The capsid of small papova viruses contains 72 pentameric capsomeres: direct evidence from cryo-electron-microscopy of simian virus 40.

The three-dimensional structure of the simian virus 40 capsid is remarkably similar to the structure of the polyoma empty capsid. This similarity is apparent despite striking differences in the methods used to determine the two structures: image analysis of electron micrographs of frozen-hydrated samples (SV40 virions) and an unconventional x-ray crystallographic analysis (polyoma empty capsids). Both methods have clearly resolved the 72 prominent capsomere units which comprise the T = 7d icosahedral capsid surface lattice. The 12 pentavalent and 60 hexavalent capsomeres consist of pentameric substructures. A pentameric morphology for hexavalent capsomeres clearly shows that the conserved bonding specificity expected from the quasi-equivalence theory is not present in either SV40 or polyoma capsids. Determination of the SV40 structure from cryo-electron microscopy supports the correctness of the polyoma structure solved crystallographically and establishes a strong complementarity of the two techniques. Similarity between the SV40 virion and the empty polyoma capsid indicates that the capsid is not detectably altered by the loss of the nucleohistone core. The unexpected pentameric substructure of the hexavalent capsomeres and the arrangement of the 72 pentamers in the SV40 and polyoma capsid lattices may be characteristic features of all members of the papova virus family, including the papilloma viruses such as human wart and rabbit papilloma.     

Discharge properties of human motor units during sustained contraction at low level force.

The characteristic of discharge behaviors of motor units (MUs) during low level contraction was investigated. The discharge of MUs in the m. vastus medialis was observed during the sustained contraction at 4 different levels below 10% MVC (2, 4, 8 and 10% MVC) for 15 min. The spike interval of all observed MUs gradually elongated during an initial several minutes of the contraction and the characteristic discharge patterns following the elongation were observed. i.e. continuous discharge throughout the contraction (CONT), decruitment (D-N), and re-recruitment following decruitment (D-REC).The relationship between recruitment threshold force (F(th)) and discharge pattern was not significant at 2% MVC but, at 10% MVC, there were significant differences in F(th) between D-N and CONT, and between D-REC and CONT MU populations.In pooled data, the MUs with the shorter mean spike interval at the beginning of the contraction (MSI(0), below 90 ms) tend to discharge continuously, but the MUs with longer MSI(0) showed various discharge patterns.In conclusion, during low level contraction MUs discharge characteristically, and the MU with high excitation levels tend to discharge continuously, but individual MU represents an intrinsic discharge pattern at not a high excitation level.     

Human motor unit recruitment and derecruitment during long lasting intermittent contractions.

Seven healthy subjects were investigated in cyclic ramp-and-hold long lasting isometric contractions. Wire branched electrodes were used for selective recording of single motor unit (MU) potentials from m. biceps brachii. MU behaviour was defined in terms of recruitment/derecruitment thresholds (RT and DT) and the duration of interspike intervals (ISI). A total of 63 MUs was investigated: 40 units were active from the beginning of the task performance and another 23 were recruited later. There were no changes in the recruitment pattern of MUs with fatigue development - a short first ISI followed by a very long second one and an almost constant firing rate after this transient phase. The tendency of RT to gradually decrease dominates the results. Thus, the required constant rate of force increase with fatigue development was maintained mostly by the mechanisms of space coding (i.e., decrease of RT and recruitment of additional MUs). Oppositely, the time behaviour of the DT changes was not uniform and rate coding was an essential mechanism in the adaptation of MU activity to muscle fatigue during relaxation phases. The recruitment pattern and fatigue related behaviour of the additionally recruited MUs were similar to those of MUs active from the first cycle of the motor task performance.     

Scar-driven shape-changes of virus capsids

We propose that certain patterns (scars) could be relevant to extend the classic Caspar and Klug construction for icosahedrally-shaped virus capsids. These scars are theoretically and numerically predicted to be formed by electrons arranged on a sphere to minimize the repulsive Coulomb potential (the Thomson problem) and are experimentally found in spherical crystals formed by self-assembled polystyrene beads (an instance of the generalized Thomson problem). Scars could be produced on the capsid at an intermediate stage of its evolution and the release of the bending energy present in scars into stretching energy could allow for shape-changes. The conjecture can be tested in experiments and/or in numerical simulations.     

Investigation of motor unit recruitment during stimulated contractions of tibialis anterior muscle

This work investigated motor unit (MU) recruitment during transcutaneous electrical stimulation (TES) of the tibialis anterior (TA) muscle, using experimental and simulated data. Surface electromyogram (EMG) and torque were measured during electrically-elicited contractions at different current intensities, on eight healthy subjects.EMG detected during stimulation (M-wave) was simulated selecting the elicited MUs on the basis of: (a) the simulated current density distribution in the territory of each MU and (b) the excitation threshold characteristic of the MU. Exerted force was simulated by adding the contribution of each of the elicited MUs. The effects of different fat layer thickness (between 2 and 8 mm), different distributions of excitation thresholds (random excitation threshold, higher threshold for larger MUs or smaller MUs), and different MU distributions within the muscle (random distribution, larger MU deeper in the muscle, smaller MU deeper) on EMG variables and torque were tested.Increase of the current intensity led to a first rapid increase of experimental M-wave amplitude, followed by a plateau. Further increases of the stimulation current determined an increase of the exerted force, without relevant changes of the M-wave. Similar results were obtained in simulations.Rate of change of conduction velocity (CV) and leading coefficient of the second order polynomial interpolating the force vs. stimulation level curve were estimated as a function of increasing current amplitudes. Experimental data showed an increase of estimated CV with increasing levels of the stimulation current (for all subjects) and a positive leading coefficient of force vs. stimulation current curve (for five of eight subjects). Simulations matched the experimental results only when larger MUs were preferably located deeper in the TA muscle (in line with a histochemical study). Marginal effect of MU excitation thresholds was observed, suggesting that MUs closer to the stimulation electrode are recruited first during TES regardless of their excitability.     

Modeling investigation of learning a fast elbow flexion in the horizontal plane--prediction of muscle forces and motor units action

Experimental investigation of practicing a dynamic, goal-directed movement reveals significant changes in kinematics. Modeling can provide insight into the alterations in muscle activity, associated with the kinematic adaptations, and reveal the potential motor unit (MU) firing patterns that underlie those changes. In this paper, a previously developed muscle model and software (Raikova and Aladjov, Journal of Biomechanics, 35, 2002) have been used to investigate changes in MU control, while practicing fast elbow flexion to a target in the horizontal plane. The first trial (before practice) and the last trial (after extensive practice) of two subjects have been simulated. The inputs for the simulation were the calculated external moments at the elbow joint. The external moments were countered by the action of three flexor muscles and two extensor ones. The muscles have been modeled as a mixture of MUs of different types. The software has chosen the MU firing times necessary to accomplish the movement. The muscle forces and MUs firing statistics were then calculated. Three hypotheses were tested and confirmed: (1) peak muscle forces and antagonist co-contraction increase during training; (2) there is an increase in the firing frequency and the synchronization between MUs; and (3) the recruitment of fast-twitch MUs dominates the action.     

Modeling investigation of learning a fast elbow flexion in the horizontal plane—prediction of muscle forces and motor units action

Experimental investigation of practicing a dynamic, goal-directed movement reveals significant changes in kinematics. Modeling can provide insight into the alterations in muscle activity, associated with the kinematic adaptations, and reveal the potential motor unit (MU) firing patterns that underlie those changes. In this paper, a previously developed muscle model and software (Raikova and Aladjov, Journal of Biomechanics, 35, 2002) have been used to investigate changes in MU control, while practicing fast elbow flexion to a target in the horizontal plane. The first trial (before practice) and the last trial (after extensive practice) of two subjects have been simulated. The inputs for the simulation were the calculated external moments at the elbow joint. The external moments were countered by the action of three flexor muscles and two extensor ones. The muscles have been modeled as a mixture of MUs of different types. The software has chosen the MU firing times necessary to accomplish the movement. The muscle forces and MUs firing statistics were then calculated. Three hypotheses were tested and confirmed: (1) peak muscle forces and antagonist co-contraction increase during training; (2) there is an increase in the firing frequency and the synchronization between MUs; and (3) the recruitment of fast-twitch MUs dominates the action.     

Estimation of the error between experimental tetanic force curves of MUs of rat medial gastrocnemius muscle and their models by summation of equal successive contractions

More accurate muscle models require appropriate modelling of individual twitches of motor units (MUs) and their unfused tetanic contractions. It was shown in our previous papers, using a few MUs, that modelling of unfused tetanic force curves by summation of equal twitches is not accurate, especially for slow MUs. The aim of this study was to evaluate this inaccuracy using a statistical number of MUs of the rat medial gastrocnemius muscle (15 of slow, 15 of fast resistant and 15 of fast fatigable type). Tetanic contractions were evoked by trains of 41 stimuli at random interpulse intervals and different mean frequencies, resembling discharge patterns observed during natural muscle activity. The tetanic curves were calculated by the summation of equal twitches according to the respective experimental patterns. The previously described 6-parameter analytical function for twitch modelling was used. Comparisons between the experimental and the modelled curves were made using two coefficients: the fit coefficient and the area coefficient. The errors between modelled and experimental tetanic forces were substantially different between the three MU types. The error was the most significant for slow MUs, which develop much higher forces in real contractions than could be predicted based on the summation of equal twitches, while the smallest error was observed for FF MUs – their recorded tetanic forces were similar to those predicted by modelling. The obtained results indicate the importance of the inclusion of the type-specific non-linearity in the summation of successive twitch-like contractions of MUs in order to increase the reliability of modelling skeletal muscle force.     

Calcium Bridge Triggers Capsid Disassembly in the Cell Entry Process of Simian Virus 40

The calcium bridge between the pentamers of polyoma viruses maintains capsid metastability. It has been shown that viral infection is profoundly inhibited by the substitution of lysine for glutamate in one calcium-binding residue of the SV40 capsid protein, VP1. However, it is unclear how the calcium bridge affects SV40 infectivity. In this in vitro study, we analyzed the influence of host cell components on SV40 capsid stability. We used an SV40 mutant capsid (E330K) in which lysine had been substituted for glutamate 330 in protein VP1. The mutant capsid retained the ability to interact with the SV40 cellular receptor GM1, and the internalized mutant capsid accumulated in caveolin-1-mediated endocytic vesicles and was then translocated to the endoplasmic reticulum (ER) region. However, when placed in ER-rich microsome, the mutant capsid retained its spherical structure in contrast to the wild type, which disassembled. Structural analysis of the mutant capsid with cryo-electron microscopy and image reconstruction revealed altered pentamer coordination, possibly as a result of electrostatic interaction, although its overall structure resembled that of the wild type. These results indicate that the calcium ion serves as a trigger at the pentamer interface, which switches on capsid disassembly, and that the failure of the E330K mutant capsid to disassemble is attributable to an inadequate triggering system. Our data also indicate that calcium depletion-induced SV40 capsid disassembly may occur in the ER region and that this is essential for successful SV40 infection.     

Musarmins: three single-chain ribosome-inactivating protein isoforms from bulbs of Muscari armeniacum L. and Miller

Three new ribosome-inactivating protein (RIP; EC 3.2.2.22) isoforms that we have named musarmins (MUs) 1, 2 and 3 have been isolated from the bulbs of Muscari armeniacum L. and Miller by ion-exchange chromatography and gel filtration. Analysis by electrophoresis revealed that they are single-chain proteins and mass spectrometry analysis afforded Mr values of 28,708, 30,003 and 27,626 for MUs 1, 2 and 3, respectively. Musarmins strongly inhibited protein synthesis carried out by mammalian ribosomes, with IC50 values in the 0.14-0.24nM range but not that carried out by plant cell-free systems or HeLa cells. MUs promote the single depurination of rabbit reticulocyte 28S rRNA. cDNA cloning of genes coding for musarmins revealed that they contain open reading frames of 298, 294 and 295 aminoacids for MU1, MU2 and MU3, respectively. Mature MU1, MU2 and MU3 contain 277, 273 and 273 aminoacids, respectively suggesting post-translational C-terminal processing. An untranslated mRNA coding for an ORF very similar to that of MU3 was detected in leaves. Each of the four MU genes contains an intron. In contrast to other RIPs, MUs are present only in bulbs and are not induced in leaves either by senescence, or by treatment of leaves with H2O2 or salicylic acid, or by growth in darkness. Therefore, these proteins could play a non-vital role in plants; for instance, as anti-pathogens and protective agents only in some stages of the plant life cycle (237).     

Impairment of human soleus motor units during ischemia

It is generally accepted that ischemia produced by limb compression affects rapidly conducting large-diameter Ia afferents in the early stage and that the motor nerve-muscle complex is blocked later. This notion, however, seems to be controversial for several reasons, so an attempt to reveal the amount of motor unit (MU) impairment during ischemia was made. Observation of human soleus muscle electromyographic (EMG) signal recorded either by bipolar needle electrode or by surface electrodes at various levels of voluntary contraction during the course of ischemia showed that low-threshold small MUs were affected first while high-threshold large MUs survived longer. The changes in EMG patterns were temporally correlated with T-reflex deterioration. It is suggested that the early loss of low-threshold MUs may play a definite role in alterations of reflexes during ischemia.     

The fatigability of two agonistic muscles in human isometric voluntary submaximal contraction: an EMG study. II. Motor unit firing rate and recruitment

The recruitment and firing rate of biceps brachii (BB) and brachioradialis (BR) motor units (MUs) were studied in the course of fatiguing isometric contractions at 20%-30% of maximal voluntary contraction (MVC). MU recruitment generally occurred throughout the maintained contraction and was similar for BB and BR muscles. Newly recruited MUs started to discharge in the form of bursts, the duration of which increased until a continuous rhythmical firing was achieved. Within each burst, the first interval between two consecutive discharges was usually the shortest. MU threshold was lowered just after the limit time of the maintained contraction. The MU's firing rate either increased or remained stable as a function of the elapsed time. It is concluded that (1) in fatiguing isometric contractions at 20%-30% MVC contractile failure is mainly compensated for by MU recruitment and a lowered MU threshold and (2) differences between in surface changes in the electromyogram of BB and BR muscles cannot easily be explained by related differences in MU firing rate and recruitment.     

Using two-dimensional spatial information in decomposition of surface EMG signals.

Recently, high-density surface EMG electrode grids and multi-channel amplifiers became available for non-invasive recording of human motor units (MUs). We present a way to decompose surface EMG signals into MU firing patterns, whereby we concentrate on the importance of two-dimensional spatial differences between the MU action potentials (MUAPs). Our method is exemplified with high-density EMG data from the vastus lateralis muscle of a single subject. Bipolar and Laplacian spatial filtering was applied to the monopolar raw signals. From the single recording in this subject six different simultaneously active MUs could be distinguished using the spatial differences between MUAPs in the direction perpendicular to the muscle fiber direction. After spike-triggered averaging, 125-channel two-dimensional MUAP templates were obtained. Template-matching allowed tracking of all MU firings. The impact of spatial information was measured by using subsets of the MUAP templates, either in parallel or perpendicular to the muscle fiber direction. The use of one-dimensional spatial information perpendicular to the muscle fiber direction was superior to the use of a linear array electrode in the longitudinal direction. However, to detect the firing events of the MUs with a high accuracy, as needed for instance for estimation of firing synchrony, two-dimensional information from the complete grid electrode appears essential.     

Selective fatigue of fast motor units after electrically elicited muscle contractions.

The aim of the present study was to elucidate the electrophysiological manifestations of selective fast motor unit (MU) activation by electrical stimulation (ES) of knee extensor muscles. In six male subjects, test contraction measurement at 40% maximal voluntary contraction (MVC) was performed before and at every 5 min (5, 10, 15 and 20 min) during 20-min low intensity intermittent exercise of either ES or voluntary contractions (VC) at 10% MVC (5-s isometric contraction and 5-s rest cycles). Both isolated intramuscular MU spikes obtained from three sets of bipolar fine-wire electrodes and surface electromyogram (EMG) were simultaneously recorded and were analyzed by means of a computer-aided intramuscular spike amplitude-frequency analysis and frequency power spectral analysis, respectively. Results indicated that mean MU spike amplitude, particularly those MUs with relatively large amplitude, was significantly reduced while those MUs with small spike amplitude increased their firing rate during the 40% MVC test contraction after the ES. This was accompanied by the increased amplitude of surface EMG (rmsEMG). However, no such significant changes in the intramuscular and surface EMGs were observed after VC. These findings indicated differential MU activation patterns in terms of MU recruitment and rate coding characteristics during ES and VC, respectively. Our data strongly suggest the possibility of "an inverse size principle" of MU recruitment during ES.     

Experimentally verified mathematical approach for the prediction of force developed by motor units at variable frequency stimulation patterns

During normal daily activity, muscle motor units (MUs) develop unfused tetanic contractions evoked by trains of motoneuronal firings at variable interpulse intervals (IPIs). The mechanical responses of a MU to successive impulses are not identical. The aim of this study was to develop a mathematical approach for the prediction of each response within the tetanus as well as the tetanic force itself. Experimental unfused tetani of fast and slow rat MUs, evoked by trains of stimuli at variable IPIs, were decomposed into series of twitch-shaped responses to successive stimuli using a previously described algorithm. The relationships between the parameters of the modeled twitches and the tetanic force level at which the next response begins were examined and regression equations were derived. Using these equations, profiles of force for the same and different stimulation patterns were mathematically predicted by summating modeled twitches. For comparison, force predictions were made by the summation of twitches equal to the first one. The recorded and the predicted tetanic forces were compared. The results revealed that it is possible to predict tetanic force with high accuracy by using regression equations. The force predicted in this way was much closer to the experimental record than the force obtained by the summation of equal twitches, especially for slow MUs. These findings are likely to have an impact on the development of realistic muscle models composed of MUs, and will assist our understanding of the significance of the neuronal code in motor control and the role of biophysical processes during MU contractions.     

Expression of Semliki Forest virus capsid protein from SV40 recombinant virus

Here, the proteolytic processing of the Semliki Forest virus (SFV) capsid protein was studied in the absence of other viral functions. Two different fragments of the SFV messenger cDNA, coding for capsid protein and 174 and 38 extra amino acids from the envelope proteins, respectively, were cloned in the late region of the SV40 viral DNA. Cells infected with the SV40 recombinant virus stocks were analyzed for the production of SFV capsid mRNA and polypeptide. Immunofluorescence staining of the infected cells indicated that the produced SFV capsid protein accumulated mainly in the nucleus. Polyacrylamide gel electrophoresis of the immunoprecipitated SFV capsid proteins showed that both recombinants yielded a labelled band equivalent in size to the SFV capsid protein. Thus the proteolytic processing takes place even under conditions where the capsid protein is the only virus-specified protein synthesized.     

Protruding Features of Viral Capsids Are Clustered on Icosahedral Great Circles

Spherical viruses are remarkably well characterized by the Triangulation (T) number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary constraints as well as bioengineering for capsid drug delivery systems. This result also suggests that in addition to biochemical attachment restrictions, there are additional geometric constraints that should be adhered to when modifying protein capsids.     

The abundant nuclear enzyme PARP participates in the life cycle of simian virus 40 and is stimulated by minor capsid protein VP3

The abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP) functions in DNA damage surveillance and repair and at the decision between apoptosis and necrosis. Here we show that PARP binds to simian virus 40 (SV40) capsid proteins VP1 and VP3. Furthermore, its enzymatic activity is stimulated by VP3 but not by VP1. Experiments with purified mutant proteins demonstrated that the PARP binding domain in VP3 is localized to the 35 carboxy-terminal amino acids, while a larger peptide of 49 amino acids was required for full stimulation of its activity. The addition of 3-aminobenzamide (3-AB), a known competitive inhibitor of PARP, demonstrated that PARP participates in the SV40 life cycle. The titer of SV40 propagated on CV-1 cells was reduced by 3-AB in a dose-dependent manner. Additional experiments showed that 3-AB did not affect viral DNA replication or capsid protein production. PARP did not modify the viral capsid proteins in in vitro poly(ADP-ribosylation) assays, implying that it does not affect SV40 infectivity. On the other hand, it greatly reduced the magnitude of the host cytopathic effects, a hallmark of SV40 infection. Additional experiments suggested that the stimulation of PARP activity by VP3 leads the infected cell to a necrotic pathway, characterized by the loss of membrane integrity, thus facilitating the release of mature SV40 virions from the cells. Our studies identified a novel function of the minor capsid protein VP3 in the recruitment of PARP for the SV40 lytic process.     

The influence of temperature on contractile properties of motor units in rat medial gastrocnemius

The effects of hypothermia and hyperthermia on mammalian skeletal muscle function have previously been reported. However, their effects on the contractile properties of different motor unit (MU) types were not described. This study aimed to explore the effect of temperature on contractile properties of MUs in rat medial gastrocnemius kept at 25 °C (hypothermia), 37 °C (normothermia), and 41 °C (hyperthermia). Hypothermia prolonged the twitch time parameters of all MU types, shifting the steep part of the force-frequency curve towards lower frequencies and increasing its steepness. In addition, it reduced the rate of force development but not the twitch and tetanus forces of slow-twitch (S) MUs. Moreover, it reduced the tetanic force of fast-twitch fatigable (FF) MUs and increased the twitch force of fast-twitch fatigue-resistant (FR) MUs. In contrast, hyperthermia had opposite effects on twitch time properties and the force-frequency relationship. The twitch-to-tetanus ratio decreased for FF and FR MUs, and the steep part of the force-frequency curve shifted towards higher frequencies and decreased in steepness. Our findings indicate that FF MUs are the most sensitive and S MUs are the least sensitive to temperature. Furthermore, force control processes involving changes in motoneuronal firing frequency were radically modified for fast MUs, especially FF MUs.     

The VP2/VP3 minor capsid protein of simian virus 40 promotes the in vitro assembly of the major capsid protein VP1 into particles

The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119-272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors.