Structural basis for therapeutic inhibition of influenza A polymerase PB2 subunit.

Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11-13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.

Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.

Base of the measles virus fusion trimer head receives the signal that triggers membrane fusion.

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.

Adherens junction protein nectin-4 is the epithelial receptor for measles virus.

Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.

MicroRNA-sensitive oncolytic measles viruses for cancer-specific vector tropism.

Oncolytic measles viruses (MV) derived from the live attenuated vaccine strain have been engineered for increased tumor-cell specificity, and are currently under investigation in clinical trials including a phase I study for glioblastoma multiforme (GBM). Recent preclinical studies have shown that the cellular tropism of several viruses can be controlled by inserting microRNA-target sequences into their genomes, thereby inhibiting spread in tissues expressing cognate microRNAs. Since neuron-specific microRNA-7 is downregulated in gliomas but highly expressed in normal brain tissue, we engineered a microRNA-sensitive virus containing target sites for microRNA-7 in the 3'-untranslated region of the viral fusion gene. In presence of microRNA-7 this modification inhibits translation of envelope proteins, restricts viral spread, and progeny production. Even though highly attenuated in presence of microRNA-7, this virus retained full efficacy against glioblastoma xenografts. Furthermore, microRNA-mediated inhibition protected genetically modified mice susceptible to MV infection from a potentially lethal intracerebral challenge. Importantly, endogenous microRNA-7 expression in primary human brain resections tightly restricted replication and spread of microRNA-sensitive virus. This is proof-of-concept that tropism restriction by tissue-specific microRNAs can be adapted to oncolytic MV to regulate viral replication and gene expression to maximize tumor specificity without compromising oncolytic efficacy.

The heads of the measles virus attachment protein move to transmit the fusion-triggering signal.

The measles virus entry system, consisting of attachment (hemagglutinin, H) and fusion proteins, operates by means of a variety of natural and targeted receptors; however, the mechanism that triggers fusion of the viral envelope with the plasma membrane is not understood. Here, we tested a model proposing that the two heads of an H dimer, which are covalently linked at their base, after binding two receptor molecules, move relative to each other to transmit the fusion-triggering signal. Indeed, stabilizing the H-dimer interface with additional intermolecular disulfide bonds prevented membrane fusion, an effect that was reversed by a reducing agent. Moreover, a membrane-anchored designated receptor efficiently triggered fusion, provided that it engaged the H dimer at locations proximal to where the natural receptors bind and distal to the H-dimer interface. We discuss how receptors may force H-protein heads to switch partners and transmit the fusion-triggering signal.

Measles virus infection of alveolar macrophages and dendritic cells precedes spread to lymphatic organs in transgenic mice expressing human signaling lymphocytic activation molecule (SLAM, CD150).

Recent studies of primate models suggest that wild-type measles virus (MV) infects immune cells located in the airways before spreading systemically, but the identity of these cells is unknown. To identify cells supporting primary MV infection, we took advantage of mice expressing the MV receptor human signaling lymphocyte activation molecule (SLAM, CD150) with human-like tissue specificity. We infected these mice intranasally (IN) with a wild-type MV expressing green fluorescent protein. One, two, or three days after inoculation, nasal-associated lymphoid tissue (NALT), the lungs, several lymph nodes (LNs), the spleen, and the thymus were collected and analyzed by microscopy and flow cytometry, and virus isolation was attempted. One day after inoculation, MV replication was documented only in the airways, in about 2.5% of alveolar macrophages (AM) and 0.5% of dendritic cells (DC). These cells expressed human SLAM, and it was observed that MV infection temporarily enhanced SLAM expression. Later, MV infected other immune cell types, including B and T lymphocytes. Virus was isolated from lymphatic tissue as early as 2 days post-IN inoculation; the mediastinal lymph node was an early site of replication and supported high levels of infection. Three days after intraperitoneal inoculation, 1 to 8% of the mediastinal LN cells were infected. Thus, MV infection of alveolar macrophages and subepithelial dendritic cells in the airways precedes infection of lymphocytes in lymphatic organs of mice expressing human SLAM with human-like tissue specificity.

Measles virus selectively blind to signaling lymphocytic activation molecule (SLAM; CD150) is attenuated and induces strong adaptive immune responses in rhesus monkeys.

The signaling lymphocytic activation molecule (SLAM; CD150) is the immune cell receptor for measles virus (MV). To assess the importance of the SLAM-MV interactions for virus spread and pathogenesis, we generated a wild-type IC-B MV selectively unable to recognize human SLAM (SLAM-blind). This virus differs from the fully virulent wild-type IC-B strain by a single arginine-to-alanine substitution at amino acid 533 of the attachment protein hemagglutinin and infects cells through SLAM about 40 times less efficiently than the isogenic wild-type strain. Ex vivo, this virus infects primary lymphocytes at low levels regardless of SLAM expression. When a group of six rhesus monkeys (Macaca mulatta) was inoculated intranasally with the SLAM-blind virus, no clinical symptoms were documented. Only one monkey had low-level viremia early after infection, whereas all the hosts in the control group had high viremia levels. Despite minimal, if any, viremia, all six hosts generated neutralizing antibody titers close to those of the control monkeys while MV-directed cellular immunity reached levels at least as high as in wild-type-infected monkeys. These findings prove formally that efficient SLAM recognition is necessary for MV virulence and pathogenesis. They also suggest that the selectively SLAM-blind wild-type MV can be developed into a vaccine vector.

The measles virus fusion protein transmembrane region modulates availability of an active glycoprotein complex and fusion efficiency.

The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F(0)) than activated (F(1+2)) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-F(L507A), the F(0)/F(1+2) ratio shifted toward F(1+2). Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex.

Measles virus blind to its epithelial cell receptor remains virulent in rhesus monkeys but cannot cross the airway epithelium and is not shed.

The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.

Interaction of Bunyamwera Orthobunyavirus NSs protein with mediator protein MED8: a mechanism for inhibiting the interferon response.

The NSs protein of Bunyamwera virus (Bunyaviridae) is an antiapoptotic interferon antagonist involved in silencing host protein expression by interfering with mRNA synthesis. Here, we show that the ability to inhibit both host transcription and the interferon response is linked to interaction of NSs with the MED8 component of Mediator, a protein complex necessary for mRNA production. The interacting domain on NSs was mapped to the C-terminal region, which contains amino acids conserved among orthobunyavirus NSs proteins. A recombinant virus in which the interacting domain in NSs was deleted had strongly reduced ability to inhibit host protein expression and was unable to inhibit the interferon response. This study provides further information on the mechanisms by which bunyavirus nonstructural proteins are involved in pathogenesis.

Requirement of the N-terminal region of orthobunyavirus nonstructural protein NSm for virus assembly and morphogenesis.

The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

Genetic elements regulating packaging of the Bunyamwera orthobunyavirus genome.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) comprises three segments of negative-sense, single-stranded RNA. The RNA segments are encapsidated by the viral nucleocapsid (N) protein and form panhandle-like structures through interaction of complementary sequences at their 5' and 3' termini. Transcription and replication of a BUN genome analogue (minireplicon), comprising the viral non-coding sequences flanking a reporter gene, requires just the viral RNA polymerase (L protein) and N protein. Here, sequences of Bunyamwera serogroup M segment RNAs were compared and conserved elements within nt 20-33 of the 3' and 5' non-coding regions that can affect packaging of minireplicons into virions were identified. RNA-folding models suggest that a conserved sequence within nt 20-33 of the 5' end of the genome segments maintains conserved structural features necessary for efficient transcription. Competitive packaging experiments using M, L and S segment-derived minireplicons that encode different reporter genes showed variable packaging efficiencies of the three segments. Packaging of a particular segment appeared to be independent of the presence of other segments and, for the S segment, packaging efficiency was unaffected by the inclusion of viral coding sequences in the minireplicon.

Homotypic interaction of Bunyamwera virus nucleocapsid protein.

The bunyavirus nucleocapsid protein, N, plays a central role in viral replication in encapsidating the three genomic RNA segments to form functional templates for transcription and replication by the viral RNA-dependent RNA polymerase. Here we report functional mapping of interacting domains of the Bunyamwera orthobunyavirus N protein by yeast and mammalian two-hybrid systems, immunoprecipitation experiments, and chemical cross-linking studies. N forms a range of multimers from dimers to high-molecular-weight structures, independently of the presence of RNA. Deletion of the N- or C-terminal domains resulted in loss of activity in a minireplicon assay and a decreased capacity for N to form higher multimers. Our data suggest a head-to-head and tail-to-tail multimerization model for the orthobunyavirus N protein.