A Second Drug Binding Site in P2X3.

Purinergic P2X3 receptors form trimeric cation-gated channels, which are activated by extracellular ATP. P2X3 plays a crucial role in chronic cough and affects over 10% of the population. Despite considerable efforts to develop drugs targeting P2X3, the highly conserved structure within the P2X receptor family presents obstacles for achieving selectivity. Camlipixant, a potent and selective P2X3 antagonist, is currently in phase III clinical trials. However, the mechanisms underlying receptor desensitization, ion permeation, principles governing antagonism, and the structure of P2X3 when bound to camlipixant remain elusive. In this study, we established a stable cell line expressing homotrimeric P2X3 and utilized a peptide scaffold to purify the complex and determine its structure using cryo-electron microscopy (cryo-EM). P2X3 binds to camlipixant at a previously unidentified drug-binding site and functions as an allosteric inhibitor. Structure-activity studies combined with modeling and simulations have shed light on the mechanisms underlying the selective targeting and inhibition of P2X3 by camlipixant, distinguishing it from other members of the P2X receptor family.

Novel peeling skin condition in neonatal Pacific walruses, Saint Lawrence Island, Alaska, USA.

We describe here a novel peeling skin condition (PSC) in 2 neonatal Pacific walruses (Odobenus rosmarus subsp. divergens). Macroscopically, calves had various degrees of peeling skin exacerbated by mechanical trauma. Lesions occurred in areas subject to friction: ventrum, fore- and hindflippers, and associated joints. Histopathologic features included pseudocarcinomatous epithelial hyperplasia with orthokeratotic hyperkeratosis. Bacterial cocci were present within the stratum corneum. A few intraepidermal clefts were present. Inflammation, epidermolysis, and vasculopathies were not observed. PCR assays were negative for vesivirus and for Staphylococcus aureus exfoliative and toxic shock syndrome toxins. Tissue samples were cultured and bacteria isolated and identified by MALDI-TOF MS as Carnobacterium maltaromaticum, Psychrobacter phenylpyruvicus, Globicatella sanguinis, Streptococcus phocae, Pseudomonas spp., Rahnella aquatilis, and Escherichia coli. Given the young age of the calves and their clinical presentation, congenital ichthyosis was suspected. No genetic differences were detected for sequenced portions of keratin genes (keratin gene K10) between diseased and normal walrus skin. This rare PSC in neonatal Pacific walruses is recognized as novel by indigenous Alaskan marine mammal hunters of the Bering Strait region. A comprehensive diagnostic work-up of future case materials is needed to characterize the underlying biochemical defect(s).

Feasibility Trial of Intensity Modulated Proton Therapy to Reduce Toxicity in Anal Cancer Patients.

PURPOSE: The purpose of this trial was to assess the patient and physician-reported toxicity in anal cancer patients undergoing definitive chemoradiation with intensity-modulated proton therapy (IMPT). METHODS: Patients with stage II and III anal cancer were treated with IMPT. All patients received 2 cycles of 5-fluorouracil and mitomycin concurrently with radiation. Toxicity was assessed at baseline, weekly during chemoradiation, and in follow-up using physician-graded common terminology criteria for adverse events (CTCAE) v 4.0 and PRO-CTCAE. The primary endpoint was to define point estimates and 95% CI for acute ≥ grade 2/3 gastrointestinal (GI), genitourinary (GU), dermatologic, and hematologic toxicity. The proportion of PRO-CTCAE questions scored ≥3 for each domain was compared with the baselinse. The proportion of ≥ grade 2 and ≥ grade 3 toxicities were compared with historic intensity-modulated radiotherapy patients treated on RTOG 0529. RESULTS: Fourteen patients were enrolled from 2017 to 2020. Rates of physician-reported GI, GU, dermatologic, and hematologic toxicity were not significantly different between patients treated with IMPT compared with patients treated with intensity-modulated radiotherapy. Rates of patient-reported dermatologic and GU toxicity were low at baseline with a peak at week 6 (91% and 58% PRO-CTCAE items ≥ grade 3, respectively) and normalization to baseline 3 months after IMPT. In contrast, the proportion of high-grade PRO-CTCAE GI scores was 40% at baseline, which persisted through 1-year posttreatment. CONCLUSIONS: Clinician-reported toxicity was not improved with IMPT in the context of this underpowered trial. High-grade GI symptoms persisted for 12 months and were similar to baseline. Additional measures are needed to minimize acute and chronic toxicity related to chemoradiation.

Paralytic shellfish toxins in Alaskan Arctic food webs during the anomalously warm ocean conditions of 2019 and estimated toxin doses to Pacific walruses and bowhead whales.

Climate change-related ocean warming and reduction in Arctic sea ice extent, duration and thickness increase the risk of toxic blooms of the dinoflagellate Alexandrium catenella in the Alaskan Arctic. This algal species produces neurotoxins that impact marine wildlife health and cause the human illness known as paralytic shellfish poisoning (PSP). This study reports Paralytic Shellfish Toxin (PST) concentrations quantified in Arctic food web samples that include phytoplankton, zooplankton, benthic clams, benthic worms, and pelagic fish collected throughout summer 2019 during anomalously warm ocean conditions. PSTs (saxitoxin equivalents, STX eq.) were detected in all trophic levels with concentrations above the seafood safety regulatory limit (80 µg STX eq. 100 g-1) in benthic clams collected offshore on the continental shelf in the Beaufort, Chukchi, and Bering Seas. Most notably, toxic benthic clams (Macoma calcarea) were found north of Saint Lawrence Island where Pacific walruses (Odobenus rosmarus) are known to forage for a variety of benthic species, including Macoma. Additionally, fecal samples collected from 13 walruses harvested for subsistence purposes near Saint Lawrence Island during March to May 2019, all contained detectable levels of STX, with fecal samples from two animals (78 and 72 µg STX eq. 100 g-1) near the seafood safety regulatory limit. In contrast, 64% of fecal samples from zooplankton-feeding bowhead whales (n = 9) harvested between March and September 2019 in coastal waters of the Beaufort Sea near Utqiagvik (formerly Barrow) and Kaktovik were toxin-positive, and those levels were significantly lower than in walruses (max bowhead 8.5 µg STX eq. 100 g-1). This was consistent with the lower concentrations of PSTs found in regional zooplankton prey. Maximum ecologically-relevant daily toxin doses to walruses feeding on clams and bowhead whales feeding on zooplankton were estimated to be 21.5 and 0.7 µg STX eq. kg body weight-1 day-1, respectively, suggesting that walruses had higher PST exposures than bowhead whales. Average and maximum STX doses in walruses were in the range reported previously to cause illness and/or death in humans and humpback whales, while bowhead whale doses were well below those levels. These findings raise concerns regarding potential increases in PST/STX exposure risks and health impacts to Arctic marine mammals as ocean warming and sea ice reduction continue.

Double life: How GRK2 and ß-arrestin signaling participate in diseases.

G-protein coupled receptor (GPCR) kinases (GRKs) and ß-arrestins play key roles in GPCR and non-GPCR cellular responses. In fact, GRKs and arrestins are involved in a plethora of pathways vital for physiological maintenance of inter- and intracellular communication. Here we review decades of research literature spanning from the discovery, identification of key structural elements, and findings supporting the diverse roles of these proteins in GPCR-mediated pathways. We then describe how GRK2 and ß-arrestins partake in non-GPCR signaling and briefly summarize their involvement in various pathologies. We conclude by presenting gaps in knowledge and our prospective on the promising pharmacological potential in targeting these proteins and/or downstream signaling. Future research is warranted and paramount for untangling these novel and promising roles for GRK2 and arrestins in metabolism and disease progression.

GRK2 contributes to glucose mediated calcium responses and insulin secretion in pancreatic islet cells.

Diabetes is a metabolic syndrome rooted in impaired insulin and/or glucagon secretory responses within the pancreatic islets of Langerhans (islets). Insulin secretion is primarily regulated by two key factors: glucose-mediated ATP production and G-protein coupled receptors (GPCRs) signaling. GPCR kinase 2 (GRK2), a key regulator of GPCRs, is reported to be downregulated in the pancreas of spontaneously obesogenic and diabetogenic mice (ob/ob). Moreover, recent studies have shown that GRK2 non-canonically localizes to the cardiac mitochondrion, where it can contribute to glucose metabolism. Thus, islet GRK2 may impact insulin secretion through either mechanism. Utilizing Min6 cells, a pancreatic ß-cell model, we knocked down GRK2 and measured glucose-mediated intracellular calcium responses and insulin secretion. Silencing of GRK2 attenuated calcium responses, which were rescued by pertussis toxin pre-treatment, suggesting a Gαi/o-dependent mechanism. Pancreatic deletion of GRK2 in mice resulted in glucose intolerance with diminished insulin secretion. These differences were due to diminished insulin release rather than decreased insulin content or gross differences in islet architecture. Furthermore, a high fat diet feeding regimen exacerbated the metabolic phenotype in this model. These results suggest a new role for pancreatic islet GRK2 in glucose-mediated insulin responses that is relevant to type 2 diabetes disease progression.

Mitochondrial Membrane Intracellular Communication in Healthy and Diseased Myocardium.

Research efforts in the twenty-first century have been paramount to the discovery and development of novel pharmacological treatments in a variety of diseases resulting in improved life expectancy. Yet, cardiac disease remains a leading cause of morbidity and mortality worldwide. Over time, there has been an expansion in conditions such as atrial fibrillation (AF) and heart failure (HF). Although past research has elucidated specific pathways that participate in the development of distinct cardiac pathologies, the exact mechanisms of action leading to disease remain to be fully characterized. Protein turnover and cellular bioenergetics are integral components of cardiac diseases, highlighting the importance of mitochondria and endoplasmic reticulum (ER) in driving cellular homeostasis. More specifically, the interactions between mitochondria and ER are crucial to calcium signaling, apoptosis induction, autophagy, and lipid biosynthesis. Here, we summarize mitochondrial and ER functions and physical interactions in healthy physiological states. We then transition to perturbations that occur in response to pathophysiological challenges and how this alters mitochondrial-ER and other intracellular organelle interactions. Finally, we discuss lifestyle interventions and innovative therapeutic targets that may be used to restore beneficial mitochondrial and ER interactions, thereby improving cardiac function.

Recognition and Management of Colonic Perforation following Endoscopy.

Although rare, perforation can be a devastating complication of colonoscopy. Incidence ranges from 0.012 to 0.65% during diagnostic procedures and is higher in therapeutic procedures. Early diagnosis and management are of paramount importance to decrease morbidity. Diagnostic imaging after colonoscopy can reveal extraintestinal air, but overall clinical status including leukocytosis, fever, pain, and peritonitis is equally important to determine management. With the expanding availability of complex endoscopic interventions, an increasing number of perforations are recognized during colonoscopy or immediately afterward based on high degree of suspicion. Colonoscopic management of these early perforations may be feasible and avoid the morbidity of surgery. Patients who require surgery may be managed with laparoscopic or open surgical techniques. Surgical management may consist of primary repair of the injury, resection with anastomosis, or resection with ostomy. Mechanical bowel preparation before endoscopy decreases fecal contamination after perforation, often obviating the need for ostomy creation.

Enhanced Recovery after Colorectal Surgery: Can We Afford Not to Use It?

BACKGROUND: Enhanced recovery pathways (ERPs) aim to reduce length of stay without adversely affecting short-term outcomes. High pharmaceutical costs associated with ERP regimens, however, remain a significant barrier to widespread implementation. We hypothesized that ERP would reduce hospital costs after elective colorectal resections, despite the use of more expensive pharmaceutical agents. STUDY DESIGN: An ERP was implemented in January 2016 at our institution. We collected data on consecutive colorectal resections for 1 year before adoption of ERP (traditional, n = 160) and compared them with consecutive resections after universal adoption of ERP (n = 146). Short-term surgical outcomes, total direct costs, and direct hospital pharmacy costs were compared between patients who received the ERP and those who did not. RESULTS: After implementation of the ERP, median length of stay decreased from 5.0 to 3.0 days (p < 0.01). There were no differences in 30-day complications (8.1% vs 8.9%) or hospital readmission (11.9% vs 11.0%). The ERP patients required significantly less narcotics during their index hospitalization (211.7 vs 720.2 morphine equivalence units; p < 0.01) and tolerated a regular diet 1 day sooner (p < 0.01). Despite a higher daily pharmacy cost ($477 per day vs $318 per day in the traditional cohort), the total direct pharmacy cost for the hospitalization was reduced in ERP patients ($1,534 vs $1,859; p = 0.016). Total direct cost was also lower in ERP patients ($9,791 vs $11,508; p = 0.004). CONCLUSIONS: Implementation of an ERP for patients undergoing elective colorectal resection substantially reduced length of stay, total hospital cost, and direct pharmacy cost without increasing complications or readmission rates. Enhanced recovery pathway after colorectal resection has both clinical and financial benefits. Widespread implementation has the potential for a dramatic impact on healthcare costs.

Advancing Coordinated Cyber-investigations and Tool Interoperability using a Community Developed Specification Language.

Any investigation can have a digital dimension, often involving information from multiple data sources, organizations and jurisdictions. Existing approaches to representing and exchanging cyber-investigation information are inadequate, particularly when combining data sources from numerous organizations or dealing with large amounts of data from various tools. To perform digital investigations effectively, there is a pressing need to harmonize how information relevant to cyber-investigations is represented and exchanged. This paper addresses this need for information exchange and tool interoperability with an open community-developed specification language called Cyber-investigation Analysis Standard Expression (CASE). To further promote a common structure, CASE aligns with and extends the Unified Cyber Ontology (UCO) construct, which provides a format for representing information in all cyber domains. This ontology abstracts objects and concepts that are not CASE-specific, so that they can be used across other cyber disciplines that may extend UCO. This work is a rational evolution of the Digital Forensic Analysis eXpression (DFAX) for representing digital forensic information and provenance. CASE is more flexible than DFAX and can be utilized in any context, including criminal, corporate and intelligence. CASE also builds on the Hansken data model developed and implemented by the Netherlands Forensic Institute (NFI). CASE enables the fusion of information from different organizations, data sources, and forensic tools to foster more comprehensive and cohesive analysis. This paper includes illustrative examples of how CASE can be implemented and used to capture information in a structured form to advance sharing, interoperability and analysis in cyber-investigations. In addition to capturing technical details and relationships between objects, CASE provides structure for representing and sharing details about how cyber-information was handled, transferred, processed, analyzed, and interpreted. CASE also supports data marking for sharing information at different levels of trust and classification, as well as protection of sensitive and private information. Furthermore, CASE supports the sharing of knowledge related to cyber-investigations, including distinctive patterns of activity/behavior that are common across cases. This paper features a proof-of-concept implementation using the open source forensic framework named plaso to export data to CASE. Community members are encouraged to participate in the development and implementation of CASE and UCO.

Prevalence of algal toxins in Alaskan marine mammals foraging in a changing arctic and subarctic environment.

Current climate trends resulting in rapid declines in sea ice and increasing water temperatures are likely to expand the northern geographic range and duration of favorable conditions for harmful algal blooms (HABs), making algal toxins a growing concern in Alaskan marine food webs. Two of the most common HAB toxins along the west coast of North America are the neurotoxins domoic acid (DA) and saxitoxin (STX). Over the last 20 years, DA toxicosis has caused significant illness and mortality in marine mammals along the west coast of the USA, but has not been reported to impact marine mammals foraging in Alaskan waters. Saxitoxin, the most potent of the paralytic shellfish poisoning toxins, has been well-documented in shellfish in the Aleutians and Gulf of Alaska for decades and associated with human illnesses and deaths due to consumption of toxic clams. There is little information regarding exposure of Alaskan marine mammals. Here, the spatial patterns and prevalence of DA and STX exposure in Alaskan marine mammals are documented in order to assess health risks to northern populations including those species that are important to the nutritional, cultural, and economic well-being of Alaskan coastal communities. In this study, 905 marine mammals from 13 species were sampled including; humpback whales, bowhead whales, beluga whales, harbor porpoises, northern fur seals, Steller sea lions, harbor seals, ringed seals, bearded seals, spotted seals, ribbon seals, Pacific walruses, and northern sea otters. Domoic acid was detected in all 13 species examined and had the greatest prevalence in bowhead whales (68%) and harbor seals (67%). Saxitoxin was detected in 10 of the 13 species, with the highest prevalence in humpback whales (50%) and bowhead whales (32%). Pacific walruses contained the highest concentrations of both STX and DA, with DA concentrations similar to those detected in California sea lions exhibiting clinical signs of DA toxicosis (seizures) off the coast of Central California, USA. Forty-six individual marine mammals contained detectable concentrations of both toxins emphasizing the potential for combined exposure risks. Additionally, fetuses from a beluga whale, a harbor porpoise and a Steller sea lion contained detectable concentrations of DA documenting maternal toxin transfer in these species. These results provide evidence that HAB toxins are present throughout Alaska waters at levels high enough to be detected in marine mammals and have the potential to impact marine mammal health in the Arctic marine environment.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

Functional characterization of the alphavirus TF protein.

Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the -1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.

Probing the early temporal and spatial interaction of the Sindbis virus capsid and E2 proteins with reverse genetics.

A 7-Å cryoelectron microscopy-based reconstruction of Sindbis virus (SINV) was recently generated. Fitting the crystal structure of the SINV capsid protein (Cp) into the density map revealed that the F2-G2 loop of the Cp was shifted away from cytoplasmic domain of E2 (cdE2) in the 7-Å reconstruction relative to its position in the Cp crystal structure. Furthermore, the reconstruction demonstrated that residue E395 in region I of the cytoplasmic domain of the E2 envelope protein (cdE2-RI) and K252 of Cp, part of the Cp F2-G2 loop, formed a putative salt bridge in the virion. We generated amino acid substitutions at residues K250 and K252 of the SINV Cp and explored the resulting phenotypes. In the context of cells infected with wild-type or mutant virus, reversing the charge of these two residues resulted in the appearance of Cp aggregates around cytopathic vacuole type I (CPV-I) structures, the absence of nucleocapsid (NC) formation, and a lack of virus particle release in the infected mammalian cell. However, expressing the same Cp mutants in the cell without the envelope proteins or expressing and purifying the mutants from an Escherichia coli expression system and assembling in vitro yielded NC assembly in all cases. In addition, second-site mutations within cdE2 restored NC assembly but not release of infectious particles. Our data suggest an early temporal and spatial interaction between cdE2-RI and the Cp F2-G2 loop that, when ablated, leads to the absence of NC assembly. This interaction also appears to be important for budding of virus particles.

Foxm1 mediates cross talk between Kras/mitogen-activated protein kinase and canonical Wnt pathways during development of respiratory epithelium.

While Kras/mitogen-activated protein kinase (MAPK) and canonical Wnt/ß-catenin are critical for lung morphogenesis, mechanisms integrating these important signaling pathways during lung development are unknown. Herein, we demonstrate that the Foxm1 transcription factor is a key downstream target of activated Kras(G12D). Deletion of Foxm1 from respiratory epithelial cells during lung formation prevented structural abnormalities caused by activated Kras(G12D). Kras/Foxm1 signaling inhibited the activity of canonical Wnt signaling in the developing lung in vivo. Foxm1 decreased T-cell factor (TCF) transcriptional activity induced by activated ß-catenin in vitro. Depletion of Foxm1 by short interfering RNA (siRNA) increased nuclear localization of ß-catenin, increased expression of ß-catenin target genes, and decreased mRNA and protein levels of the ß-catenin inhibitor Axin2. Axin2 mRNA was reduced in distal lung epithelium of Foxm1-deficient mice. Foxm1 directly bound to and increased transcriptional activity of the Axin2 promoter region. Foxm1 is required for Kras signaling in distal lung epithelium and provides a mechanism integrating Kras and canonical Wnt/ß-catenin signaling during lung development.

Direct and indirect impact of influenza vaccination of young children on school absenteeism.

Special mass influenza vaccination programs of elementary school-aged children (ESAC) in some or all Maryland Counties were conducted during the falls of 2005-2007. From 3% to 46% of ESAC received live attenuated influenza vaccine during these county programs, which were in addition to routine influenza vaccination efforts conducted in county medical offices. Anonymous, all cause public school absentee data for all grades was available from 11 of Maryland's 24 counties. Binomial regression was used to estimate associations between the percentage of children vaccinated in each county and the degree of increase in absenteeism rates during influenza outbreaks. We estimated that, for every 20% increase in vaccination rates for ESAC during these special programs, a 4% decrease in the rise in absentee rates occurred during influenza outbreak periods in both elementary and upper schools (P<0.05). These results suggest both direct and indirect benefits of influenza vaccination of young children.

Operative decision-making for follicular thyroid lesions: a community hospital system experience.

Follicular neoplasms of the thyroid are a frequent indication for surgery of the thyroid gland. We evaluated the use of frozen sections on intraoperative decision-making, possible avoidance of reoperative surgery, and histologic findings in a retrospective cohort. A database was created of all thyroid operations from 2001 to 2007. Data collected included age, gender, preoperative cytology, indication for surgery, surgeon, intraoperative decision-making, and histologic findings. Of the 723 thyroidectomies, 203 were performed for follicular neoplasms diagnosed by fine needle aspiration. Of these, 135 had cytology reports available within our electronic medical record; 44 per cent (59 of 135) of these patients had an intraoperative frozen section. Only two of 59 (3.4%) were positive for carcinoma, both of which were papillary carcinomas. One was interpreted as "suspicious" for carcinoma by the pathologist. In these three cases, the surgeon proceeded with total thyroidectomy at the time of initial surgery. The results of frozen section altered the operation in only three of 59 cases (5.1%). Intraoperative frozen section rarely impacts the conduct of thyroidectomy for follicular neoplasms.

Rescue of infectious particles from preassembled alphavirus nucleocapsid cores.

Alphaviruses are small, spherical, enveloped, positive-sense, single-stranded, RNA viruses responsible for considerable human and animal disease. Using microinjection of preassembled cores as a tool, a system has been established to study the assembly and budding process of Sindbis virus, the type member of the alphaviruses. We demonstrate the release of infectious virus-like particles from cells expressing Sindbis virus envelope glycoproteins following microinjection of Sindbis virus nucleocapsids purified from the cytoplasm of infected cells. Furthermore, it is shown that nucleocapsids assembled in vitro mimic those isolated in the cytoplasm of infected cells with respect to their ability to be incorporated into enveloped virions following microinjection. This system allows for the study of the alphavirus budding process independent of an authentic infection and provides a platform to study viral and host requirements for budding.