LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

A critical role for alternative polyadenylation factor CPSF6 in targeting HIV-1 integration to transcriptionally active chromatin.

Integration is vital to retroviral replication and influences the establishment of the latent HIV reservoir. HIV-1 integration favors active genes, which is in part determined by the interaction between integrase and lens epithelium-derived growth factor (LEDGF)/p75. Because gene targeting remains significantly enriched, relative to random in LEDGF/p75 deficient cells, other host factors likely contribute to gene-tropic integration. Nucleoporins 153 and 358, which bind HIV-1 capsid, play comparatively minor roles in integration targeting, but the influence of another capsid binding protein, cleavage and polyadenylation specificity factor 6 (CPSF6), has not been reported. In this study we knocked down or knocked out CPSF6 in parallel or in tandem with LEDGF/p75. CPSF6 knockout changed viral infectivity kinetics, decreased proviral formation, and preferentially decreased integration into transcriptionally active genes, spliced genes, and regions of chromatin enriched in genes and activating histone modifications. LEDGF/p75 depletion by contrast preferentially altered positional integration targeting within gene bodies. Dual factor knockout reduced integration into genes to below the levels observed with either single knockout and revealed that CPSF6 played a more dominant role than LEDGF/p75 in directing integration to euchromatin. CPSF6 complementation rescued HIV-1 integration site distribution in CPSF6 knockout cells, but complementation with a capsid binding mutant of CPSF6 did not. We conclude that integration targeting proceeds via two distinct mechanisms: capsid-CPSF6 binding directs HIV-1 to actively transcribed euchromatin, where the integrase-LEDGF/p75 interaction drives integration into gene bodies.

A cyclophilin homology domain-independent role for Nup358 in HIV-1 infection.

The large nucleoporin Nup358/RanBP2 forms eight filaments that project from the nuclear pore into the cytoplasm where they function as docking platforms for nucleocytoplasmic transport receptors. RNAi screens have implicated Nup358 in the HIV-1 life cycle. The 164 C-terminal amino acids of this 3,224 amino acid protein are a cyclophilin homology domain (Nup358Cyp), which has potential to bind the HIV-1 capsid and regulate viral progress to integration. Here we examined the virological role of Nup358 in conditional knockout mouse cells and in RNAi-depleted human CD4⁺ T cells. Cre-mediated gene knockout was toxic and diminished HIV-1 infectivity. However, cellular health and HIV-1 susceptibility were coordinately preserved if, prior to gene inactivation, a transposon was used to express all of Nup358 or only the N-terminal 1340 amino acids that contain three FG repeats and a Ran-binding domain. HIV-1, but not N74D capsid-mutant HIV-1, was markedly sensitive to TNPO3 depletion, but they infected 1-1340 segment-complemented Nup358 knockout cells equivalently. Human and mouse CypA both rescued HIV-1 in CypA gene⁻/⁻ Jurkat cells and TRIM-Nup358Cyp fusions derived from each species were equally antiviral; each also inhibited both WT and N74D virus. In the human CD4⁺T cell line SupT1, abrupt Nup358 depletion reduced viral replication but stable Nup358-depleted cells replicated HIV-1 normally. Thus, human CD4⁺ T cells can accommodate to loss of Nup358 and preserve HIV-1 susceptibility. Experiments with cylosporine, viruses with capsids that do not bind cyclophilins, and growth arrest did not uncover viral dependency on the C-terminal domains of Nup358. Our data reinforce the virological importance of TNPO3 and show that Nup358 supports nuclear transport functions important for cellular homeostasis and for HIV-1 nuclear import. However, the results do not suggest direct roles for the Nup358 cyclophilin or SUMO E3 ligase domains in engaging the HIV-1 capsid prior to nuclear translocation.

A new class of multimerization selective inhibitors of HIV-1 integrase.

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.

Feline immunodeficiency virus envelope glycoproteins antagonize tetherin through a distinctive mechanism that requires virion incorporation.

UNLABELLED: BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated that the tiger and cat share a start codon mutation that truncated most of the tetherin cytoplasmic tail early in the Feliformia lineage (19 of 27 amino acids, including the dual tyrosine motif). Alpha interferon (IFN-α) induced tetherin and blocked feline immunodeficiency virus (FIV) replication in lymphoid and nonlymphoid feline cells. Budding of bald FIV and HIV particles was blocked by carnivore tetherins. However, infectious FIV particles were resistant, and spreading FIV replication was uninhibited. Antagonism mapped to the envelope glycoprotein (Env), which rescued FIV from carnivore tetherin restriction when expressed in trans but, in contrast to known antagonists, did not rescue noncognate particles. Also unlike the primate lentiviral antagonists, but similar to the Ebola virus glycoprotein, FIV Env did not reduce intracellular or cell surface tetherin levels. Furthermore, FIV-enveloped FIV particles actually required tetherin for optimal release from cells. The results show that FIV Envs mediate a distinctive tetherin evasion. Well adapted to a phylogenetically ancient tetherin tail truncation in the Felidae, it requires functional virion incorporation of Env, and it shields the budding particle without downregulating plasma membrane tetherin. Moreover, FIV has evolved dependence on this protein: particles containing FIV Env need tetherin for optimal release from the cell, while Env(-) particles do not. IMPORTANCE: HIV-1 antagonizes the restriction factor tetherin with the accessory protein Vpu, while HIV-2 and the filovirus Ebola use their envelope (Env) glycoproteins for this purpose. It turns out that the FIV tetherin antagonist is also its Env protein, but the mechanism is distinctive. Unlike other tetherin antagonists, FIV Env cannot act in trans to rescue vpu-deficient HIV-1. It must be incorporated specifically into FIV virions to be active. Also unlike other retroviral antagonists, but similar to Ebola virus Env, it does not act by downregulating or degrading tetherin. FIV Env might exclude tetherin locally or direct assembly to tetherin-negative membrane domains. Other distinctive features are apparent, including evidence that this virus evolved an equilibrium in which tetherin is both restriction factor and cofactor, as FIV requires tetherin for optimal particle release.

TALEN knockout of the PSIP1 gene in human cells: analyses of HIV-1 replication and allosteric integrase inhibitor mechanism.

UNLABELLED: HIV-1 utilizes the cellular protein LEDGF/p75 as a chromosome docking and integration cofactor. The LEDGF/p75 gene, PSIP1, is a potential therapeutic target because, like CCR5, depletion of LEDGF/p75 is tolerated well by human CD4+ T cells, and knockout mice have normal immune systems. RNA interference (RNAi) has been useful for studying LEDGF/p75, but the potent cofactor activity of small protein residua can be confounding. Here, in human cells with utility for HIV research (293T and Jurkat), we used transcription activator-like effector nucleases (TALENs) to completely eradicate all LEDGF/p75 expression. We performed two kinds of PSIP1 knockouts: whole-gene deletion and deletion of the integrase binding domain (IBD)-encoding exons. HIV-1 integration was inhibited, and spreading viral replication was severely impaired in PSIP1-/- Jurkat cells infected at high multiplicity. Furthermore, frameshifting the gene in the first coding exon with a single TALEN pair yielded trace LEDGF/p75 levels that were virologically active, affirming the cofactor's potency and the value of definitive gene or IBD exon segment deletion. Some recent studies have suggested that LEDGF/p75 may participate in HIV-1 assembly. However, we determined that assembly of infectious viral particles is normal in PSIP1-/- cells. The potency of an allosteric integrase inhibitor, ALLINI-2, for rendering produced virions noninfectious was also unaffected by total eradication of cellular LEDGF/p75. We conclude that HIV-1 particle assembly and the main ALLINI mechanism are LEDGF/p75 independent. The block to HIV-1 propagation in PSIP1-/- human CD4+ T cells raises the possibility of gene targeting PSIP1 combinatorially with CCR5 for HIV-1 cure. IMPORTANCE: LEDGF/p75 dependence is universally conserved in the retroviral genus Lentivirus. Once inside the nucleus, lentiviral preintegration complexes are thought to attach to the chromosome when integrase binds to LEDGF/p75. This tethering process is largely responsible for the 2-fold preference for integration into active genes, but the cofactor's full role in the lentiviral life cycle is not yet clear. Effective knockdowns are difficult because even trace residua of this tightly chromatin-bound protein can support integration cofactor function. Here, in experimentally useful human cell lines, we used TALENs to definitively eradicate LEDGF/p75 by deleting either all of PSIP1 or the exons that code for the integrase binding domain. HIV-1 replication was severely impaired in these PSIP1 knockout cells. Experiments in these cells also excluded a role for LEDGF/p75 in HIV-1 assembly and showed that the main ALLINI mechanism is LEDGF/p75 independent. Site-specific gene targeting of PSIP1 may have therapeutic potential for HIV-1 disease.

Relationships Among Rheological, Sensory Texture, and Swallowing Pressure Measurements of Hydrocolloid-Thickened Fluids.

The objective of this study was to examine the relationships among three categories of measurements (rheological, sensory texture, and swallowing pressure) from fluids thickened to two different viscosities with 15 different hydrocolloids. Fluids at viscosities of 300 and 1500 cP (at 30 s(-1)) were targeted because these are the viscosities corresponding to the barium standards used in radiographic dysphagia diagnosis. Within the low viscosity (nectar) fluids (300 cP), the sensory properties thickness, stickiness, adhesiveness, mouth coating, and number of swallows were highly positively correlated with each other and highly positively correlated with the flow behavior index, n value (an indicator of shear-thinning behavior). Within the higher viscosity (thin honey) fluids (1500 cP), the sensory textures of adhesiveness, stickiness, mouth coating, and number of swallows correlated positively with rheological measures of n value. Swallowing pressures measured in the anterior oral cavity correlated negatively with the consistency coefficient k [shear stress/(shear rate) (n) ]. Samples that were more shear thinning (lower n values, higher k values) were generally perceived as less thick, with less adhesive properties (stickiness, adhesiveness, mouthcoating, and number of swallows). This information can be useful for selecting thickeners for people with dysphagia. A desirable thickener for many dysphagic patients would be one that allowed for a safe swallow by being viscous enough to reduce airway penetration, yet pleasant to drink, having the minimal perceived thickness and mouthcoating associated with greater shear thinning.

The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase.

BACKGROUND: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. RESULTS: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. CONCLUSIONS: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.

Indications and successes of intestinal transplantation in children in the 21st century: A retrospective cohort study.

AIMS: To report the results and successes of intestinal transplantation (ITx) in the most active European centres, to emphasize that, although it is a difficult procedure, it should remain a therapeutic option for children with total, definitive and complicated intestinal failure when intestinal rehabilitation fails. METHODS: We retrospectively collected data about all patients less than 18 receiving an ITx from 2010 to 2022 in 8 centres, and outcomes in July 2022. RESULTS: ITx was performed in 155 patients, median age 6.9 years, in 45% for short bowel syndromes, 22% congenital enteropathies, 25% motility disorders, and 15% re-transplantations. Indications were multiple in most patients, intestinal failure-associated liver disease in half. The graft was in 70% liver-containing. At last follow up 64% were alive, weaned from parenteral nutrition, for 7.9 years; 27% had died and the graft was removed in 8%, mostly early after ITx. DISCUSSION: ITx, despite its difficulties, can give a future to children with complicated intestinal failure. It should be considered among the therapeutic options offered to patients with a predicted survival rate lower than that after ITx. Patients should be early discussed within multidisciplinary teams in ITx centres, to avoid severe complications impacting the results of ITx, or even to avoid ITx.

Productive replication and evolution of HIV-1 in ferret cells.

A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1 animal model.

An image-based eyelid lesion management service-evaluation of a pilot.

BACKGROUND/AIMS: Oculoplastics is a predominantly visual specialty and many of the pathologies can be diagnosed based on external appearance. An image-based eyelid lesion management service was piloted to reduce the number of patients who would require outpatient clinic review. The aim of this study was to determine its accuracy and feasibility, both as a hospital-based and community optometrist-based service. If successful, the service was envisaged to significantly reduce the number of patients that require face-to-face (F2F) review, in accordance with current post-COVID-19 principles of social distancing. METHODS: Patients with lid lesions attending an oculoplastics clinic were assessed by consultant oculoplastic surgeons in an F2F consultation (Arm A). The lesions were photographed by a professional clinical photographer (Arm B) and by an optometrist with a handheld digital camera (Arm C). These images were reviewed by independent consultants masked to the outcome of the F2F clinical encounter. Data were collected prospectively including patient demographics, diagnosis, suspicion of malignancy and management. The image-based clinic results were compared to the F2F clinic results. RESULTS: Ninety-five patients were included. Clinical diagnoses were compared for intra-observer variability and substantial agreement was demonstrated between gold-standard F2F clinic visit (Arm A) and Arm B (Ƙ = 0.708) and C (Ƙ = 0.776). There was no statistically significant difference in the rate of discharge and all cases of malignancy were either identified or flagged for F2F review in the image-based arms. CONCLUSION: This pilot demonstrated substantial diagnostic agreement of image-based diagnoses with F2F consultation and image review alone did not miss any cases of malignancy.

Allosteric Integrase Inhibitor Influences on HIV-1 Integration and Roles of LEDGF/p75 and HDGFL2 Host Factors.

Allosteric integrase (IN) inhibitors (ALLINIs), which are promising preclinical compounds that engage the lens epithelium-derived growth factor (LEDGF)/p75 binding site on IN, can inhibit different aspects of human immunodeficiency virus 1 (HIV-1) replication. During the late phase of replication, ALLINIs induce aberrant IN hyper-multimerization, the consequences of which disrupt IN binding to genomic RNA and virus particle morphogenesis. During the early phase of infection, ALLINIs can suppress HIV-1 integration into host genes, which is also observed in LEDGF/p75-depelted cells. Despite this similarity, the roles of LEDGF/p75 and its paralog hepatoma-derived growth factor like 2 (HDGFL2) in ALLINI-mediated integration retargeting are untested. Herein, we mapped integration sites in cells knocked out for LEDGF/p75, HDGFL2, or both factors, which revealed that these two proteins in large part account for ALLINI-mediated integration retargeting during the early phase of infection. We also determined that ALLINI-treated viruses are defective during the subsequent round of infection for integration into genes associated with speckle-associated domains, which are naturally highly targeted for HIV-1 integration. Class II IN mutant viruses with alterations distal from the LEDGF/p75 binding site moreover shared this integration retargeting phenotype. Altogether, our findings help to inform the molecular bases and consequences of ALLINI action.

Towards rainbow portable Cytophone with laser diodes for global disease diagnostics.

In vivo, Cytophone has demonstrated the capability for the early diagnosis of cancer, infection, and cardiovascular disorders through photoacoustic detection of circulating disease markers directly in the bloodstream with an unprecedented 1,000-fold improvement in sensitivity. Nevertheless, a Cytophone with higher specificity and portability is urgently needed. Here, we introduce a novel Cytophone platform that integrates a miniature multispectral laser diode array, time-color coding, and high-speed time-resolved signal processing. Using two-color (808 nm/915 nm) laser diodes, we demonstrated spectral identification of white and red clots, melanoma cells, and hemozoin in malaria-infected erythrocytes against a blood background and artifacts. Data from a Plasmodium yoelii murine model and cultured human P. falciparum were verified in vitro with confocal photothermal and fluorescent microscopy. With these techniques, we detected infected cells within 4 h after invasion, which makes hemozoin promising as a spectrally selective marker at the earliest stages of malaria progression. Along with the findings from our previous application of Cytophone with conventional lasers for the diagnosis of melanoma, bacteremia, sickle anemia, thrombosis, stroke, and abnormal hemoglobin forms, this current finding suggests the potential for the development of a portable rainbow Cytophone with multispectral laser diodes for the identification of these and other diseases.

Multivalent interactions essential for lentiviral integrase function.

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits1. Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 Å resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the α-helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching reveals that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 results in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

Optimized pathogen detection with 30- compared to 20-milliliter blood culture draws.

Using data from 23,313 patients, we assessed whether two blood culture sets of three bottles per set would detect more pathogens than two sets of two bottles per set and achieve similar sensitivity to collecting three sets of two bottles per set. We also compared the yield of aerobic and anaerobic bottles. Thirty milliliters of blood was distributed to one anaerobic and two aerobic bottles. Among 26,855 collections of ≥ 60 ml within 30 min, 1,379 (5.1%) were positive for a pathogen not requiring detection in more than one set to be considered a pathogen, with 72 additional distinct pathogens detected using two 30-ml compared to two 20-ml sets of one aerobic and one anaerobic bottle (increased yield, 7.9%; 95% confidence interval [CI], 6.2 to 9.8%). For conditional pathogens requiring detection in at least two positive blood cultures for classification as pathogens (i.e., otherwise classified as contaminants), there were 162 positive detections with two 30-ml sets, of which 16 would not have been detected by two 20-ml sets (increased yield, 11.0% [95% CI, 6.4 to 17.2%]). Among 134 subjects who had three sets of 30 ml each within a 30-min interval, there was complete concordance between 60 ml of blood drawn in the first two sets of 30 ml and three 20-ml sets (P = 1.0). One aerobic bottle plus one anaerobic bottle yielded more pathogens than two aerobic bottles for organisms requiring a single (P < 0.001) and two (P = 0.04) positive sets to be defined as pathogens. In conclusion, we showed that collection of two aerobic and one anaerobic blood culture bottles per set results in improved yield compared to two bottles per set. We also confirmed that an anaerobic bottle should be included in blood culture sets.

The HIV-1 central polypurine tract functions as a second line of defense against APOBEC3G/F.

HIV-1 and certain other retroviruses initiate plus-strand synthesis in the center of the genome as well as at the standard retroviral 3' polypurine tract. This peculiarity of reverse transcription results in a central DNA "flap" structure that has been of controversial functional significance. We mutated both HIV-1 flap-generating elements, the central polypurine tract (cPPT) and the central termination sequence (CTS). To avoid an ambiguity of previous studies, we did so without affecting integrase coding. DNA flap formation was disrupted but single-cycle infection was unaffected in all target cells tested, regardless of cell cycle status. Spreading HIV-1 infection was also normal in most T cell lines, and flap mutant viruses replicated equivalently to the wild type in nondividing cells, including macrophages. However, spreading infection of flap mutant HIV-1 was impaired in non-vif-permissive cells (HuT78, H9, and primary human peripheral blood mononuclear cells [PBMCs]), suggesting APOBEC3G (A3G) restriction. Single-cycle infections confirmed that vif-intact flap mutant HIV-1 is restricted by producer cell A3G/F. Combining the Δvif and cPPT-CTS mutations increased A3G restriction synergistically. Moreover, RNA interference knockdown of A3G in HuT78 cells released the block to flap mutant HIV-1 replication. Flap mutant HIV-1 also accrued markedly increased A3G-mediated G→A hypermutation compared to that of wild-type HIV-1 (a full log(10) in the 0.36 kb downstream of the mutant cPPT). We suggest that the triple-stranded DNA structure, the flap, is not the consequential outcome. The salient functional feature is central plus-strand initiation, which functions as a second line of defense against single-stranded DNA editing by A3 proteins that survive producer cell degradation by Vif.

Productive replication of Vif-chimeric HIV-1 in feline cells.

Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-cell lines, and primary peripheral blood mononuclear cells supported early and late HIV-1 life cycle phases in a manner equivalent to that of human cells, except that produced virions had low infectivity. Stable expression of feline immunodeficiency virus (FIV) Vif-green fluorescent protein (GFP) in HIV-1 entry receptor-complemented feline (CrFK) cells enabled robust spreading HIV-1 replication. FIV Vif colocalized with feline APOBEC3 (fA3) proteins, targeted them for degradation, and prevented G-->A hypermutation of the HIV-1 cDNA by fA3CH and fA3H. HIV-1 Vif was inactive against fA3s as expected and even paradoxically augmented restriction in some assays. In an interesting contrast, simian immunodeficiency virus SIVmac Vif had substantial anti-fA3 activities, which were complete against fA3CH and partial against fA3H. Moreover, both primate lentiviral Vifs colocalized with fA3s and could be pulled down from cell lysates by fA3CH. HIV-1 molecular clones that encode FIV Vif or SIVmac Vif (HIV-1(VF) and HIV-1(VS)) were then constructed. These viruses replicated productively in HIV-1 receptor-expressing CrFK cells and could be passaged serially to uninfected cells. Thus, with the exception of entry receptors, the cat genome can supply the dependency factors needed by HIV-1, and a main restriction can be countered by vif chimerism. The results raise the possibility that the domestic cat could yield an animal model of HIV-1 infection.

Outcomes in Patients with SARS-CoV-2 and Clostridioides difficile Coinfection.

BACKGROUND: Coronavirus infectious disease 2019 (COVID-19) is primarily a respiratory disease. However, it may manifest with gastrointestinal symptoms that may overlap with Clostridioides difficile infection (CDI). COVID-19 appears to have higher mortality in those with comorbidities. We aimed to assess the outcomes of coinfection in these patients. METHODS: A retrospective chart review was conducted to identify patients with CDI and COVID-19 from January 1st, 2020 to November 17th, 2020. Both infections were diagnosed via PCR. Clinical features, treatment for COVID-19 and CDI and outcomes including intensive care unit admission, colectomy, 30 day-mortality and long-term complications were analyzed. RESULTS: Overall, 21 patients (20 hospitalized) with median age 70.9 years (range 51.8-90.7 years) had CDI and COVID-19 within 4 weeks of each other. Of these, 4 patients (19%) with CDI were diagnosed with COVID-19 at the time of admission, 12 (57%) had CDI diagnosed after COVID-19, and 5 (23.9%) developed COVID-19 within 4 weeks after CDI. Fourteen patients (66.7%) were treated with medications directed against COVID-19 including remdesivir and dexamethasone (n=7), remdesivir with convalescent plasma (n= 1), remdesivir (n= 5) and dexamethasone (n=1). The most common treatment for CDI was oral vancomycin in 20 patients (95.2%), and 1 patient received intravenous metronidazole. No patient required colectomy for CDI but 2 (9.5%) required ICU admission. Four patients (19%) died likely due to COVID-19 with median age 80 years (range 61-90 years). CONCLUSION: The relationship between COVID-19 and CDI is poorly understood, and studies are required to further investigate this association. Whether coinfection results in a worsening of outcomes, including mortality and clinical course, are questions that should be answered in future research studies. Diagnosing both infections for appropriate management is vital in light of overlapping symptoms.

Outcomes of COVID-19 With the Mayo Clinic Model of Care and Research.

OBJECTIVE: To report the Mayo Clinic experience with coronavirus disease 2019 (COVID-19) related to patient outcomes. METHODS: We conducted a retrospective chart review of patients with COVID-19 diagnosed between March 1, 2020, and July 31, 2020, at any of the Mayo Clinic sites. We abstracted pertinent comorbid conditions such as age, sex, body mass index, Charlson Comorbidity Index variables, and treatments received. Factors associated with hospitalization and mortality were assessed in univariate and multivariate models. RESULTS: A total of 7891 patients with confirmed COVID-19 infection with research authorization on file received care across the Mayo Clinic sites during the study period. Of these, 7217 patients were adults 18 years or older who were analyzed further. A total of 897 (11.4%) patients required hospitalization, and 354 (4.9%) received care in the intensive care unit (ICU). All hospitalized patients were reviewed by a COVID-19 Treatment Review Panel, and 77.5% (695 of 897) of inpatients received a COVID-19-directed therapy. Overall mortality was 1.2% (94 of 7891), with 7.1% (64 of 897) mortality in hospitalized patients and 11.3% (40 of 354) in patients requiring ICU care. CONCLUSION: Mayo Clinic outcomes of patients with COVID-19 infection in the ICU, hospital, and community compare favorably with those reported nationally. This likely reflects the impact of interprofessional multidisciplinary team evaluation, effective leveraging of clinical trials and available treatments, deployment of remote monitoring tools, and maintenance of adequate operating capacity to not require surge adjustments. These best practices can help guide other health care systems with the continuing response to the COVID-19 pandemic.

Lupus-associated endogenous retroviral LTR polymorphism and epigenetic imprinting promote HRES-1/RAB4 expression and mTOR activation.

Overexpression and long terminal repeat (LTR) polymorphism of the HRES­1/Rab4 human endogenous retrovirus locus have been associated with T cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is diminished overall in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus affect T cell activation. The results showed that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR enhancer are hypomethylated in SLE. Pharmacologic or genetic inactivation of DNA methyltransferase 1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR enhancer and exerted control over HRES-1/Rab4 expression in rs451401 genotype- and methylation-dependent manners. The LTR enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation- and rs451401 allele-dependent transducer of environmental stress and controller of T cell activation.