First Metatarsophalangeal Arthrodesis for Hallux Rigidus and Hallux Valgus: A Comparison of Patient Reported and Clinical Outcomes.

Few prior studies have compared the patient reported outcomes of first metatarsophalangeal arthrodesis between hallux rigidus and hallux valgus patients. Furthermore, we sought to examine the impact of postoperative radiographic hallux alignment on outcomes scores within each group. A retrospective review of 98 patients who a received primary metatarsophalangeal arthrodesis from January 2010 to March 2020. Clinical complications including nonunion were collected. Patient Reported Outcomes Measurement Information Systems (PROMIS) Physical Function, PROMIS Pain Interference, and the foot function index (FFI) revised short form scores were obtained via telephone. Patients were grouped based on review of preoperative radiographs of the foot and this grouping 37 hallux rigidus and 61 hallux valgus patients. Clinical and patient reported outcomes were compared between these pathologies. No differences in the rate of wound complications, radiographic union, and revision surgery were found between the 2 subgroups. At a median of 2.4 years (3.9 IQR) postoperatively, PROMIS and FFI scores did not vary by pathology group. For both groups, PROMIS scores were similar to the general population of the United States. The postoperative first MTP dorsiflexion angle in the hallux rigidus group was correlated with decreased FFI Pain, FFI Total, and PROMIS Pain Interference domain scores (|r| ≥ 0.40, p < .05 for all). When performing MTP arthrodesis in patients with hallux rigidus, increasing the first MTP dorsiflexion angle may correlate with improved intermediate term patient reported outcomes. However, further studies will need to be done to confirm this theoretical relationship.

Achilles tendon disorders: An overview of diagnosis and conservative treatment.

ABSTRACT: Achilles tendon-related pain affects up to 6% of the US population during their lifetime and is commonly encountered by primary care providers. An accurate diagnosis and early conservative management can improve patient quality of life and reduce unnecessary surgical consultations, saving healthcare dollars. Achilles tendon pathologies can be categorized into acute (pain lasting less than 6 weeks), chronic (pain lasting more than 6 weeks), and acute on chronic (worsening of pain with preexisting chronic Achilles tendon pathology). This article describes the diagnosis, conservative management, indications for imaging, and indications for surgical referral for acute and chronic Achilles tendon rupture, Achilles tendinitis, gastrocnemius strain, plantaris rupture, insertional Achilles tendinopathy, Haglund deformity, and noninsertional Achilles tendinopathy.

Which surgical approach is optimal for joint preparation in talonavicular fusion - A cadaver study.

BACKGROUND: Fusion of the talonavicular joint has proven challenging in literature. The optimal surgical approach for talonavicular arthrodesis is still uncertain. This study compares the amount of physical joint preparation between dorsal and medial approaches to the talonavicular joint. METHODS: Twenty fresh frozen cadaver specimens were randomly assigned to receive either a dorsal or medial operative approach to the talonavicular joint. The joint surface was prepared, and the joint was disarticulated. Image analysis, using ImageJ, was performed by two blinded reviewers to assess the joint surface preparation and this was compared by surgical approach. RESULTS: The dorsal approach had a higher median percentage of talar and total talonavicular joint surface area prepared (75% vs. 59% (p = .007) and 82% vs. 70% (p = .005)). Irrespective of approach, the talus was significantly more difficult to prepare than the navicular (62% vs 88% (p = .001)). CONCLUSION: The dorsal approach provides superior talonavicular joint preparation. The lateral »th of the talar head was the most difficult surface to prepare, and surgeons performing double or triple arthrodesis may prepare the lateral talar head from the lateral approach. LEVEL OF EVIDENCE: Level V.

A Novel Plating System for First Metatarsophalangeal Joint Arthrodesis: A Retrospective Comparison of Hybrid and Traditional Locking Plate Constructs.

BACKGROUND: Dorsal plate fixation is commonly used for first metatarsophalangeal joint (1st MTPJ) arthrodesis and plate design continues to evolve. A new staple compression plate (SCP) design attempts to utilize the continuous compression of a nitinol staple across the fusion site while simultaneously providing the stability of a dorsal locked plate. Herein, we compare the radiographic, clinical, and patient-reported outcomes of 1st MTPJ joint arthrodesis using 2 dorsal locking plate constructs including a novel SCP construct. METHODS: Forty-four patients who underwent 1st MTPJ arthrodesis between 2016 and 2020 were retrospectively evaluated. There were 2 group cohorts. Group 1 cohort included 23 patients who received a CrossRoads Extremity SCP, and Group 2 cohort included 21 patients who received a Stryker dorsal locking precontoured titanium plate (LPP). All patients were evaluated with radiographs, Patient-Reported Outcomes Measures Information System (PROMIS) outcome scores, and Foot Function Index (FFI). RESULTS: The complication and union rates did not vary between groups with a fusion rate of 95.7% in the SCP group and 90.5 % in the LPP group. Similarly, we found no significant differences in PROMIS or FFI scores between the SCP and LPP plates. CONCLUSION: Use of either dorsal locking plate construct for 1st MTPJ arthrodesis was associated with high union rates and comparable functional outcomes. As locked plate technology continues to evolve for 1st MTPJ arthrodesis, it is important that clinical outcomes are reported. LEVELS OF EVIDENCE: Level IV.

Prevalence and Risk Factors of Postoperative Falls Following Foot and Ankle Surgery.

BACKGROUND: No study has examined the incidence of risk factors for postoperative falls following foot and ankle surgery. We investigated the incidence and risk factors for postoperative falls in foot and ankle surgery using inpatient and outpatient population. METHODS: A single fellowship-trained foot and ankle surgeon instituted collection of a postoperative fall questionnaire at 2 and 6 weeks postoperatively. A retrospective review of 135 patients with complete prospectively collected fall questionnaire data was performed. Patient demographic information, injury characteristics, comorbidities, baseline medications, length of hospital stay, visual analog scale (VAS) pain scores were collected. After univariable analysis, a multivariable binary logistic regression was conducted to assess independent risk factors for postoperative falls. RESULTS: The median (interquartile range) age was 52 (21) and body mass index was 32.7 (11.1). A total of 108 patients (80%) underwent outpatient procedures. Thirty-nine of the 135 patients (28.9%) reported experiencing a fall in the first 6 weeks after surgery. In multivariable analysis, antidepressant use (adjusted odds ratio 3.41, 95% CI 1.19-9.81) and higher VAS pain scores at 2 weeks postoperatively (adjusted odds ratio 1.27, 95% CI 1.08-1.50) were found to be independent risk factors for postoperative falls. CONCLUSION: This study found a high incidence of postoperative falls in the first 6 weeks after foot and ankle surgery. Baseline antidepressant use and higher 2-week VAS pain scores were associated with postoperative falls. Foot and ankle surgeons should discuss the risk of falling with patients especially those with risk factors. LEVEL OF EVIDENCE: Level III, retrospective cohort study at a single institution.

Suture Anchor-Enhanced Medial Capsulorrhaphy of the Great Toe.

The suture anchor-enhanced medial capsulorrhaphy of the great toe is utilized as an adjuvant procedure to proximal and distal osteotomies for the treatment of hallux valgus. In traditional open techniques, hallux valgus repair requires both osseous correction along with shortening of the capsule on the medial side of the metatarsophalangeal joint. Osseous correction typically corrects the intermetatarsal angle, whereas capsular correction maintains the hallux valgus angle1. Description: A standard medial approach to the 1st metatarsophalangeal joint is performed. A medial midline horizontal capsulotomy is performed starting just proximal to the medial eminence and extending distally to the base of the proximal phalanx. Once the concomitant osseous and soft-tissue procedures are completed, a vertical capsulotomy is made in the inferior capsular flap at the level of the metatarsophalangeal joint in a manner perpendicular to the first ray in order to form an L shape. A 3 to 4-mm wedge of capsule is formed near the base of the vertical limb, running obliquely to the horizontal limb, and is excised. Optionally, the free limbs of the inferior capsule are imbricated. A unicortical hole is then drilled in the first metatarsal head, and a 2.7-mm outer diameter by 7-mm deep suture anchor with 2-0 FiberWire (Arthrex) is placed. The free ends of the suture are then utilized to close the horizontal capsulotomy in a running-locking interrupted fashion. Fluoroscopic imaging is performed throughout the procedure to prevent overcorrection and varus malignment. Alternatives: Alternative treatments include L-shaped capsulorrhaphy without suture anchor augmentation, dorsolinear capsulorrhaphy, Y-shaped capsulorrhaphy, and proximal hallux osteotomy or distal hallux osteotomy without capsulorrhaphy. Rationale: Anchor-enhanced capsulorrhaphy has been proven to assist in early maintenance of hallux valgus angle correction when combined with relevant distal osteotomy techniques. The anchor-enhanced capsulorrhaphy has an advantage over traditional capsulorrhaphy methods because it allows enhanced tightening of the capsule to the bone and, therefore, the potential for enhanced short-term maintenance. Additionally, the use of a running-locking interrupted suture technique reduces the number of suture knots required for capsular closure, potentially reducing the chance of complications such as suture granuloma formation. This technique is useful in all patients with hallux valgus deformity because it helps to provide durable deformity correction through additional modification of the soft tissues surrounding the 1st metatarsophalangeal joint. Expected Outcomes: Medial capsulorrhaphy has been shown to help with short-term reduction of the hallux valgus angle, both with and without the use of suture anchors1-3. Gould et al. demonstrated the superiority of adding suture anchors to the L-shaped medial capsulorrhaphy in order to aid in prevention of early postoperative relapse of the valgus deformity in patients undergoing chevron or modified McBride osteotomy1. We have utilized this suture anchor-enhanced capsulorrhaphy technique as an adjuvant procedure in most patients receiving osteotomies or Lapidus procedures for hallux valgus correction with consistent, reproducible results. In our experience, the suture anchor-enhanced medial capsulorrhaphy is an effective and time-efficient adjunctive soft-tissue corrective procedure in hallux valgus patients. Important Tips: Always excise a small capsular wedge to start with.Throughout the capsular tightening process, utilize clinical judgment and fluoroscopy to avoid pulling the hallux into varus malalignment.If varus is noted during plication of the plantar capsule, simply undo the tightening stitch.Because the majority of capsular tightening occurs at the first distal knot during the running horizontal capsular closure, if varus is noted, untie the knot and proceed with less correction.The extra cost of the suture anchor is a drawback but should be weighed against the enhanced durability of capsular correction compared with a traditional capsulorrhaphy.Always check the position of the suture anchor under fluoroscopy before proceeding with capsular closure in order to ensure proper deployment and adequate osseous purchase.Suture anchor failure can cause misleading radiographic presentation or joint impingement. Acronyms and Abbreviations: VAS = Visual analog scaleAOFAS = American Orthopaedic Foot & Ankle SocietyHV = Hallux valgusHVA = Hallux valgus angleMTP = Metatarsophalangeal jointDVT = Deep venous thrombosis.

First Metatarsophalangeal Joint Arthrodesis: A Narrative Review of Fixation Constructs and Their Evolution.

First metatarsophalangeal (MTP) joint arthrodesis is a surgical procedure in which the first metatarsal head is fused to the proximal phalanx of the great toe in order to permanently stiffen the first MTP joint. It was originally proposed as a treatment for severe cases of hallux valgus deformity, but the procedure's indications and utilization have expanded since its initial development. Despite a wide variety of indications, first MTP arthrodesis has been shown to have reliable, satisfactory outcomes. As a result, the development of a wide array of surgical approaches, joint preparation techniques, and fixation devices used in the procedure has occurred. In this narrative review, we highlight the evolution of fixation constructs used in first MTP arthrodesis in order to provide a frame of reference for the various types of fixation constructs available.

Management of acute lesser toe pain.

Patients with foot pain commonly present to their primary care physicians for their initial management and treatment. These patients and their respective foot or lesser toe pain can present the physician with a complex problem with a long differential list. Depending on the timing of the pain and underlying pathology, these differentials can be divided into acute and acute exacerbation of chronic conditions. This review categorizes the history, physical exam, radiological findings, conservative treatment, and surgical management for each major cause of lesser toe pain, whether acute or chronic. The acute conditions surrounding lesser toe pain in the adult population discussed are toe fractures, toe dislocations, and metatarsal head and neck fractures. The chronic pathologies surrounding lesser toe pain in the adult population evaluated in this review include metatarsalgia, Morton's neuroma, Freiberg infraction, brachymetatarsia, bunionettes, and lesser toe disorders.

Impact of Patient Resilience on Outcomes of Open Brostrom-Gould Lateral Ligament Repair.

INTRODUCTION: Little is known about the factors affecting the intermediate outcomes of the Brostrom-Gould repair as measured by new patient-reported outcome instruments and the impact of patient resilience on postoperative outcomes. This is the first study to investigate the impact of resilience on the outcomes of lateral ligament repair. METHODS: Retrospectively, 173 patients undergoing Brostrom-Gould at single institution from January 2013 to June 2020 were identified. Patient characteristics, participation in athletic activities, surgical variables, and complications were recorded. Patient-Reported Outcome Measurement Information System (PROMIS) Pain Interference v1.1 (PI), Physical Function v1.2 (PF), and the Foot Ankle Ability Measure (FAAM) were collected. The Brief Resilience Scale was used to quantify resilience. A linear regression model was constructed to evaluate the independent effect of resilience on each PROMIS and FAAM outcome instrument. Variables were included in the regression model based on an a priori significance threshold of P <0.05 in bivariate analysis. RESULTS: Resilience's independent effect on outcome measures was as follows: PROMIS PF (unstandardized ß 8.2, 95% confidence interval [CI] 3.9 to 12.6), PROMIS PI (unstandardized ß -4.8, 95% CI -7.9 to -1.7), FAAM Activities of Daily Living (unstandardized ß 16.6, 95% CI 8.7 to 24.6), and FAAM Sports (unstandardized ß 28.4, 95% CI 15.9 to 40.9). Preoperative participation in athletic activities also had a positive independent effect on multiple outcome metrics including PROMIS PF (unstandardized ß 9.4, 95% CI 2.8 to 16.0), PROMIS PI (unstandardized ß -5.3, 95% CI -10.0 to -0.582), and FAAM Sport scores (unstandardized ß 34.4, 95% CI 15.4 to 53.4). CONCLUSIONS: Resilience and patient participation in athletic activities are independent predictors of improved postoperative functional outcomes as measured by PROMIS and FAAM instruments at intermediate term follow-up. Resilient patients and athletes reported markedly higher PF and less pain burden postoperatively. Preoperative quantification of resilience could enable improved prognostication of patients undergoing lateral ligament repair of the ankle.

Diagnosis and conservative management of great toe pathologies: a review.

Acute great toe (Hallux) pain is a common complaint encountered by the primary care physician. Pathological conditions can vary from acute trauma to acute exacerbation of underlying chronic conditions. Delay in treatment or misdiagnosis can lead to debilitating loss of function and long-lasting pain. This review endeavors to discuss the pertinent history, physical exam findings, radiographic evidence, conservative treatment options, and surgical management for the musculoskeletal causes of acute and acute on chronic great toe pain in the adult population. The acute pathologies discussed in this review are hallux fractures and dislocations, turf toe, sand toe, and sesamoid disorders. The chronic pathologies discussed include hallux rigidus, hallux valgus, and chronic sesamoiditis.

Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein.

Murine leukemia viruses (MuLV) have been adapted for use as gene transfer vectors for experimental and human gene therapy applications. Their utility for these purposes has been circumscribed by the limited host range and relatively low titer of available producer clones. Pseudotyping of MuLV particles with the vesicular stomatitis virus envelope protein (VSV-G), expressed transiently in cells producing MuLV Gag and Pol proteins, has yielded vector preparations with a broader host range that can be concentrated by ultracentrifugation. We have explored the use of steroid-inducible and tetracycline-modulated promoter systems (necessary because the VSV-G protein is toxic to cells when constitutively expressed) to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MuLV particles. A packaging cell line and producer clones capable of expressing a chimeric transcription factor, composed of the tetracycline repressor (tetR) and the VP16 trans-activating sequences of herpes simplex virus VP16 gene and containing the VSV-G coding sequences linked to a minimal promoter having seven tandem copies of the tetracycline responsive operator (tetO), exhibited high levels of VSV-G protein expression when cultured in the absence of tetracycline. Vector particles, produced at titers of 10(5)-10(6) infectious colony forming units per ml (cfu/ml), could be concentrated effectively by ultracentrifugation yielding vector preparations having a titer of 10(9) cfu/ml. These cell lines grew normally when VSV-G protein expression was repressed by tetracycline. Such producer clones hold promise for future human gene therapy applications.

A new cationic liposome reagent mediating nearly quantitative transfection of animal cells.

One of the most efficient systems for the expression of genes in the cytoplasm of animal cells utilizes a recombinant vaccinia virus encoding the bacteriophage T7 RNA polymerase. Cells infected with this virus are transfected with plasmid DNAs containing the gene to be expressed under T7 promoter control. The major limitation of this system is the efficiency with which DNA is introduced into the cell. Recently, a cationic liposome-mediated transfection reagent has yielded transfection frequencies of greater than 80%. To determine if commercially available cationic lipids could form liposomes that would yield similar transfection efficiencies, we tested liposomes prepared with five different cationic lipids. When used at appropriate concentrations in liposomes that also contained a neutral lipid, four of the five cationic lipids were effective in the transfection of HeLa cells. However, liposomes formed with the neutral lipid and one of the cationic lipids, dimethyldioctadecylammonium bromide (DDAB), gave transfection frequencies of greater than 95% and had a broad spectrum of effectiveness on a variety of cell lines. Liposomes containing DDAB are an inexpensive, highly efficient and reproducible alternative for the transfection of animal cells and are well suited for use with the vaccinia virus/T7 expression system.

Induction of hepatic aromatase by p,p'-DDE in adult male rats.

1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE, DDE) is a stable metabolite of the pesticide DDT and a persistent environmental pollutant. Earlier reports have demonstrated that DDE is an endocrine-active compound capable of affecting early-stage sexual differentiation in male rats. Experiments based on receptor binding affinity and receptor-mediated transcriptional activation have identified DDE as an androgen receptor antagonist. Other effects of DDE include modulation of the expression and activity of cytochrome P450 (CYP) enzymes, some of which function as steroid hydroxylases, and elevation of serum estrogen levels in treated male rats. These effects suggest the possibility of DDE-caused induction of aromatase, a member of CYP family that catalyzes the conversion of C19 steroids to estrogens. The present study was conducted to determine whether hepatic aromatase was responsive to DDE treatment. We found that hepatic aromatase protein in adult male rats was greatly increased after seven daily oral treatments of DDE at a dosage of 100 mg/kg wt. per day. This induction was seen in both immunoblot and immunohistochemistry of liver tissue sections. Distribution of the aromatase in the liver corresponded to the distribution of hypertrophic hepatocytes in the tissue. Furthermore, we found a large increase in hepatic microsomal aromatase activity in DDE-treated animals, although the difference in serum 17beta-estradiol concentrations between treated animals and controls was not statistically significant. However, an in vitro experiment using primary culture of rat hepatocytes did not show a change in aromatase level after DDE treatment at four concentrations ranging from 0 to 5x10(-6) M for 24 h. Meanwhile, CYP 2B1 induction, a known DDE effect in primary rat hepatocyte culture, was seen in those cells. This study supports the notion that induction of aromatase by DDE is a contributory factor to its reproductive developmental effects.

Compendium of physical activities: an update of activity codes and MET intensities.

We provide an updated version of the Compendium of Physical Activities, a coding scheme that classifies specific physical activity (PA) by rate of energy expenditure. It was developed to enhance the comparability of results across studies using self-reports of PA. The Compendium coding scheme links a five-digit code that describes physical activities by major headings (e.g., occupation, transportation, etc.) and specific activities within each major heading with its intensity, defined as the ratio of work metabolic rate to a standard resting metabolic rate (MET). Energy expenditure in MET-minutes, MET-hours, kcal, or kcal per kilogram body weight can be estimated for specific activities by type or MET intensity. Additions to the Compendium were obtained from studies describing daily PA patterns of adults and studies measuring the energy cost of specific physical activities in field settings. The updated version includes two new major headings of volunteer and religious activities, extends the number of specific activities from 477 to 605, and provides updated MET intensity levels for selected activities.

Outcomes of elective home births: a series of 1,146 cases.

Medical records of 1,146 elective home births from five home delivery services in northern California were reviewed. Three of the services consisted of family physicians and nurses, whereas two consisted of lay midwives without immediate physician supervision. Rates of medical complications in both groups were low. Perinatal morbidity and mortality were lower than California averages. Fifteen premature infants (1.3%) were delivered successfully. Apgar scores were high. Four infants (0.3%) were neurologically abnormal at follow-up. The perinatal mortality rate was 9.5 per 1,000 total births. There were no maternal deaths. These figures demonstrate that in a self-selected, medically screened, low-risk population, home delivery with medical facility back-up can be a reasonable alternative to hospital delivery. Possible reasons for the good results obtained are cited.

Molecular determinants of antiviral potency of paramyxovirus entry inhibitors.

Hendra virus (HeV) and Nipah virus (NiV) constitute the Henipavirus genus of paramyxoviruses, both fatal in humans and with the potential for subversion as agents of bioterrorism. Binding of the HeV/NiV attachment protein (G) to its receptor triggers a series of conformational changes in the fusion protein (F), ultimately leading to formation of a postfusion six-helix bundle (6HB) structure and fusion of the viral and cellular membranes. The ectodomain of paramyxovirus F proteins contains two conserved heptad repeat regions, the first (the N-terminal heptad repeat [HRN]) adjacent to the fusion peptide and the second (the C-terminal heptad repeat [HRC]) immediately preceding the transmembrane domain. Peptides derived from the HRN and HRC regions of F are proposed to inhibit fusion by preventing activated F molecules from forming the 6HB structure that is required for fusion. We previously reported that a human parainfluenza virus 3 (HPIV3) F peptide effectively inhibits infection mediated by the HeV glycoproteins in pseudotyped-HeV entry assays more effectively than the comparable HeV-derived peptide, and we now show that this peptide inhibits live-HeV and -NiV infection. HPIV3 F peptides were also effective in inhibiting HeV pseudotype virus entry in a new assay that mimics multicycle replication. This anti-HeV/NiV efficacy can be correlated with the greater potential of the HPIV3 C peptide to interact with the HeV F N peptide coiled-coil trimer, as evaluated by thermal unfolding experiments. Furthermore, replacement of a buried glutamic acid (glutamic acid 459) in the C peptide with valine enhances antiviral potency and stabilizes the 6HB conformation. Our results strongly suggest that conserved interhelical packing interactions in the F protein fusion core are important determinants of C peptide inhibitory activity and offer a strategy for the development of more-potent analogs of F peptide inhibitors.

Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity.

We have introduced amino acid substitutions into two regions of the extracellular domain of the vesicular stomatitis virus (VSV) glycoprotein (G protein) and examined the effect of these mutations on protein transport, low-pH-induced stability of G protein oligomers, and membrane fusion activity. We suggested previously that the region between amino acids 118 and 139 may be important for the membrane fusion activity of G protein, on the basis of the characterization of a fusion-defective G protein mutant (M. A. Whitt, P. Zagouras, B. Crise, and J. K. Rose, J. Virol. 64:4907-4913, 1990). It has also been postulated by others that this region as well as the region between amino acids 181 and 212 may constitute putative internal fusion domains of VSV G protein. In this report, we show that three different amino acids substitutions between residues 118 and 139 (G-124-->E, P-127-->D, and A-133-->K) either altered or abolished low-pH-dependent membrane fusion activity. In contrast, substitutions between residues 192 and 212 resulted either in G proteins that had wild-type fusion activity or in mutant proteins in which the mutation prevented transport of G protein to the cell surface. Two of the substitutions between residues 118 and 139 (G-124-->E and P-127-->D) resulted in G proteins that were fusion defective at pH 5.7, although syncytia were observed after cells were treated with fusion buffer at pH 5.5, albeit at levels significantly less than that induced by wild-type G protein. Interestingly, when either G-124-->E or P-127-->D was incorporated into tsO45 virions, the resulting particles were not infectious, presumably because the viral envelope was not able to fuse with the proper intracellular membrane. These results support the hypothesis that the region between amino acids 118 and 139 is important for the membrane fusion activity of VSV G protein and may constitute an internal fusion domain.

A phosphorylated 34-kDa protein and a subpopulation of polyhedrin are thiol linked to the carbohydrate layer surrounding a baculovirus occlusion body.

Surrounding baculovirus occlusion bodies is an electron-dense layer reported to be composed of carbohydrate which we term calyx. Incubation of Autographa californica nuclear polyhedrosis virus occlusion bodies (AcMNPV OBs) with dilute alkaline saline (DAS) followed by centrifugation at 12,000 g resulted in the sedimentation of calyx material which contained pp34, residual polyhedrin (p32), and entrapped occluded virions (DAS P-12 fraction). Incubation of the DAS P-12 fraction with sodium dodecyl sulfate (SDS) resulted in solubilization of the entrapped virions and the majority of p32, while calyx material, pp34, and some p32 remained sedimentable at 12,000 g. Immunofluorescence microscopy of DAS-solubilized OBs using monoclonal antibody to pp34 and p32 revealed that both pp34 and p32 are closely associated with the calyx. When DAS P-12 fractions were resuspended in SDS and reducing agent, not only were the entrapped virions solubilized, but pp34 and the remaining p32 were also liberated, indicating that pp34 and a subpopulation of p32 are associated with the calyx via thiol linkages. Immunoblot analysis and peptide mapping demonstrated that pp34 is neither immunologically nor structurally related to p32. The kinetics of pp34 synthesis were also examined by immunoprecipitation of infected cell polypeptides using pp34-specific monoclonal antibody. pp34 was detected initially 15 hr postinfection (p.i.) and continued to be phosphorylated until 60-70 hr p.i. This study demonstrates that the AcMNPV calyx has a proteinaceous component and we propose that other occluded baculoviruses may also have a calyx-associated protein analogous to pp34.

Mutational analyses of the intergenic dinucleotide and the transcriptional start sequence of vesicular stomatitis virus (VSV) define sequences required for efficient termination and initiation of VSV transcripts.

We have used dicistronic vesicular stomatitis virus (VSV) minigenomes to dissect the functional importance of the nontranscribed intergenic dinucleotide and the conserved transcription start sequence found at the beginning of all VSV genes. The minigenomes were generated entirely from cDNA and contained the G and M protein genes, flanked by the leader and trailer regions from the Indiana serotype of VSV. All mutations were made either within the nontranscribed M-G intergenic dinucleotide or within the transcription start sequence of the downstream G gene. Immunofluorescence microscopy and immunoprecipitation analysis of the mutated minigenomes indicated that the first three nucleotides of the transcriptional start sequence are the most critical for efficient VSV gene expression, whereas the nontranscribed, intergenic dinucleotide and the other conserved nucleotides found at the 5' mRNA start sequence can tolerate significant sequence variability without affecting G protein production. RNA analysis indicated that nucleotide changes in the transcriptional start sequence which resulted in reduced G protein expression correlated with the amount of transcript present. Therefore, this conserved sequence appears to be required for efficient transcript initiation following polyadenylation of the upstream mRNA. While the minimum sequence for efficient transcription (3'-UYGnn-5') is similar to that of other rhabdoviruses, it is not homologous to the start sites for viruses from the Paramyxoviridae or Filoviridae families. Using Northern blot analysis, we also found that some nucleotide changes in the nontranscribed intergenic region resulted in higher levels of read-through transcription. Therefore, the nontranscribed intergenic dinucleotide plays a role in transcript termination.

The membrane-proximal stem region of vesicular stomatitis virus G protein confers efficient virus assembly.

In this report, we show that the glycoprotein of vesicular stomatitis virus (VSV G) contains within its extracellular membrane-proximal stem (GS) a domain that is required for efficient VSV budding. To determine a minimal sequence in GS that provides for high-level virus assembly, we have generated a series of recombinant DeltaG-VSVs which express chimeric glycoproteins having truncated stem sequences. The recombinant viruses having chimeras with 12 or more membrane-proximal residues of the G stem, and including the G protein transmembrane-cytoplasmic tail domains, produced near-wild-type levels of particles. In contrast, viruses encoding chimeras with shorter or no G-stem sequences produced approximately 10- to 20-fold less. This budding domain when present in chimeric glycoproteins also promoted their incorporation into the VSV envelope. We suggest that the G-stem budding domain promotes virus release by inducing membrane curvature at sites where virus budding occurs or by recruiting condensed nucleocapsids to sites on the plasma membrane which are competent for efficient virus budding.