The influenza virus RNA-dependent RNA polymerase (RdRP) cleaves the 5′ end of nascent capped host RNAs and uses the capped RNA fragment to prime viral transcription in a mechanism called ‘cap ...snatching’. Cap snatching requires an intimate association between influenza RdRP and cellular RNA polymerase II (Pol II), which is the source of nascent capped host RNAs targeted by influenza virus. Recent structural studies have revealed how influenza RdRP binds to Pol II and how this binding promotes the initiation of viral transcription by influenza RdRP. In this review we focus on these recent insights into the mechanism of cap snatching by influenza virus and the impact of cap snatching on host gene expression during influenza virus infection.
Influenza virus RdRP associates with cellular Pol II. This provides influenza RdRP with a source of nascent capped host RNAs which it targets in a process called ‘cap snatching’ to generate primers for viral transcription.
Influenza RdRP makes direct interactions with the Pol II C terminal domain (CTD). Molecular details of these interactions have recently been determined, and there is evidence that Pol II CTD binding promotes viral transcription by modulating the conformation of influenza RdRP.
Influenza RdRP cap snatches small nuclear/nucleolar RNAs most frequently. When cap snatching generates suboptimal capped RNA fragments, influenza RdRP can use a prime-and-realign mechanism to promote transcription initiation.
Influenza virus infection reduces Pol II abundance in gene bodies, suggesting that cap snatching contributes to host shut-off.
Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge
. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by ...the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes
. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity
. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.
Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans
. Influenza A viruses contain a segmented ...negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol
) composed of PB1, PB2 and PA subunits
. Although the high-resolution crystal structure of FluPol
of bat influenza A virus has previously been reported
, there are no complete structures available for human and avian FluPol
. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase
-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol
from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol
forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol
bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol
dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol
dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol
, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol
that could be targeted in the development of antiviral drugs.
Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using ...a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription
but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.
Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.
Abstract
SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. ...SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5′ triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.
Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral ...RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
A noninvasive and accurate method of determining fluid responsiveness in ventilated patients would help to mitigate unnecessary fluid administration. Although carotid ultrasound has been previously ...studied for this purpose, several studies have recently been published. We performed an updated systematic review and meta-analysis to evaluate the accuracy of carotid ultrasound as a tool to predict fluid responsiveness in ventilated patients.
Studies eligible for review investigated the accuracy of carotid ultrasound parameters in predicting fluid responsiveness in ventilated patients, using sensitivity and specificity as markers of diagnostic accuracy (International Prospective Register of Systematic Reviews PROSPERO CRD42022380284). All included studies had to use an independent method of determining cardiac output and exclude spontaneously ventilated patients. Six bibliographic databases and 2 trial registries were searched. Medline, Embase, Emcare, APA PsycInfo, CINAHL, and the Cochrane Library were searched on November 4, 2022. Clinicaltrials.gov and Australian New Zealand Clinical Trials Registry were searched on February 24, 2023. Results were pooled, meta-analysis was conducted where possible, and hierarchical summary receiver operating characteristic models were used to compare carotid ultrasound parameters. Bias and evidence quality were assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) tool and the Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) guidelines.
Thirteen prospective clinical studies were included (n = 648 patients), representing 677 deliveries of volume expansion, with 378 episodes of fluid responsiveness (58.3%). A meta-analysis of change in carotid Doppler peak velocity (∆CDPV) yielded a sensitivity of 0.79 (95% confidence interval CI, 0.74-0.84) and a specificity of 0.85 (95% CI, 0.76-0.90). Risk of bias relating to recruitment methodology, the independence of index testing to reference standards and exclusionary clinical criteria were evaluated. Overall quality of evidence was low. Study design heterogeneity, including a lack of clear parameter cutoffs, limited the generalizability of our results.
In this meta-analysis, we found that existing literature supports the ability of carotid ultrasound to predict fluid responsiveness in mechanically ventilated adults. ∆CDPV may be an accurate carotid parameter in certain contexts. Further high-quality studies with more homogenous designs are needed to further validate this technology.
There are currently no proven treatments to reduce the risk of mild cognitive impairment and dementia.
To evaluate the effect of intensive blood pressure control on risk of dementia.
Randomized ...clinical trial conducted at 102 sites in the United States and Puerto Rico among adults aged 50 years or older with hypertension but without diabetes or history of stroke. Randomization began on November 8, 2010. The trial was stopped early for benefit on its primary outcome (a composite of cardiovascular events) and all-cause mortality on August 20, 2015. The final date for follow-up of cognitive outcomes was July 22, 2018.
Participants were randomized to a systolic blood pressure goal of either less than 120 mm Hg (intensive treatment group; n = 4678) or less than 140 mm Hg (standard treatment group; n = 4683).
The primary cognitive outcome was occurrence of adjudicated probable dementia. Secondary cognitive outcomes included adjudicated mild cognitive impairment and a composite outcome of mild cognitive impairment or probable dementia.
Among 9361 randomized participants (mean age, 67.9 years; 3332 women 35.6%), 8563 (91.5%) completed at least 1 follow-up cognitive assessment. The median intervention period was 3.34 years. During a total median follow-up of 5.11 years, adjudicated probable dementia occurred in 149 participants in the intensive treatment group vs 176 in the standard treatment group (7.2 vs 8.6 cases per 1000 person-years; hazard ratio HR, 0.83; 95% CI, 0.67-1.04). Intensive BP control significantly reduced the risk of mild cognitive impairment (14.6 vs 18.3 cases per 1000 person-years; HR, 0.81; 95% CI, 0.69-0.95) and the combined rate of mild cognitive impairment or probable dementia (20.2 vs 24.1 cases per 1000 person-years; HR, 0.85; 95% CI, 0.74-0.97).
Among ambulatory adults with hypertension, treating to a systolic blood pressure goal of less than 120 mm Hg compared with a goal of less than 140 mm Hg did not result in a significant reduction in the risk of probable dementia. Because of early study termination and fewer than expected cases of dementia, the study may have been underpowered for this end point.
ClinicalTrials.gov Identifier: NCT01206062.
Abstract
The SARS-CoV-2 coronavirus is the causal agent of the current global pandemic. SARS-CoV-2 belongs to an order, Nidovirales, with very large RNA genomes. It is proposed that the fidelity of ...coronavirus (CoV) genome replication is aided by an RNA nuclease complex, comprising the non-structural proteins 14 and 10 (nsp14–nsp10), an attractive target for antiviral inhibition. Our results validate reports that the SARS-CoV-2 nsp14–nsp10 complex has RNase activity. Detailed functional characterization reveals nsp14–nsp10 is a versatile nuclease capable of digesting a wide variety of RNA structures, including those with a blocked 3′-terminus. Consistent with a role in maintaining viral genome integrity during replication, we find that nsp14–nsp10 activity is enhanced by the viral RNA-dependent RNA polymerase complex (RdRp) consisting of nsp12–nsp7–nsp8 (nsp12–7–8) and demonstrate that this stimulation is mediated by nsp8. We propose that the role of nsp14–nsp10 in maintaining replication fidelity goes beyond classical proofreading by purging the nascent replicating RNA strand of a range of potentially replication-terminating aberrations. Using our developed assays, we identify drug and drug-like molecules that inhibit nsp14–nsp10, including the known SARS-CoV-2 major protease (Mpro) inhibitor ebselen and the HIV integrase inhibitor raltegravir, revealing the potential for multifunctional inhibitors in COVID-19 treatment.
Purpose
This article examines the incidence and management of vascular injury during Lateral Lumbar Interbody Fusion (LLIF). The details of the mini-open access technique are presented.
Methods
A ...total of 900 patients who underwent a LLIF at an average 1.94 levels (range: 1–5 levels) by one of six fellowship trained surgeons on 1,754 levels from 2006 to 2013 were identified. The incidence of intraoperative vascular injury was retrospectively determined from the Operative Records. The management of vascular injury was evaluated. The mini-open access adapted by our institution for LLIF is described.
Results
The incidence of major vascular complication in our series was 1/900. The incidence of minor vascular injury was 4/900. The overall incidence of vascular injury was calculated to be 0.056 % per case and 0.029 % per level. All minor vascular injuries were identified to be segmental vessel lacerations, which were readily ligated under direct visualization without further extension of the incision with no clinical sequelae. The laceration of the abdominal aorta, the major vascular complication of this series, was emergently repaired through an exploratory laparotomy. None of the patients suffered long-term sequelae from their intraoperative vascular injuries.
Conclusions
The mini-open lateral access technique for LLIF provides for minimal risk of vascular injury to the lumbar spine. In the rare event of minor vascular injury, the mini-open access approach allows for immediate visualization, confirmation and repair of the vessel with no long-term sequelae.
PDF
