Grafting Tomato to Manage Bacterial Wilt Caused by Ralstonia solanacearum in the Southeastern United States.

Bacterial wilt, caused by Ralstonia solanacearum, can result in severe losses to tomato (Solanum lycopersicum) growers in the southeastern United States, and grafting with resistant rootstocks may be an effective strategy for managing this disease. However, R. solanacearum populations maintain considerable diversity, and little information is known regarding the efficacy of commercially available rootstocks to reduce bacterial wilt incidence and subsequent crop loss in the United States. In this study, tomato plants grafted onto 'Dai Honmei' and 'RST-04-105-T' rootstocks had significantly lower area under the disease progress curve (AUDPC) values compared with nongrafted plants (P < 0.05). Across three locations in North Carolina, final bacterial wilt incidence for non- and self-grafted plants was 82 ± 14 to 100%. In contrast, bacterial wilt incidence for the grafted plants was 0 to 65 ± 21%. Final bacterial wilt incidence of plants grafted with Dai Honmei rootstock was 0 and 13 ± 3% at two locations in western North Carolina but 50 ± 3% at a third site in eastern North Carolina. Similarly, grafting onto RST-04-105-T rootstock significantly reduced AUDPC values at two of the three locations (P < 0.05) compared with that of the nongrafted plants, but performed poorly at the third site. Total fruit yields were significantly increased by grafting onto resistant rootstocks at all three sites (P < 0.05). Regression analyses indicated that yield was significantly negatively correlated with bacterial wilt AUDPC values (R2 was 0.4048 to 0.8034), and the use of resistant rootstocks enabled economically viable tomato production in soils naturally infested with R. solanacearum.

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore.

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

A new RNA element located in the coding region of a murine endogenous retrovirus can functionally replace the Rev/Rev-responsive element system in human immunodeficiency virus type 1 Gag expression.

Nuclear export of incompletely spliced RNAs is a prerequisite for retroviral replication. Complex retroviruses like human immunodeficiency virus (HIV) encode a viral transport factor (Rev), which binds to its target sequence on the RNA genome and directs it into the Crm-1-mediated export pathway. Other retroviruses, like Mason-Pfizer monkey virus, contain cis-acting constitutive RNA transport elements (CTE) which achieve nuclear export of intron-containing RNA via cellular transport factors. Here, we describe the identification and characterization of a novel cis-acting orientation-dependent RNA expression element in the coding region of the murine intracisternal A-type particle (IAP) MIA14. This IAP expression element (IAPE) can functionally replace the Rev system in the expression of HIV-1 Gag proteins but functions independently of Crm-1. The presence of this element is needed for the expression of the IAP Gag proteins, indicating its biological significance. The IAPE can be functionally replaced by placing a CTE on the MIA14 RNA, further supporting its role in mRNA export. Northern blot analysis revealed that total RNA, as well as cytoplasmic RNA, was increased when the element was present. The element was mapped to a predicted stem-loop structure in the 3' part of the pol open reading frame. There was no overall homology between the IAPE and the CTE, but there was complete sequence identity between short putative single-stranded loops. Deletion of these loops from the IAPE severely reduced Rev-independent Gag expression.

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

Organization of immature human immunodeficiency virus type 1.

Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particle's center. We related image features to the polyprotein domain structure by combining mutagenesis with cryoelectron microscopy and image analysis. The matrix (MA) domain appears as a thin layer tightly associated with the inner face of the viral membrane, separated from the capsid (CA) layer by a low-density region corresponding to its C terminus. Deletion of the entire p6 domain has no effect on the width or spacing of the density layers, suggesting that p6 is not ordered in immature human immunodeficiency virus type 1 (HIV-1). In vitro assembly of a recombinant Gag polyprotein containing only capsid (CA) and nucleocapsid (NC) domains results in the formation of nonenveloped spherical particles which display two layers with density matching that of the CA-NC portion of immature HIV-1 Gag particles. Authentic, immature HIV-1 displays additional surface features and an increased density between the lipid bilayers which reflect the presence of gp41. The other internal features match those of virus-like particles.

Proteasome inhibition interferes with gag polyprotein processing, release, and maturation of HIV-1 and HIV-2.

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6(gag), a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6(gag). Consistent with this, viruses with mutations in PR or p6(gag) were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Biochemical and structural analysis of isolated mature cores of human immunodeficiency virus type 1.

Mature human immunodeficiency virus type 1 (HIV-1) particles contain a cone-shaped core structure consisting of the internal ribonucleoprotein complex encased in a proteinaceous shell derived from the viral capsid protein. Because of their very low stability after membrane removal, HIV-1 cores have not been purified in quantities sufficient for structural and biochemical analysis. Based on our in vitro assembly experiments, we have developed a novel method for isolation of intact mature HIV-1 cores. Concentrated virus suspensions were briefly treated with nonionic detergent and immediately centrifuged in a microcentrifuge for short periods of time. The resuspended pellet was subsequently analyzed by negative-stain and thin-section electron microscopy and by immunoelectron microscopy. Abundant cone-shaped cores as well as tubular and aberrant structures were observed. Stereo images showed that core structures preserved their three-dimensional architecture and exhibited a regular substructure. Detailed analysis of 155 cores revealed an average length of ca. 103 nm, an average diameter at the base of ca. 52 nm, and an average angle of 21.3 degrees. There was significant variability in all parameters, indicating that HIV cores are not homogeneous. Immunoblot analysis of core preparations allowed semiquantitative estimation of the relative amounts of viral and cellular proteins inside the HIV-1 core, yielding a model for the topology of various proteins inside the virion.

Toxins that are activated by HIV type-1 protease through removal of a signal for degradation by the N-end-rule pathway.

Diphtheria toxin enters the cytosol of mammalian cells where it inhibits cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work we describe the construction of toxins that contain a signal for N-end-rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed.

Update on myocardial contrast echocardiography: a surgeon's perspective.

The ability to evaluate myocardial perfusion and microvascular structural integrity can help surgeons predict the necessity for surgical intervention, the sequence of intraoperative interventions, the risk of perioperative infarction, the likelihood of successful surgical recovery, and the degree of long-term clinical benefit. The ability to directly assess perfusion intraoperatively may allow surgeons to reliably evaluate a patient's myocardial perfusion at any time during the operative procedure. As this article will discuss, surgeons may use myocardial contrast echocardiography intraoperatively to evaluate myocardial function and integrity, to determine the order of graft placement, to determine the success of bypass graft patency, and to help predict those patients who will experience successful cardiac function after recovering from surgery.

Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity.

The nef gene of primate immunodeficiency viruses is essential for high-titer virus replication and AIDS pathogenesis in vivo. In tissue culture, Nef is not required for human immunodeficiency virus (HIV) infection but enhances viral infectivity. We and others have shown that Nef is incorporated into HIV-1 particles and cleaved by the viral proteinase. To determine the signal for Nef incorporation and to analyze whether virion-associated Nef is responsible for enhancement of infectivity, we generated a panel of nef mutants and analyzed them for virion incorporation of Nef and for their relative infectivities. We report that N-terminal truncations of Nef abolished its incorporation into HIV particles. Incorporation was reconstituted by targeting the respective proteins to the plasma membrane by using a heterologous signal. Mutational analysis revealed that both myristoylation and an N-terminal cluster of basic amino acids were required for virion incorporation and for plasma membrane targeting of Nef. Grafting the N-terminal anchor domain of Nef onto the green fluorescent protein led to membrane targeting and virion incorporation of the resulting fusion protein. These results indicate that Nef incorporation into HIV-1 particles is mediated by plasma membrane targeting via an N-terminal bipartite signal which is reminiscent of a Src homology region 4. Virion incorporation of Nef correlated with enhanced infectivity of the respective viruses in a single-round replication assay. However, the phenotypes of HIV mutants with reduced Nef incorporation only partly correlated with their ability to replicate in primary lymphocytes, indicating that additional or different mechanisms may be involved in this system.

Intracisternal A-type particles express their proteinase in a separate reading frame by translational frameshifting, similar to D-type retroviruses.

Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum of rodent cells. IAP genomes share extensive sequence homologies with D-type retroviruses, but were presumed to express the viral proteinase (PR) as part of the gag open reading frame (ORF) while D-type retroviruses express PR in a separate ORF. Here we show that expression of the murine IAP element MIA14 yields three major translation products, corresponding to the Gag, Gag-PR, and Gag-PR-Pol polyproteins. Sequence analysis revealed that MIA14 PR is encoded in its own reading frame, separate from gag and pol. Frameshifting occurred with an efficiency of approximately 25% between the gag and pro ORFs and 35% between pro and pol. The region containing the putative gag-pro frameshift signal consists of a heptanucleotide slippery sequence (A6C) and a stem-loop structure probably forming a pseudoknot. Deletion of this structure element almost completely abolished frameshifting. Insertion of an additional base next to the frameshift signal placed gag and pro in the same ORF and resulted in predominant formation of Gag-PR and Gag-PR-Pol polyproteins which were not processed following in vitro translation. Expression of a similar construct in tissue culture cells, on the other hand, led to efficient intracellular processing of the mutant polyproteins.

Plasma membrane targeting of chimeric intracisternal A-type particle polyproteins leads to particle release and specific activation of the viral proteinase.

Retrovirus morphogenesis involves assembly of structural Gag polyproteins with subsequent budding from the plasma membrane, followed by proteolytic cleavage by the viral proteinase (PR) and extracellular maturation to the infectious virion. Intracisternal A-type particles (IAPs) are defective retroviruses that assemble and bud at the membranes of the endoplasmic reticulum (ER), where they remain as immature particles consisting exclusively of uncleaved polyproteins. To analyze requirements for intracellular polyprotein transport and PR activation, we constructed deletion and substitution mutations in the IAP gag gene, including the putative ER-targeting signal. Mutant polyproteins were transported to various intracellular locations, including the nucleus, the cytoplasm, the ER, and the plasma membrane. Interestingly, assembly of capsid-like particle structures occurred at almost all sites. However, only those polyproteins transported to the plasma membrane were efficiently and specifically cleaved by viral PR, with cleavage occurring predominantly within the virus particle. Thus, at least in the experimental system presented here, retroviral particle assembly can occur at almost any location within the cell, while polyprotein processing and, consequently, virion maturation are confined to a specific cellular site. These results suggest that a factor restricted to the plasma membrane is required to trigger PR activation and maturation of infectious retroviruses.

Analysis of hepatitis C virus core protein interaction domains.

Hepatitis C virus (HCV) core protein forms the internal viral coat that encapsidates the genomic RNA and is enveloped in a host cell-derived lipid membrane. As the single capsid protein, core should be capable of multimerization but attempts to produce virus-like particles following expression of HCV structural proteins have not been successful. In this study, we have analysed the interaction capacity of full-length and truncated HCV core using the yeast two-hybrid system. Full-length core containing or lacking the translocation signal for the E1 glycoprotein did not interact with full-length or truncated core proteins. Truncation to the N-terminal 122 aa revealed an interaction domain which was mapped to the tryptophan-rich sequence from aa 82-102 and was termed the main homotypic interaction domain. The C-terminal hydrophobic fragment of core (aa 122-172) was incapable of interacting with itself but interacted with the main homotypic interaction domain in trans (the weak heterotypic interaction domain). Core proteins truncated at their N and C termini (aa 46-102) were trans-activating when fused to the DNA-binding domain of GAL4. Based on our results, we suggest that the C-terminal segment may interact in cis with the main homotypic interaction domain and thereby prevent multimerization. Core-core interaction was also observed for in vitro-translated proteins bound to truncated immobilized core 102. However, interaction was less specific in this system suggesting that protein interaction and possibly conformational alteration of core may be dependent on the experimental system.

Human immunodeficiency virus type 1 Nef protein is incorporated into virus particles and specifically cleaved by the viral proteinase.

The Nef protein of primate immunodeficiency viruses is essential for establishing a highly productive pathogenic infection in vivo. In tissue culture, Nef is not required for infection but enhances viral infectivity. This effect is most pronounced in unstimulated primary lymphocytes and occurs in the early phase of infection prior to viral gene expression. Since Nef expression does not lead to obvious changes in virus composition, it was of interest to analyze whether Nef is incorporated into virus particles. Here, we show that Nef is specifically immunoprecipitated from radioactively labeled human immunodeficiency virus type 1 (HIV-1)-infected cells and virus particle preparations. Quantitative analysis revealed Nef to be incorporated on the order of 10% of reverse transcriptase incorporation, which corresponds to 5 to 10 molecules of Nef per virion. In infected cells, Nef was detected as a full-length 27-kDa protein. In contrast, approximately 50% of particle-associated Nef corresponded to an 18-kDa species which comigrated with the larger product after in vitro cleavage of purified HIV-1 Nef by the viral proteinase. Nef cleavage in particle preparations was completely abolished by a specific inhibitor of HIV-1 proteinase. Most likely, Nef is cleaved concomitantly with viral structural proteins on maturation of virus particles. This cleavage is likely to be functionally significant because it dissociates the conserved core domain from the N-terminal membrane attachment region. Our results suggest that the profound influence of Nef on establishing infection of unstimulated cells in tissue culture and in vivo is mediated by virion-associated Nef which functions in early infection before viral gene expression.

An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity.

Infectious retrovirus particles are derived from structural polyproteins which are cleaved by the viral proteinase (PR) during virion morphogenesis. Besides cleaving viral polyproteins, which is essential for infectivity, PR of human immunodeficiency virus (HIV) also cleaves cellular proteins and PR expression causes a pronounced cytotoxic effect. Retroviral PRs are aspartic proteases and contain two copies of the triplet Asp-Thr-Gly in the active center with the threonine adjacent to the catalytic aspartic acid presumed to have an important structural role. We have changed this threonine in HIV type 1 PR to a serine. The purified mutant enzyme had an approximately 5- to 10-fold lower activity against HIV type 1 polyprotein and peptide substrates compared with the wild-type enzyme. It did not induce toxicity on bacterial expression and yielded significantly reduced cleavage of cytoskeletal proteins in vitro. Cleavage of vimentin in mutant-infected T-cell lines was also markedly reduced. Mutant virus did, however, elicit productive infection of several T-cell lines and of primary human lymphocytes with no significant difference in polyprotein cleavage and with similar infection kinetics and titer compared with wild-type virus. The discrepancy between reduced processing in vitro and normal virion maturation can be explained by the observation that reduced activity was due to an increase in Km which may not be relevant at the high substrate concentration in the virus particle. This mutation enables us therefore to dissociate the essential function of PR in viral maturation from its cytotoxic effect.

Lyme disease acquired in Europe and presenting in CONUS.

Lyme disease is recognized in many parts of the world, including large areas of North America, Europe, Asia and Australia. Diagnosis and treatment of the disease is essential to avoid the debilitating and potentially life-threatening long-term effects of the infection; however, many physicians may not be aware of the international scope of the disease. This is particularly important for military physicians whose patients may visit or live in endemic areas and whose activities may bring them in contact with the organism. We report here the case of a soldier with near-fatal Lyme carditis acquired in Europe and presenting in Massachusetts.

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.

Interpersonal concomitants and antecedents of depression among college students.

Depressed college students were compared with other-psychopathology and normal controls regarding the relationship they developed with dormitory roommates during a 9-month period. Diagnostic status was periodically assessed via SADS interviews, thus also permitting identification of new cases of depression during the year. Psychosocial characteristics found to be uniquely associated with current depression were: (a) low social contact with roommates, (b) low enjoyability of these contacts, and (c) high life-event stress. Roommates of depressives reported low enjoyability of the relationship and high levels of aggressive behavior towards the depressive. No features were found to be uniquely associated with new cases before they became depressed; however, several antecedents of general psychopathology were identified.