Comparison of polymerase chain reaction of polymorphonuclear leukocytes and plasma identifies patients who control cytomegalovirus infection after hematopoietic cell transplantation.

By use of an automated polymerase chain reaction test of plasma and a qualitative polymerase chain reaction assay on polymorphonuclear leukocytes, we identified a subgroup of hematopoietic cell transplant recipients who were able to control cytomegalovirus infection early after hematopoietic cell transplantation without antiviral therapy. Thirty-one percent of patients had cytomegalovirus DNA detected by qualitative polymerase chain reaction assay but had no cytomegalovirus DNA detected by the automated test; this group maintained a lower peak cytomegalovirus load, compared with the group of patients who had cytomegalovirus DNA detected by both tests (P = .03), suggesting a greater degree of functional immune reconstitution.

Changes in coronary arterial dimensions early after cardiac transplantation.

BACKGROUND: Significant changes in coronary artery structure, including intimal thickening and vessel remodeling, occur early after cardiac transplantation. The degree to which these changes compromise coronary lumen dimensions, and the clinical factors that affect these changes, remain controversial. METHODS: Thirty-eight adult cardiac transplant recipients underwent coronary angiography and volumetric intravascular ultrasound (IVUS) evaluation of the left anterior descending artery within 8 weeks of transplantation and at 1 year. Clinical parameters including donor and recipient characteristics, rejection episodes, and serology were prospectively recorded. Two-dimensional IVUS measurements and vessel, lumen and plaque volume were calculated at both time points and compared. Multivariate regression analysis was performed to reveal clinical predictors of change in coronary dimensions. RESULTS: During the first year after transplantation, significant decreases in vessel size (negative remodeling) and lumen size were observed with significant increases in plaque burden based on IVUS analyses. Loss of lumen volume correlated significantly with the degree of negative remodeling (R=0.82, P<0.0001), but not with changes in plaque burden (R=0.08, P=0.64). Patients with the greatest increase in plaque volume had significantly less negative remodeling (R=0.53, P=0.0006). Transplant recipient cytomegalovirus (CMV) antibody seropositivity and lack of aggressive prophylaxis against CMV infection/reactivation were significant independent predictors of greater negative remodeling (P<0.01 and P=0.03, respectively) and greater lumen loss (P=0.02 and P=0.03, respectively). CONCLUSION: Negative remodeling is primarily responsible for coronary artery lumen loss during the first year after cardiac transplantation. CMV seropositivity and lack of aggressive CMV prophylaxis correlate with increased negative remodeling, resulting in greater lumen loss.

Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication.

A series of amino acid substitutions (M239F, M239G, P240F, V241G) were placed in the p10-CA protease cleavage site (VVAM*PVVI) to change the rate of cleavage of the junction. The effects of these substitutions on p10-CA cleavage by RSV PR were confirmed by measuring the kinetics of cleavage of model peptide substrates containing the wild type and mutant p10-CA sites. The effects of these substitutions on processing of the Gag polyprotein were determined by labeling Gag transfected COS-1 cells with 35S-Met and -Cys, and immunoprecipitation of Gag and its cleavage products from the media and lysate fractions. All substitutions except M239F caused decreases in detectable Gag processing and subsequent release from cells. Several of the mutants also caused defects in production of the three CA proteins. The p10-CA mutations were subcloned into an RSV proviral vector (RCAN) and introduced into a chick embryo fibroblast cell line (DF-1). All of the mutations except M239F blocked RSV replication. In addition, the effects of the M239F and M239G substitutions on the morphology of released virus particles were examined by electron microscopy. While the M239F particles appeared similar to wild type particles, M239G particles contained cores that were large and misshapen. These results suggest that mutations affecting cleavage at the p10-CA protease cleavage site block RSV replication and can have a negative impact on virus particle morphology.

Analysis of Rous sarcoma virus capsid protein variants assembled on lipid monolayers.

During assembly and morphogenesis of Rous sarcoma virus (RSV), proteolytic processing of the structural precursor (Pr76Gag) protein generates three capsid (CA) protein variants, CA476, CA479, and CA488. The proteins share identical N-terminal domains (NTDs), but are truncated at residues corresponding to gag codons 476, 479, and 488 in their CA C-terminal domains (CTDs). To characterize oligomeric forms of the RSV CA variants, we examined 2D crystals of the capsid proteins, assembled on lipid monolayers. Using electron microscopy and image analysis approaches, the CA proteins were observed to organize in hexagonal (p6) arrangements, where rings of membrane-proximal NTD hexamers were spaced at 95 A intervals. Differences between the oligomeric structures of the CA variants were most evident in membrane-distal regions, where apparent CTDs interconnect hexamer rings. In this region, CA488 connections were observed readily, while CA476 and CA479 contacts were resolved poorly, suggesting that in vivo processing of CA488 to the shorter forms may permit virions to adopt a dissembly-competent conformation. In addition to crystalline arrays, the CA479 and CA488 proteins formed small spherical particles with diameters of 165-175 A. The spheres appear to be arranged from hexamer or hexamer plus pentamer ring subunits that are related to the 2D crystal forms. Our results implicate RSV CA hexamer rings as basic elements in the assembly of RSV virus cores.

Frequent occult infection with Cytomegalovirus in cardiac transplant recipients despite antiviral prophylaxis.

Despite antiviral prophylaxis, a high percentage (over 90%) of heart transplant patients experience active cytomegalovirus (CMV) infection, diagnosed by detection of viral DNA in peripheral blood polymorphonuclear leukocytes within the first few months posttransplantation. Viral DNA was detected in mononuclear cells prior to detection in granulocytes from CMV-seropositive recipients (R+) receiving a heart from a CMV-seropositive donor (D+). Based on assessment of systemic infection in leukocyte populations, both R+ subgroups (R+/D- and R+/D+) experienced a greater infection burden than the R-/D+ subgroup, which was aggressively treated because of a higher risk of acute CMV disease. Despite widespread systemic infection in all at-risk patient subgroups, CMV DNA was rarely (< 3% of patients) detected in transplanted heart biopsy specimens. The R+ patients more frequently exceeded the 75th percentile of the CMV DNA copy number distribution in leukocytes (110 copies/10(5) polymorphonuclear leukocytes) than the R-/D+ subgroup. Therefore, active systemic CMV infection involving leukocytes is common in heart transplant recipients receiving prophylaxis to reduce acute disease. Infection of the transplanted organ is rare, suggesting that chronic vascular disease attributed to CMV may be driven by the consequences of systemic infection.

The functionally exchangeable L domains in RSV and HIV-1 Gag direct particle release through pathways linked by Tsg101.

The functionally exchangeable L domains of HIV-1 and Rous sarcoma virus (RSV) Gag bind Tsg101 and Nedd4, respectively. Tsg101 and Nedd4 function in endocytic trafficking, and studies show that expression of Tsg101 or Nedd4 fragments interfere with release of HIV-1 or RSV Gag, respectively, as virus-like particles (VLPs). To determine whether functional exchangeability reflects use of the same trafficking pathway, we tested the effect on RSV Gag release of co-expression with mutated forms of Vps4, Nedd4 and Tsg101. A dominant-negative mutant of Vps4A, an AAA ATPase required for utilization of endosomal sorting proteins that was shown previously to interfere with HIV-1 budding, also inhibited RSV Gag release, indicating that RSV uses the endocytic trafficking machinery, as does HIV. Nedd4 and Tsg101 interacted in the presence or absence of Gag and, through its binding of Nedd4, RSV Gag interacted with Tsg101. Deletion of the N-terminal region of Tsg101 or the HECT domain of Nedd4 did not prevent interaction; however, three-dimensional spatial imaging suggested that the interaction of RSV Gag with full-length Tsg101 and N-terminally truncated Tsg101 was not the same. Co-expression of RSV Gag with the Tsg101 C-terminal fragment interfered with VLP release minimally; however, a significant fraction of the released VLPs was tethered to each other. The results suggest that, while Tsg101 is not required for RSV VLP release, alterations in the protein interfere with VLP budding/fission events. We conclude that RSV and HIV-1 Gag direct particle release through independent ESCRT-mediated pathways that are linked through Tsg101-Nedd4 interaction.

Role of Nedd4 and ubiquitination of Rous sarcoma virus Gag in budding of virus-like particles from cells.

Rous sarcoma virus (RSV) budding requires an interaction of the L domain within the p2b region of Gag with cellular Nedd4-family E3 ubiquitin protein ligases. Members of our laboratories previously demonstrated that overexpression of a fragment of the chicken Nedd4-like protein (LDI-1 WW) inhibits Gag release in a dominant-negative manner (A. Kikonyogo, F. Bouamr, M. L. Vana, Y. Xiang, A. Aiyar, C. Carter, and J. Leis, Proc. Natl. Acad. Sci. USA 98:11199-11204, 2001). We have now identified the complete 3' end of LDI-1 and determined that it has a C-terminal ubiquitin ligase HECT domain, similar to other Nedd4 family members. While overexpression of the full-length LDI-1 clone (LDI-1 FL) had little effect on Gag budding, an LDI-1 FL mutant with a substitution in the HECT domain catalytic site blocked Gag release, similar to LDI-1 WW. The coexpression of Gag and hemagglutinin-tagged ubiquitin (HA-Ub) resulted in the detection of mono- and polyubiquitinated forms of Gag in cells and mostly monoubiquitinated Gag in virus-like particles (VLPs). When the Nedd4-binding site (L domain) was deleted, ubiquitinated Gag was not detected. Interestingly, the release of Gag with ubiquitin covalently linked to the C terminus (Gag-Ub) was still blocked by LDI-1 WW. To understand the mechanism of this inhibition, we examined cells expressing Gag and LDI-1 WW by electron microscopy. In the presence of LDI-1 WW, VLPs were found in electron-dense inclusion bodies in the cytoplasm of transfected cells. In contrast, when cells that coexpressed Gag-Ub and LDI-1 WW were examined, inclusion bodies were detected but did not contain VLPs. These results indicate that the ubiquitination of Gag is dependent upon Nedd4 binding to the L domain and suggest that Nedd4 has additional functions during RSV release besides the ubiquitination of Gag.