradigm for a long term persistent as opposed to chronic infection by an RNA virus. We have reviewed the mechanisms of persistence of the virus and discuss specific mutations associated with CNS infection affecting the matrix and fusion protein genes. These are placed in the context of our current understanding of the viral replication cycle. We also consider the proposed mechanisms of persistence of the virus in replicating cell cultures and conclude that no general mechanistic model can be derived from our current state of knowledge. Finally, we indicate how reverse genetics approaches and the use of mouse models with specific knock-out and knock-in modifications can further our understanding of measles virus persistence.

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Nidovirales: evolving the largest RNA virus genome.

This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32 kb) and will therefore be called ‘large’ nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13–16 kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3'-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.

ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Replication of the ~30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.