Structure and function of the nairovirus cap-snatching endonuclease

Nairoviruses, such as Crimean-Congo hemorrhagic fever virus (CCHFV) initiate viral mRNA synthesis using an N-terminal cap-snatching endonuclease domain within their viral L protein. The endonuclease of this nairoviruses have an extended domain architecture of 350 residues with flexible insertion loops, an a7 support helix and a hybrid active site motif. Biochemical assays show that nairovirus EN activity must require manganese ions and not magnesium ions and prefer uridine rich single stranded RNA substrates.  Antibody assisted crystallization with antigen binding fragments of CCHF virus has shown a distinct two metal ion binding mode. 
Mutation analysis and viral mini replicon assays have confirmed that the dual metal ion structure is necessary for the endonuclease activity and viral transcription. Inhibitor assay shows that metal chelating compounds DPBA, L742001 and baloxavir acid inhibit the EN activity. The authors also show using co-crystal structure that baloxavir acid has higher inhibition potency and higher binding affinity.
(MCW)

Antagonistic activity against innate immunity determines virulence in mammalian bornaviruses

Mammalian Bornaviruses include the classic Borna disease virus isolated from schizophrenic patients (BoDV-1) and the emerging and highly fatal zoonotic Variegated squirrel bornavirus (VSBV-1). The two share similar cell tropisms and core viral ribonucleic compatibility but the cause and underlying reason for the  VSBV-1 higher virulence have remained unclear.By use of Minigenome and functional assays, VSBV-1 phosphoprotein (P) was shown to boost BoDV-1 polymerase activity but lacked the innate immune-suppressive capabilities of BODV-1 P. BoDV-1 P requires X co-expression to transport to the cytoplasm but VSBV-1 P mostly localizes to the cytoplasm independently. This is driven by specific amino acid differences in its N-terminal nuclear localization signal. In the investigation of invivo pathogenesis, a recombinant chimeric virus (rBoDV-1 XPV) which carried the VSBV-1 X/P genes was engineered. rBoDV-1 XPV showed replication kinetics in vitro similar to wild-type BoDV-1 (Wt), but it caused 100% mortality in adult rats following intranasal infection. Histopathological analysis showed that rBoDV-1 XPV caused severe neuroinflammatory response with microgliosis, perivascular cuffing, high viral titers, and marked upregulation of pro-inflammatory cytokines in rat brains. VSBV-1 virulence is driven by weak innate immune antagonisms and cytoplasmic viral replication that causes severe and lethal immunopathology.
(MCW)

Discovery of three small-molecule inhibitors targeting Ebolavirusgenome replication and transcription

Using established ligand discovery pipeline, three small molecule inhibitors were identified to target hydrophobic binding pocket of the Ebola virus nucleoprotein. These are MCCB5, CB6  and MJM364. The compounds interfere with the NP VP35 protein protein interaction required for viral replication and transcription. Further evaluation using EboV minigenome assay showed a potent dose dependent inhibition with EC50 values of 4.41 µM, 0.194 µM and 0.834 µM for MCCB5, CB6 and MJM364 respectively in BSR-T7 cells. These concentrations also showed low cytotoxicity The drugs were confirmed to be specific for EBOV as they showed a lack of efficacy against Bunyamwera virus. The inhibitory activity was also shown  and validated in human hepatoma cells, Huh7-Lunet-T7. Time of addition studies showed that MCCB5 suppressed minigenome activity when administered posttransfection about 24-48 hpt showing its capacity to target already established NP VP35 replication complexes which is a key therapeutic requirement for post exposure treatment. Mutation targeting hotspot 2 of the NP hydrophobic pocket caused  a shift to higher EC 50 values for all the three compounds showing that this pocket is their functional target. Since these NP residues are highly confirmed in filoviruses, these compounds are potential broad spectrum filoviral therapeutics. 
(MCW)

Evidence that Human parainfluenza virus type 2 paramyxovirus antigenomes are edited during infection

Mononegaviruses mRNA synthesis relies on cis-acting sequences for gene start and Gene end and non-templated nucleotide insertions at editing sites. Gene start and Gene end are silent during genome replication but editing site signals are actively utilized during antigenome synthesis. Amplicon sequencing of wildtype and NP mutants infection rPIV219 showed that antigenomes are edited at frequencies similar to mRNA with 2G insertions. Paramyxovirus replications always adheres to rule of six with genomes length being always multiples of 6 and because of this, edited genomes with 2 more insertions serve as poor replication templates. The NP-Q202A mutation helped relieve this constraint and tripled the accumulation of edited pre-geniomes intracellularly providing genetic evidence that antigenomes are indeed edited during infection. Despite the intracellular accumulation of non 6n+-o genomes, supernatant virions still selectively packaged only unedited 6n+0 genomes. This demonstrates that the rule of six is still kept true even by a combination of replication inhibition of non hexamer templates and exclusion at viral assembly and budding off. 
(MCW)

Vesicular Stomatitis Virus-Based Oncolytic Virotherapy: Recent Progress and Emerging Trends

Vesicular Stomatitis virus (VSV) is a prototype Rhabdovirus which is characterised by rapid cytoplasmic replication and robust cell lysis but is normally non pathogenic in humans. It has shown a prominent oncolytic virus Potential. VSV uses low density lipoprotein receptors for entry and its tumor selectivity relies mainly on widespread type I interferon (IFN) signaling defects in cancer cells other than tumor-restricted cell binding. Recently, engineering strategies have focused on limiting wildtype M protein mediated neurotoxicity and off target replication. Selectivity and safety has been enhanced by use of chimeric envelope swapping, attenuation via different mutations and insertions of IFN transgenes, and microRNA mediated suppression in normal tissues. To overcome resistance in IFN competent tumors, VSV has been combined with JAK/STAT inhibitors (such as ruxolitinib), epigenetic modulators (targeting SIRT1, CHD1, MAP3K7), and metabolic reprogramming agents. Direct oncolysis has been also amplified by giving vectors pro-apoptotic/PANoptotic drugs or tumor suppressors and also using  radiation and chemotherapeutics. To evade rapid immune/complement clearance during delivery, cellular carriers like naïve T cells, MSCs are being used. Related vesiculoviruses like Maraba, Morreton, and Jurona viruses are also being studied due to their complement resistance, distinct receptor usage, and reduced neurovirulence.
(MCW) 

Maternal vaccination protects dams and prevents in utero transmission of Rift Valley fever virus in rats

Rift Valley fever virus (RVFV) infection during pregnancy can cause abortion, fetal death, and vertical transmission, making pregnant individuals particularly susceptible to severe disease. This study evaluated the safety and efficacy of a live-attenuated RVFV vaccine lacking the NSs and NSm genes (RVFV-delNSs/NSm) in a pregnant rat model. Female rats were vaccinated either before or during pregnancy and later challenged with a virulent wild-type RVFV strain. Maternal survival, viral distribution, and fetal outcomes were then assessed. Vaccination, even at a high dose during early pregnancy, caused no adverse effects in either the dams or fetuses. Although low levels of viral RNA were occasionally detected in fetal tissues, no infectious virus was recovered. After challenge with wild-type RVFV, all vaccinated rats survived, whereas approximately 80% of unvaccinated animals died. No virus was detected in the organs of fetuses from vaccinated dams, indicating complete prevention of vertical transmission. Nearly all offspring from vaccinated mothers were born healthy without congenital abnormalities or low birth weight, while unvaccinated animals frequently experienced stillbirths and hemorrhagic lesions. Protection was also achieved when vaccination was performed before pregnancy. These findings suggest that deletion of NSs and NSm effectively attenuates RVFV while maintaining protective immunity, making this vaccine a promising candidate for preventing maternal infection and fetal transmission during pregnancy.
(AI)

The polymerase of negative stranded RNA viruses

Negative-sense RNA viruses are divided into non segmented viruses such as Ebola and segmented viruses such as influenza. Both groups protect their genomic RNA inside a nucleocapsid protein(NP) that the viral RNA-Dependent RNA polymerase must navigate during transcription and replication. Both classes share similar polymerase core architecture but their capping and structural mechanisms are different. NNS viruses use internal PRNTase and MTase domains to synthesize fully modified 5’ caps and have rigid locked nucleoplasmids. SNS viruses have loosely organized nucleocaspids and hijack host mRNA caps via a cap dependent endonuclease domain. In NNs systems the phosphoprotein (P) is an important cofactor that changes the L proteins structural appendage and acts as a processivity factor whereas SNS polymerases  accesses templates directly. Recent Invitro assays have shown that polymerases can initiate denovo or using primers on naked RNA through precise 3’ terminal base pairing. Also template associated protein NP remains important to achieve full processivity. 
(MCW)