Towards a resolution of the proton form factor problem: new electron and positron scattering data.

There is a significant discrepancy between the values of the proton electric form factor, G(E)(p), extracted using unpolarized and polarized electron scattering. Calculations predict that small two-photon exchange (TPE) contributions can significantly affect the extraction of G(E)(p) from the unpolarized electron-proton cross sections. We determined the TPE contribution by measuring the ratio of positron-proton to electron-proton elastic scattering cross sections using a simultaneous, tertiary electron-positron beam incident on a liquid hydrogen target and detecting the scattered particles in the Jefferson Lab CLAS detector. This novel technique allowed us to cover a wide range in virtual photon polarization (ϵ) and momentum transfer (Q(2)) simultaneously, as well as to cancel luminosity-related systematic errors. The cross section ratio increases with decreasing ϵ at Q(2)=1.45 GeV(2). This measurement is consistent with the size of the form factor discrepancy at Q(2)≈1.75 GeV(2) and with hadronic calculations including nucleon and Δ intermediate states, which have been shown to resolve the discrepancy up to 2-3 GeV(2).

Measurement of exclusive π(0) electroproduction structure functions and their relationship to transverse generalized parton distributions.

Exclusive π(0) electroproduction at a beam energy of 5.75 GeV has been measured with the Jefferson Lab CLAS spectrometer. Differential cross sections were measured at more than 1800 kinematic values in Q(2), x(B), t, and ϕ(π), in the Q(2) range from 1.0 to 4.6 GeV(2), -t up to 2 GeV(2), and x(B) from 0.1 to 0.58. Structure functions σ(T)+ϵσ(L), σ(TT), and σ(LT) were extracted as functions of t for each of 17 combinations of Q(2) and x(B). The data were compared directly with two handbag-based calculations including both longitudinal and transversity generalized parton distributions (GPDs). Inclusion of only longitudinal GPDs very strongly underestimates σ(T)+ϵσ(L) and fails to account for σ(TT) and σ(LT), while inclusion of transversity GPDs brings the calculations into substantially better agreement with the data. There is very strong sensitivity to the relative contributions of nucleon helicity-flip and helicity nonflip processes. The results confirm that exclusive π(0) electroproduction offers direct experimental access to the transversity GPDs.

Tensor correlations measured in 3He(e,e' pp)n.

We have measured the 3He(e,e' pp)n reaction at an incident energy of 4.7 GeV over a wide kinematic range. We identified spectator correlated pp and pn nucleon pairs by using kinematic cuts and measured their relative and total momentum distributions. This is the first measurement of the ratio of pp to pn pairs as a function of pair total momentum p(tot). For pair relative momenta between 0.3 and 0.5 GeV/c, the ratio is very small at low p(tot) and rises to approximately 0.5 at large p(tot). This shows the dominance of tensor over central correlations at this relative momentum.

Use of a ventilatory support system (BiPAP) for acute respiratory failure in the emergency department.

STUDY OBJECTIVES: Bilevel pressure ventilation has had proven success in the treatment of acute respiratory failure (ARF). The purpose of this study was to identify patient characteristics early in the course of acute illness that can predict the successful use of bilevel pressure ventilation. METHODS: Ventilatory assistance using a ventilatory support system (BiPAP model ST-D; Respironics; Murrysville, PA) was considered a treatment option for stable patients with ARF. The system was titrated to patient comfort. Once stable settings had been achieved for 30 min, a posttrial arterial blood gas (ABG) measurement was obtained. Patient charts were reviewed for pretrial and posttrial ABG levels, along with demographics, APACHE (acute physiology and chronic health evaluation) II score, Glasgow Coma Scale (GCS), and length of stay (LOS) data. RESULTS: Bilevel pressure ventilation trials were performed on 58 patients. In 43 patients (74.1%), the trials were successful. Of the 15 patients (25.9%) in whom the trials were not successful, 13 patients required intubation. The pretrial ABG levels did not predict success, as there were no significant differences between the success and failure groups for pH and PaCO2, respectively: 7.26 vs 7.26 mm Hg and 75.3 vs 72.8 mm Hg. After 30 min, posttrial ABG levels for pH and PaCO2 predicted successful avoidance of intubation: 7.34 vs 7.27 mm Hg (p < 0.002) and 61.9 vs 73.0 mm Hg (p < 0.04), respectively. There were no significant differences between the success and failure groups in age, gender, GCS, or APACHE II. There were differences between the success and failure groups for LOS data (ventilator days, ICU days, and hospital days): 1.8 vs 10.4 days (p < 0.01), 4.2 vs 12.3 days (p < 0.02), and 7.5 vs 15.6 days (p < 0.02), respectively. CONCLUSION: Successful treatment with bilevel pressure ventilation could not be predicted by pretrial data (including pH and PaCO2) obtained in the emergency department; however, a successful outcome could be determined quickly with a 30-min trial. Successful treatment with bilevel pressure ventilation significantly reduced LOS data. CLINICAL IMPLICATIONS: Our inability to predict success based on initial data supports the use of bilevel pressure ventilation trials for all stable patients with ARF. If the patient's condition fails to improve within 30 min, intubation and mechanical ventilation is indicated.

Conditions for copackaging rous sarcoma virus and murine leukemia virus Gag proteins during retroviral budding.

Rous sarcoma virus (RSV) and murine leukemia virus (MLV) are examples of distantly related retroviruses that normally do not encounter one another in nature. Their Gag proteins direct particle assembly at the plasma membrane but possess very little sequence similarity. As expected, coexpression of these two Gag proteins did not result in particles that contain both. However, when the N-terminal membrane-binding domain of each molecule was replaced with that of the Src oncoprotein, which is also targeted to the cytoplasmic face of the plasma membrane, efficient copackaging was observed in genetic complementation and coimmunoprecipitation assays. We hypothesize that the RSV and MLV Gag proteins normally use distinct locations on the plasma membrane for particle assembly but otherwise have assembly domains that are sufficiently similar in function (but not sequence) to allow heterologous interactions when these proteins are redirected to a common membrane location.

Importance of basic residues in the nucleocapsid sequence for retrovirus Gag assembly and complementation rescue.

The Gag proteins of Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) contain small interaction (I) domains within their nucleocapsid (NC) sequences. These overlap the zinc finger motifs and function to provide the proper density to viral particles. There are two zinc fingers and at least two I domains within these Gag proteins. To more thoroughly characterize the important sequence features and properties of I domains, we analyzed Gag proteins that contain one or no zinc finger motifs. Chimeric proteins containing the amino-terminal half of RSV Gag and various portions of the carboxy terminus of murine leukemia virus (MLV) (containing one zinc finger) Gag had only one I domain, whereas similar chimeras with human foamy virus (HFV) (containing no zinc fingers) Gag had at least two. Mutational analysis of the MLV NC sequence and inspection of I domain sequences within the zinc-fingerless C terminus of HFV Gag suggested that clusters of basic residues, but not the zinc finger motif residues themselves, are required for the formation of particles of proper density. In support of this, a simple string of strongly basic residues was found to be able to substitute for the RSV I domains. We also explored the possibility that differences in I domains (e.g., their number) account for differences in the ability of Gag proteins to be rescued into particles when they are unable to bind to membranes. Previously published experiments have shown that such membrane-binding mutants of RSV and HIV (two I domains) can be rescued but that those of MLV (one I domain) cannot. Complementation rescue experiments with RSV-MLV chimeras now map this difference to the NC sequence of MLV. Importantly, the same RSV-MLV chimeras could be rescued by complementation when the block to budding was after, rather than before, transport to the membrane. These results suggest that MLV Gag molecules begin to interact at a much later time after synthesis than those of RSV and HIV.

Fusion of green fluorescent protein with the Zeocin-resistance marker allows visual screening and drug selection of transfected eukaryotic cells.

Green fluorescent protein (GFP) and the Zeocin-resistance gene Sh ble (ZeoR) were fused together to generate a bifunctional protein for the identification and selection of transfected mammalian cells. Expression of this hybrid selectable marker, GFP-ZeoR, was visually detected and conferred Zeocin resistance in prokaryotes and eukaryotes. This selectable marker provides a way to determine transient transfection efficiencies in tissue culture cells using fluorescence microscopy. Expression of the GFP-ZeoR was also used to identify and select stable mammalian cell lines expressing a heterologous gene. Selection was efficient and GFP fluorescence provides an excellent, noninvasive technique to monitor the success of Zeocin selection during the development of the stable cell lines. This hybrid resistance gene combines the functional properties of the Zeocin-resistance marker and GFP and should be useful for combined selection and fluorescence in a variety of organisms.

A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles.

Incorporation of Vpr into human immunodeficiency virus type 1 (HIV-1) virions is mediated by the Gag protein, independently of other viral components. We have coexpressed Vpr and Gag constructs in a vaccinia virus expression system in order to map the region of Gag involved in Vpr packaging. Deletion of the carboxyl-terminal p6 region of Gag impaired the ability of Gag to package Vpr. To confirm the role of p6 in Vpr packaging, Rous sarcoma virus (RSV)-HIV chimeras containing HIV-1 p6 were constructed. Although RSV Gag does not package Vpr into virus particles, a chimera containing HIV-1 p6 is sufficient for Vpr incorporation. To map the region of p6 involved in Vpr packaging, a series of p6 point mutations and deletion mutations was analyzed. Mutations in the N-terminal p6 proline-rich domain, for which preliminary evidence shows a marked decrease in virion incorporated RNA, did not affect Vpr incorporation. Deletion of residues 1 to 31 of HIV-1 p6 did not affect Vpr packaging, but residues 35 to 47, including an (LXX)4 domain, were required for Vpr incorporation into virus particles.

Positionally independent and exchangeable late budding functions of the Rous sarcoma virus and human immunodeficiency virus Gag proteins.

The Gag proteins of Rous sarcoma virus and human immunodeficiency virus (HIV) each contain a function involved in a late step in budding, defects in which result in the accumulation of these molecules at the plasma membrane. In the Rous sarcoma virus Gag protein (Pr76gag), this assembly domain is associated with a PPPY motif, which is located at an internal position between the MA and CA sequences. This motif is not contained anywhere within the HIV Gag protein (Pr55gag), and the MA sequence is linked directly to CA. Instead, a late assembly function of HIV has been associated with the p6 sequence situated at the C terminus of Gag. Here we demonstrate the remarkable finding that the late assembly domains from these two unrelated Gag proteins are exchangeable between retroviruses and can function in a positionally independent manner.

An assembly domain of the Rous sarcoma virus Gag protein required late in budding.

The Gag protein of Rous sarcoma virus has the ability to direct particle assembly at the plasma membrane in the absence of all the other virus-encoded components. An extensive deletion analysis has revealed that very large regions of this protein can be deleted without impairing budding and has suggested that the essential functions map to three discrete regions. In the studies reported here, we establish the location of assembly domain 2 (AD2) within the proline-rich p2b sequence of this Gag protein. AD2 mutants lacking the p2b sequence were completely defective for particle release even though their Gag proteins were tightly associated with the membrane fraction and exhibited high levels of protease activity. Mutations that inactivate the viral protease did not restore budding to wild-type levels for these mutants, indicating that the defect is not due simply to a loss of protease regulation. AD2 mutants could be rescued into dense particles in genetic complementation assays, indicating that their defect is not due to a gross alteration of the overall conformation of the protein and that the assembly function is not needed on every Gag molecule in the population. Several mutants with amino acid substitutions in the p2b sequence were found to have an intermediate capacity for budding. Inactivation of the protease of these mutants stabilized the Gag polyprotein within the cells and allowed an increase in particle release; however, the rate of budding remained slow. We favor the idea that AD2 is a dynamic region of movement, perhaps serving as a molecular hinge to allow the particle to emerge from the surface of the cell during budding.

Functional chimeras of the Rous sarcoma virus and human immunodeficiency virus gag proteins.

The Gag protein encoded by Rous sarcoma virus (RSV) is the only viral product required for the process of budding whereby virus particles are formed at the plasma membrane. Deletion analysis of this Gag molecule has revealed several regions (assembly domains) that are important for budding. One of these domains is located at the amino terminus and is needed for membrane binding. Another is located within the carboxy-terminal third of the protein. Though there is little sequence homology among the Gag proteins of unrelated retroviruses, it seemed possible that their assembly domains might be functionally conserved, and to explore this idea, numerous Gag chimeras were made. The results indicate that the first 10 amino acids of the human immunodeficiency virus (HIV) Gag protein can suppress the block to budding caused by deletions in the RSV MA sequence, much as described previously for the first 10 residues from the Src oncoprotein (J.W. Wills, R.C. Craven, R. A. Weldon, Jr., T. D. Nelle, and C.R. Erdie, J. Virol. 65:3804-3812, 1991). In addition, the carboxy-terminal half of the HIV Gag protein was fused to a truncated RSV Gag molecule, mutant Bg-Bs, which is unable to direct core assembly. This chimera was able to produce particles at a rate identical to that of RSV and of a density similar to that of authentic virions. Deletion analysis of the carboxy-terminal chimera revealed two small regions within the HIV NC protein that were sufficient for endowing mutant Bg-Bs with these properties. Chimeras lacking both regions produced particles of a low density, suggesting that these sequences may be involved in the tight packing of Gag molecules during assembly. In a related set of experiments, replacement of the RSV protease with that of HIV resulted in premature processing within the RSV sequence and a block to budding. Particle assembly was restored when the HIV PR activity was inactivated by mutagenesis. Collectively, the data presented here illustrate the functional similarities of Gag proteins from unrelated retroviruses.

Role of the avian retroviral protease in the activation of reverse transcriptase during virion assembly.

The retroviruses of the avian sarcoma-leukosis virus group synthesize their viral protease (PR) in two precursor forms--as a carboxy-terminal domain of the Gag precursor and as an embedded domain within the Gag-Pol precursor. We have shown previously that the Gag-derived PR is fully capable of processing the Gag precursor in the absence of the embedded PR (R.P. Bennett, S. Rhee, R.C. Craven, E. Hunter, and J.W. Wills, J. Virol. 65:272-280, 1991). In this study, we examined the question of whether or not the PR domain of Gag-Pol has an essential role in the maturation of the Pol proteins. The Gag-Pol precursor was expressed in the absence of Gag by use of a simian virus 40-based vector in which the gag and pol reading frames were fused. The fusion protein accumulated to high levels in transfected cells without being released into the medium but could be rescued into particles by coexpression of the Gag protein from a second vector. The resulting particles contained mature Gag and Pol proteins and active reverse transcriptase (RT). Using this complementation system, the effects of PR defects in the Gag and/or Gag-Pol proteins on the activation of RT were examined. The results showed that the presence of a functional PR on the Gag precursor, but not on Gag-Pol, was required for full activation of RT. The embedded PR of Gag-Pol was unable to carry out any detectable processing of the Gag precursor and was able to activate RT to only a low level in the absence of a functional Gag PR domain. Finally, some point mutations in the Gag-Pol PR domain inhibited activation of RT in trans by a wild-type PR, suggesting that the correct conformation of the PR domain in Gag-Pol is prerequisite for activation of RT.

Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly.

Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the active protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.