RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study

The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors.     

Endocytosis optimizes the dynamic localization of membrane proteins that regulate cortical polarity

Diverse cell types require the ability to maintain dynamically polarized membrane-protein distributions through balancing transport and diffusion. However, design principles underlying dynamically maintained cortical polarity are not well understood. Here we constructed a mathematical model for characterizing the morphology of dynamically polarized protein distributions. We developed analytical approaches for measuring all model parameters from single-cell experiments. We applied our methods to a well-characterized system for studying polarized membrane proteins: budding yeast cells expressing activated Cdc42. We found that a balance of diffusion, directed transport, and endocytosis was sufficient for accurately describing polarization morphologies. Surprisingly, the model predicts that polarized regions are defined with a precision that is nearly optimal for measured endocytosis rates and that polarity can be dynamically stabilized through positive feedback with directed transport. Our approach provides a step toward understanding how biological systems shape spatially precise, unambiguous cortical polarity domains using dynamic processes.     

The modular structure of haemagglutinin/adhesin regions in gingipains of Porphyromonas gingivalis

High-molecular-weight arginine- and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct β-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.     

San Francisco Syncope Rule to predict short-term serious outcomes: a systematic review

Background:

The San Francisco Syncope Rule has been proposed as a clinical decision rule for risk stratification of patients presenting to the emergency department with syncope. It has been validated across various populations and settings. We undertook a systematic review of its accuracy in predicting short-term serious outcomes.

Methods:

We identified studies by means of systematic searches in seven electronic databases from inception to January 2011. We extracted study data in duplicate and used a bivariate random-effects model to assess the predictive accuracy and test characteristics.

Results:

We included 12 studies with a total of 5316 patients, of whom 596 (11%) experienced a serious outcome. The prevalence of serious outcomes across the studies varied between 5% and 26%. The pooled estimate of sensitivity of the San Francisco Syncope Rule was 0.87 (95% confidence interval [CI] 0.79–0.93), and the pooled estimate of specificity was 0.52 (95% CI 0.43–0.62). There was substantial between-study heterogeneity (resulting in a 95% prediction interval for sensitivity of 0.55–0.98). The probability of a serious outcome given a negative score with the San Francisco Syncope Rule was 5% or lower, and the probability was 2% or lower when the rule was applied only to patients for whom no cause of syncope was identified after initial evaluation in the emergency department. The most common cause of false-negative classification for a serious outcome was cardiac arrhythmia.

Interpretation:

The San Francisco Syncope Rule should be applied only for patients in whom no cause of syncope is evident after initial evaluation in the emergency department. Consideration of all available electrocardiograms, as well as arrhythmia monitoring, should be included in application of the San Francisco Syncope Rule. Between-study heterogeneity was likely due to inconsistent classification of arrhythmia.Syncope is defined as sudden, transient loss of consciousness with the inability to maintain postural tone, followed by spontaneous recovery and return to pre-existing neurologic function.^1–5 It represents a common clinical problem, accounting for 1%–3% of visits to the emergency department and up to 6% of admissions to acute care hospitals.^6,7Assessment of syncope in patients presenting to the emergency department is challenging because of the heterogeneity of underlying pathophysiologic processes and diseases. Although many underlying causes of syncope are benign, others are associated with substantial morbidity or mortality, including cardiac arrhythmia, myocardial infarction, pulmonary embolism and occult hemorrhage.^4,8–10 Consequently, a considerable proportion of patients with benign causes of syncope are admitted for inpatient evaluation.^11,12 Therefore, risk stratification that allows for the safe discharge of patients at low risk of a serious outcome is important for efficient management of patients in emergency departments and for reduction of costs associated with unnecessary diagnostic workup.^12,13In recent years, various prediction rules based on the probability of an adverse outcome after an episode of syncope have been proposed.^3,14–16 However, the San Francisco Syncope Rule, derived by Quinn and colleagues in 2004,³ is the only prediction rule for serious outcomes that has been validated in a variety of populations and settings. This simple, five-step clinical decision rule is intended to identify patients at low risk of short-term serious outcomes^3,17 (Box 1).

Box 1:

San Francisco Syncope Rule³

AimPrediction of short-term (within 30 days) serious outcomes in patients presenting to the emergency department with syncope.DefinitionsSyncope: Transient loss of consciousness with return to baseline neurologic function. Trauma-associated and alcohol- or drug-related loss of consciousness excluded, as is definite seizure or altered mental status.Serious outcome: Death, myocardial infarction, arrhythmia, pulmonary embolism, stroke, subarachnoid hemorrhage, significant hemorrhage or any condition causing or likely to cause a return visit to the emergency department and admission to hospital for a related event.Selection of predictors in multivariable analysis: Fifty predictor variables were evaluated for significant associations with a serious outcome and combined to create a minimal set of predictors that are highly sensitive and specific for prediction of a serious outcome.Clinical decision ruleFive risk factors, indicated by the mnemonic “CHESS,” were identified to predict patients at high risk of a serious outcome:

• C – History of congestive heart failure
• H – Hematocrit < 30%
• E – Abnormal findings on 12-lead ECG or cardiac monitoring¹⁷ (new changes or nonsinus rhythm)
• S – History of shortness of breath
• S – Systolic blood pressure < 90 mm Hg at triage

Note: ECG = electrocardiogram.The aim of this study was to conduct a systematic review and meta-analysis of the accuracy of the San Francisco Syncope Rule in predicting short-term serious outcome for patients presenting to the emergency department with syncope.     

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.     

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.     

High cell-free virus load and robust autologous humoral immune responses in breast milk of simian immunodeficiency virus-infected african green monkeys

The design of immunologic interventions to prevent postnatal transmission of human immunodeficiency virus (HIV) will require identification of protective immune responses in this setting. Simian immunodeficiency virus (SIV)-infected rhesus monkeys (RMs), a species that develops an AIDS-like illness following experimental infection, transmit the virus at a high rate during breastfeeding. In contrast, postnatal transmission of SIV occurs rarely or not at all in natural, asymptomatic primate hosts of SIV. These contrasting transmission patterns provide a unique opportunity to study mechanisms that evolved to protect suckling infants from SIV infection. We compared the virologic and immunologic properties of milk of SIV-infected and uninfected natural hosts of SIV, African green monkeys (AGMs), to that of RMs. Interestingly, despite a low number of milk CD4(+) T lymphocytes in uninfected AGMs, milk virus RNA load in SIV-infected AGMs was comparable to that of SIV-infected RMs and that in AGM plasma. This observation is in contrast to the relatively low virus load in milk compared to that in plasma of SIV-infected RMs and HIV-infected women. Milk of SIV-infected AGMs also displayed robust virus-specific cellular immune responses. Importantly, an autologous challenge virus-specific neutralization response was detected in milk of five of six SIV-infected AGMs that was comparable in magnitude to that in plasma. In contrast, autologous challenge virus neutralization was not detectable in milk of SIV-infected RMs. The autologous virus-specific adaptive immune responses in breast milk of AGMs may contribute to impedance of virus transmission in the infant oral/gastrointestinal tract and the rarity of postnatal virus transmission in natural hosts of SIV.     

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus

Influenza C virus spike glycoprotein HEF specifically recognizesglycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid.The same protein also contains an esterase activity. Takingadvantage of these two properties, influenza C virus was usedas a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminicacid in human leucocytes. The binding of influenza C virus toleucocyte glycoproteins and gangliosides separated by sodiumdodecyl sulphate–polyacrylamide gel electrophoresis andthin-layer chromatography, respectively, was assayed using achromogenic esterase substrate. In this way, glycoproteins ofB-lymphocytes and T-lymphocytes were found to contain 9-O-acetylatedsialic acids. Of the various 9-O-acetylated gangliosides detected,one had the characteristics of 9-O-acetylated G^D3. The identificationof 9-O-acetylated sialic acids on distinct glycoproteins andglycolipids should be helpful in assigning a physiological roleto this sugar. O-acetylation gangliosides influenza C virus lymphocytes sialic acids