Targeted sequencing for hereditary breast and ovarian cancer in BRCA1/2-negative families reveals complex genetic architecture and phenocopies.

Approximately 20% of breast cancer cases are attributed to increased family risk, yet variation in BRCA1/2 can only explain 20%-25% of cases. Historically, only single gene or single variant testing were common in at-risk family members, and further sequencing studies were rarely offered after negative results. In this study, we applied an efficient and inexpensive targeted sequencing approach to provide molecular diagnoses in 245 human samples representing 134 BRCA mutation-negative (BRCAX) hereditary breast and ovarian cancer (HBOC) families recruited from 1973 to 2019 by Dr. Henry Lynch. Sequencing identified 391 variants, which were functionally annotated and ranked based on their predicted clinical impact. Known pathogenic CHEK2 breast cancer variants were identified in five BRCAX families in this study. While BRCAX was an inclusion criterion for this study, we still identified a pathogenic BRCA2 variant (p.Met192ValfsTer13) in one family. A portion of BRCAX families could be explained by other hereditary cancer syndromes that increase HBOC risk: Li-Fraumeni syndrome (gene: TP53) and Lynch syndrome (gene: MSH6). Interestingly, many families carried additional variants of undetermined significance (VOUSs) that may further modify phenotypes of syndromic family members. Ten families carried more than one potential VOUS, suggesting the presence of complex multi-variant families. Overall, nine BRCAX HBOC families in our study may be explained by known likely pathogenic/pathogenic variants, and six families carried potential VOUSs, which require further functional testing. To address this, we developed a functional assay where we successfully re-classified one family's PMS2 VOUS as benign.

In vitro data suggest a role for PMS2 Kozak sequence mutations in Lynch syndrome risk.

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Heterozygous loss-of-function variants in PMS2 are linked to LS. While these variants are not directly cancer causing, reduced PMS2 function results in the accumulation of somatic variants and increased cancer risk over time due to DNA mismatch repair dysfunction. It is reasonable that other types of genetic variation that impact the expression of PMS2 may also contribute to cancer risk. The Kozak sequence is a highly conserved translation initiation motif among higher eukaryotes and is defined as the nine base pairs upstream of the translation start codon through the first four bases of the translated sequence (5'-[GTT]GCATCCATGG-3'; human PMS2: NM_000535.7). While Kozak sequence variants in PMS2 have been reported in ClinVar in patients with suspected hereditary cancer, all variants upstream of the translation start site are currently classified as variants of undetermined significance (VUSs). We hypothesized that variants significantly disrupting the Kozak sequence of PMS2 would decrease PMS2 protein expression, contributing to increased cancer risk over time. Using a dual-luciferase reporter plasmid and site-directed mutagenesis, we generated the wild-type human PMS2 and the ClinVar VUSs within the PMS2 Kozak sequence. Besides the c.1A>C variant, which is already known to be pathogenic, we implicate six additional variants as American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) pathogenic supporting (PP) variants and classify ten as benign supporting (BP). In summary, we present a method developed for the classification of human PMS2 Kozak sequence variants that can contribute to the re-classification of VUSs identified in patients.

Cryptosporidium uses CSpV1 to activate host type I interferon and attenuate antiparasitic defenses.

Cryptosporidium infects gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. There are no vaccines and no fully effective therapy available for the infection. Type II and III interferon (IFN) responses are important determinants of susceptibility to infection but the role for type I IFN response remains obscure. Cryptosporidium parvum virus 1 (CSpV1) is a double-stranded RNA (dsRNA) virus harbored by Cryptosporidium spp. Here we show that intestinal epithelial conditional Ifnar1-/- mice (deficient in type I IFN receptor) are resistant to C. parvum infection. CSpV1-dsRNAs are delivered into host cells and trigger type I IFN response in infected cells. Whereas C. parvum infection attenuates epithelial response to IFN-γ, loss of type I IFN signaling or inhibition of CSpV1-dsRNA delivery can restore IFN-γ-mediated protective response. Our findings demonstrate that type I IFN signaling in intestinal epithelial cells is detrimental to intestinal anti-C. parvum defense and Cryptosporidium uses CSpV1 to activate type I IFN signaling to evade epithelial antiparasitic response.

Targeted Immuno-Antiretroviral to Promote Dual Protection against HIV: A Proof-of-Concept Study.

The C-C motif chemokine receptor-5 (CCR5) expression on the T-cell surface is the prime barrier to HIV/AIDS eradication, as it promotes both active human immunodeficiency virus (HIV)-infection and latency; however, antiretrovirals (ARVs) suppress plasma viral loads to non-detectable levels. Keeping this in mind, we strategically designed a targeted ARVs-loaded nanoformulation that targets CCR5 expressing T-cells (e.g., CD4+ cells). Conceptually, CCR5-blocking and targeted ARV delivery would be a dual protection strategy to prevent HIV infection. For targeting CCR5+ T-cells, the nanoformulation was surface conjugated with anti-CCR5 monoclonal antibodies (CCR5 mAb) and loaded with dolutegravir+tenofovir alafenamide (D+T) ARVs to block HIV replication. The result demonstrated that the targeted-ARV nanoparticle's multimeric CCR5 binding property improved its antigen-binding affinity, prolonged receptor binding, and ARV intracellular retention. Further, nanoformulation demonstrated high binding affinity to CCR5 expressing CD4+ cells, monocytes, and other CCR5+ T-cells. Finally, the short-term pre-exposure prophylaxis study demonstrated that prolonged CCR5 blockage and ARV presence further induced a "protective immune phenotype" with a boosted T-helper (Th), temporary memory (TM), and effector (E) sub-population. The proof-of-concept study that the targeted-ARV nanoformulation dual-action mechanism could provide a multifactorial solution toward achieving HIV "functional cure."

Epidemiologic and Genomic Analysis of the Severe Acute Respiratory Syndrome Coronavirus 2 Epidemic in the Nebraska Region of the United States, March 2020-2021.

COVID-19 emerged at varying intervals in different regions of the United States in 2020. This report details the epidemiologic and genetic evolution of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the first year of the epidemic in the state of Nebraska using data collected from the Creighton Catholic Health Initiatives (CHI) health system. Statistical modelling identified age, gender, and previous history of diabetes and/or stroke as significant risk factors associated with mortality in COVID-19 patients. In parallel, the viral genomes of over 1,000 samples were sequenced. The overall rate of viral variation in the population was 0.07 mutations/day. Genetically, the first 9 months of the outbreak, which include the initial outbreak, a small surge in August and a major outbreak in November 2020 were primarily characterized by B.1. lineage viruses. In early 2021, the United Kingdom variant (B.1.1.7 or alpha) quickly became the dominant variant. Notably, surveillance of non-consensus variants detected B.1.1.7 defining mutations months earlier in Fall 2020. This work provides insights into the regional variance and evolution of SARS-CoV-2 in the Nebraska region during the first year of the pandemic.

Characterization of Ag-Ion Releasing Zeolite Filled 3D Printed Resins.

There has been profound growth in the use of 3D printed materials in dentistry in general, including orthodontics. The opportunity to impart antimicrobial properties to 3D printed parts from existing resins requires the capability of forming a stable colloid incorporating antimicrobial fillers. The objective of this research was to characterize a colloid consisting of a 3D printable resin mixed with Ag-ion releasing zeolites and fumed silica to create 3D printed parts with antiviral properties. The final composite was tested for antiviral properties against SARS-CoV-2 and HIV-1. Antiviral activity was measured in terms of the half-life of SARS-CoV-2 and HIV-1 on the composite surface. The inclusion of the zeolite did not interfere with the kinetics measured on the surface of the ATR crystal. While the depth of cure, measured following ISO4049 guidelines, was reduced from 3.8 mm to 1.4 mm in 5 s, this greatly exceeded the resolution required for 3D printing. The colloid was stable for at least 6 months and the rheological behavior was dependent upon the fumed silica loading. The inclusion of zeolites and fumed silica significantly increased the flexural strength of the composite as measured by a 3 point bend test. The composite released approximately 2500 µg/L of silver ion per gram of composite as determined by potentiometry. There was a significant reduction of the average half-life of SARS-CoV-2 (1.9 fold) and HIV-1 (2.7 fold) on the surface of the composite. The inclusion of Ag-ion releasing zeolites into 3D-printable resin can result in stable colloids that generate composites with improved mechanical properties and antiviral properties.

SWATH-MS and MRM: Quantification of Ras-related proteins in HIV-1 infected and methamphetamine-exposed human monocyte-derived macrophages (hMDM).

HIV-1 infection of macrophages is a multistep and multifactorial process that has been shown to be enhanced by exposure to methamphetamine (Meth). In this study, we sought to identify the underlying mechanisms of this effect by quantifying the effect of Meth on the proteome of HIV-1-infected macrophages using sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) approach. The analyses identified several members of the Rab family of proteins as being dysregulated by Meth treatment, which was confirmed by bioinformatic analyses that indicated substantial alteration of vesicular transport pathways. Validation of the SWATH-MS was performed using an MRM based approach, which confirmed that Meth exposure affects expression of the Rab proteins. However, the pattern of expression changes were highly dynamic, and displayed high donor-to-donor variability. Surprisingly a similar phenomenon was observed for Actin. Our results demonstrate that Meth affects vesicular transport pathways, suggesting a possible molecular mechanism underlying its effect on HIV infection hMDM and a potential broader effect of Meth on cellular homeostasis.

Presence of antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 in COVID-19 plasma.

BACKGROUND: Neutralizing-antibody (nAb) is the major focus of most ongoing COVID-19 vaccine trials. However, nAb response against SARS-CoV-2, when present, decays rapidly. Given the myriad roles of antibodies in immune responses, it is possible that antibodies could also mediate protection against SARS-CoV-2 via effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), which we sought to explore here. METHODS: Plasma of 3 uninfected controls and 20 subjects exposed to, or recovering from, SARS-CoV-2 infection were collected from U.S. and sub-Saharan Africa. Immunofluorescence assay was used to detect the presence of SARS-CoV-2 specific IgG antibodies in the plasma samples. SARS-CoV-2 specific neutralizing capability of these plasmas was assessed with SARS-CoV-2 spike pseudotyped virus. ADCC activity was assessed with a calcein release assay. RESULTS: SARS-CoV-2 specific IgG antibodies were detected in all COVID-19 subjects studied. All but three COVID-19 subjects contained nAb at high potency (>80% neutralization). Plasma from 19/20 of COVID-19 subjects also demonstrated strong ADCC activity against SARS-CoV-2 spike glycoprotein, including two individuals without nAb against SARS-CoV-2. CONCLUSION: Both neutralizing and non-neutralizing COVID-19 plasmas can mediate ADCC. Our findings argue that evaluation of potential vaccines against SARS-CoV-2 should include investigation of the magnitude and durability of ADCC, in addition to nAb.

Discovery of candidate HIV-1 latency biomarkers using an OMICs approach.

Infection with HIV-1 remains uncurable due to reservoirs of latently infected cells. Any potential cure for HIV will require a mechanism to identify and target these cells in vivo. We created a panel of Jurkat cell lines latently infected with the HIV DuoFlo virus to identify candidate biomarkers of latency. SWATH mass spectrometry was used to compare the membrane proteomes of one of the cell lines to parental Jurkat cells. Several candidate proteins with significantly altered expression were identified. The differential expression of several candidates was validated in multiple latently infected cell lines. Three factors (LAG-3, CD147,CD231) were altered across numerous cell lines, but the expression of most candidate biomarkers was variable. These results confirm that phenotypic differences in latently infected cells exists and identify additional novel biomarkers. The variable expression of biomarkers across different cell clones suggests universal antigen-based detection of latently infected cells may require a multiplex approach.

CD4+ T cell activation and associated susceptibility to HIV-1 infection in vitro increased following acute resistance exercise in human subjects.

Early studies in exercise immunology suggested acute bouts of exercise had an immunosuppressive effect in human subjects. However, recent data, show acute bouts of combined aerobic and resistance training increase both lymphocyte activation and proliferation. We quantified resistance exercise-induced changes in the activation state of CD4+ T lymphocytes via surface protein expression and using a medically relevant model of infection (HIV-1). Using a randomized cross-over design, 10 untrained subjects completed a control and exercise session. The control session consisted of 30-min seated rest while the exercise session entailed 3 sets × 10 repetitions of back squat, leg press, and leg extensions at 70% 1-RM with 2-min rest between each set. Venous blood samples were obtained pre/post each session. CD4+ T lymphocytes were isolated from whole blood by negative selection. Expression of activation markers (CD69 & CD25) in both nonstimulated and stimulated (costimulation through CD3+ CD28) cells were assessed by flow cytometry. Resistance exercised-induced effects on intracellular activation was further evaluated via in vitro infection with HIV-1. Nonstimulated CD4+ T lymphocytes obtained postexercise exhibited elevated CD25 expression following 24 h in culture. Enhanced HIV-1 replication was observed in cells obtained postexercise. Our results demonstrate that an acute bout of resistance exercise increases the activation state of CD4+ T lymphocytes and results in a greater susceptibility to HIV-1 infection in vitro. These findings offer further evidence that exercise induces activation of T lymphocytes and provides a foundation for the use of medically relevant pathogens as indirect measures of intracellular activation.

A potential long-acting bictegravir loaded nano-drug delivery system for HIV-1 infection: A proof-of-concept study.

Bictegravir (BIC), a newly FDA-approved integrase strand transfer inhibitor (INSTI), as a single tablet regimen has proven efficacious in treating HIV-1 and SIV viruses, with reduced resistance. BIC clinical trials have not investigated its prophylaxis potency. This study investigates the HIV prevention potency of a novel long-acting BIC nano-formulation aimed to improve adherence. Poly (lactic-co-glycolic acid) loaded BIC nanoparticles (BIC NPs) were formulated using an oil-in-water emulsion methodology. BIC NPs were <200 nm in size, with 47.9 ±â€¯6.9% encapsulation efficiency. A novel, sensitive and high throughput LC-MS/MS method was used to estimate intracellular pharmacokinetics (PK) of BIC NPs and compared to BIC solution demonstrated prolonged intracellular BIC retention. BIC NPs safety was assessed based on cytotoxicity. Further, in-vitro prevention study of BIC NPs vs BIC solution was assessed against HIV-1NLX and HIV-1ADA on TZM-bl cell line and PBMCs, respectively. BIC nanoencapsulation demonstrated elevated cellular cytotoxicity concentration (CC50: 2.25 µM (BIC solution) to 820.4 µM (BIC NPs)] and lowers HIV-1 inhibitory concentration [EC50: 0.604 µM (BIC solution) to 0.0038 µM (BIC NPs)) thereby improving selectivity index (SI) from 3.7 (BIC solution) to 215,789 (BIC NP) for TZM-bl cells. Comparable results in PBMCs were obtained where BIC NPs improved SI from 0.29 (BIC solution) to 523.33 (BIC NPs). This demonstrates long-acting BIC nano-formulation with sustained drug-release potency, improved BIC cytotoxicity and enhanced HIV-1 protection compared to BIC in solution.

Proteomic profiling of HIV-infected T-cells by SWATH mass spectrometry.

Viral pathogenesis results from changes in host cells due to virus usurpation of the host cell and the innate cellular responses to thwart infection. We measured global changes in protein expression and localization in HIV-1 infected T-cells using subcellular fractionation and the Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS) proteomic platform. Eight biological replicates were performed in two independent experimental series. In silico merging of both experiments identified 287 proteins with altered expression (p < .05) between control and infected cells- 172 in the cytoplasm, 84 in the membrane, and 31 in nuclei. 170 of the proteins are components of the NIH HIV interaction database. Multiple Reaction Monitoring and traditional immunoblotting validated the altered expression of several factors during infection. Numerous factors were found to affect HIV infection in gain- and loss-of-expression infection assays, including the intermediate filament vimentin which was found to be required for efficient infection.

An Enhanced Emtricitabine-Loaded Long-Acting Nanoformulation for Prevention or Treatment of HIV Infection.

Among various FDA-approved combination antiretroviral drugs (cARVs), emtricitabine (FTC) has been a very effective nucleoside reverse transcriptase inhibitor. Thus far, FTC is the only deoxycytidine nucleoside analog. However, a major drawback of FTC is its large volume distribution (averaging 1.4 liters/kg) and short plasma half-life (8 to 10 h), necessitating a high daily dosage. Thus, we propose an innovative fabrication method of loading FTC in poly(lactic-co-glycolic acid) polymeric nanoparticles (FTC-NPs), potentially overcoming these drawbacks. Our nanoformulation demonstrated enhanced FTC loading (size of <200 nm and surface charge of -23 mV) and no to low cytotoxicity with improved biocompatibility compared to those with FTC solution. An ex vivo endosomal release assay illustrated that NP entrapment prolongs FTC release over a month. Intracellular retention studies demonstrate sustained FTC retention over time, with approximately 8% (24 h) to 68% (96 h) release with a mean retention of ∼0.74 µg of FTC/105 cells after 4 days. An in vitro HIV-1 inhibition study demonstrated that FTC-NP treatment results in a 50% inhibitory concentration (IC50) ∼43 times lower in TZM-bl cells (0.00043 µg/ml) and ∼3.7 times lower (0.009 µg/ml) in peripheral blood mononuclear cells (PBMCs) than with FTC solution (TZM-bl cells, 0.01861, and PBMCs, 0.033 µg/ml). Further, on primary PBMCs, FTC-NPs also illustrate an HIV-1 infection blocking efficacy comparable to that of FTC solution. All the above-described studies substantiate that FTC nanoformulation prolongs intracellular FTC concentration and inhibition of HIV infection. Therefore, FTC-NPs potentially could be a long-acting, stable formulation to ensure once-biweekly dosing to prevent or treat HIV infection.

NF45 and NF90 Bind HIV-1 RNA and Modulate HIV Gene Expression.

A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1) replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies indicate that both NF45 and NF90 potentiate HIV infection through their RNA binding domains.

Cyclophilin B enhances HIV-1 infection.

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import.

Investigation of the HIV-1 matrix interactome during virus replication.

PURPOSE: Like all viruses, human immunodeficiency virus type 1 (HIV-1) requires host cellular factors for productive replication. Identification of these factors may lead to the development of novel cell-based inhibitors. EXPERIMENTAL DESIGN: A Strep-tag was inserted into the C-terminus of the matrix (MA) region of the HIV-1 gag gene. The resultant virus was replication competent and used to infect Jurkat T-cells. MA complexes were affinity purified with Strep-Tactin agarose. Protein quantification was performed using sequential window acquisition of all theoretical fragment ion spectra (SWATH) MS, data were log2 -transformed, and Student t-tests with Bonferroni correction used to determine statistical significance. Several candidate proteins were validated by immunoblot and investigated for their role in virus infection by siRNA knockdown assays. RESULTS: A total of 17 proteins were found to be statistically different between the infected versus uninfected and untagged control samples. X-ray repair cross-complementing protein 6 (Ku70), X-ray repair cross-complementing protein 5 (Ku80), and Y-box binding protein 1 (YB-1) were confirmed to interact with MA by immunoblot. Knockdown of two candidates, EZRIN and Y-box binding protein 1, enhanced HIV infection in vitro. CONCLUSIONS AND CLINICAL RELEVANCE: The Strep-tag allowed for the capture of viral protein complexes in the context of virus replication. Several previously described factors were identified and at least two candidate proteins were found to play a role in HIV-1 infection. These data further increase our understanding of HIV host -cell interactions.

Nanoformulations of Rilpivirine for Topical Pericoital and Systemic Coitus-Independent Administration Efficiently Prevent HIV Transmission.

Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection.

Thermosensitive Gel Containing Cellulose Acetate Phthalate-Efavirenz Combination Nanoparticles for Prevention of HIV-1 Infection.

The objective of this investigation was to develop and evaluate a nano-microbicide containing a combination of cellulose acetate phthalate (HIV-1 entry inhibitor) and efavirenz (anti-HIV agent) for HIV prophylaxis. Cellulose acetate phthalate-efavirenz combination nanoparticles (CAP-EFV-NPs) were fabricated by the nanoprecipitation method and were characterized for particle size, zeta potential and encapsulation efficiency of efavirenz. CAP-EFV-NPs were incorporated into a thermosensitive gel (CAP-EFV-NP-Gel). CAP-EFV-NPs, CAP-EFV-NP-Gel and efavirenz solution were evaluated for cytotoxicity to HeLa cells and for in vitro short-term (1-day) and long-term (3-day) prophylaxis against HIV-1 infection in TZM-bl cells. CAP-EFV-NPs had size < 100 nm, negative surface charge and encapsulation efficiency of efavirenz was > 98%. CAP-EFV-NPs and CAP-EFV-NP-Gel were significantly less toxic (P < 0.01) to HeLa cells as compared to efavirenz solution. CAP-EFV-NPs showed significantly higher prophylactic activity (P < 0.01) against HIV-1 infection to TZM-bl cells as compared to efavirenz solution and blank CAP nanoparticles. CAP-EFV-NP-Gel can be a promising nano-microbicide for long-term HIV prophylaxis.

Deletions in the fifth alpha helix of HIV-1 matrix block virus release.

The matrix (MA) protein of HIV-1 is the N-terminal component of the Gag structural protein and is critical for the early and late stages of viral replication. MA contains five α-helices (α1-α5). Deletions in the N-terminus of α5 as small as three amino acids impaired virus release. Electron microscopy of one deletion mutant (MA∆96-120) showed that its particles were tethered to the surface of cells by membranous stalks. Immunoblots indicated all mutants were processed completely, but mutants with large deletions had alternative processing intermediates. Consistent with the EM data, MA∆96-120 retained membrane association and multimerization capability. Co-expression of this mutant inhibited wild type particle release. Alanine scanning mutation in this region did not affect virus release, although the progeny virions were poorly infectious. Combined, these data demonstrate that structural ablation of the α5 of MA inhibits virus release.