The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment.

The endosomal sorting complex I required for transport (ESCRT-I) is composed of the three subunits Vps23/Tsg101, Vps28 and Vps37. ESCRT-I is recruited to cellular membranes during multivesicular endosome biogenesis and by enveloped viruses such as HIV-1 to mediate budding from the cell. Here, we describe the crystal structure of a conserved C-terminal domain from Sacharomyces cerevisiae Vps28 (Vps28-CTD) at 3.05 A resolution which folds independently into a four-helical bundle structure. Co-expression experiments of Vps28-CTD, Vps23 and Vps37 suggest that Vps28-CTD does not directly participate in ESCRT-I assembly and may thus act as an adaptor module for downstream interaction partners. We show through mutagenesis studies that Vps28-CTD employs its strictly conserved surface in the interaction with the ESCRT-III factor Vps20. Furthermore, we present evidence that Vps28-CTD is sufficient to rescue an equine infectious anaemia virus (EIAV) Gag late domain deletion. Vps28-CTD mutations abolishing Vps20 interaction in vitro also prevent the rescue of the EIAV Gag late domain mutant consistent with a potential direct Vps28-ESCRT-III Vps20 recruitment. Therefore, the physiological relevant EIAV Gag-Alix interaction can be functionally replaced by a Gag-Vps28-CTD fusion. Because both Alix and Vps28-CTD can directly recruit ESCRT-III proteins, ESCRT-III assembly coupled to Vps4 action may therefore constitute the minimal budding machinery for EIAV release.

Emerging drug targets for antiretroviral therapy.

Current targets for antiretroviral therapy (ART) include the viral enzymes reverse transcriptase and protease. The use of a combination of inhibitors targeting these enzymes can reduce viral load for a prolonged period and delay disease progression. However, complications of ART, including the emergence of viruses resistant to current drugs, are driving the development of new antiretroviral agents targeting not only the reverse transcriptase and protease enzymes but novel targets as well. Indeed, enfuvirtide, an inhibitor targeting the viral envelope protein (Env) was recently approved for use in combination therapy in individuals not responding to current antiretroviral regimens. Emerging drug targets for ART include: (i) inhibitors that directly or indirectly target Env; (ii) the HIV enzyme integrase; and (iii) inhibitors of maturation that target the substrate of the protease enzyme. Env mediates entry of HIV into target cells via a multistep process that presents three distinct targets for inhibition by viral and cellular-specific agents. First, attachment of virions to the cell surface via nonspecific interactions and CD4 binding can be blocked by inhibitors that include cyanovirin-N, cyclotriazadisulfonamide analogues, PRO 2000, TNX 355 and PRO 542. In addition, BMS 806 can block CD4-induced conformational changes. Secondly, Env interactions with the co-receptor molecules can be targeted by CCR5 antagonists including SCH-D, maraviroc (UK 427857) and aplaviroc (GW 873140), and the CXCR4 antagonist AMD 070. Thirdly, fusion of viral and cellular membranes can be inhibited by peptides such as enfuvirtide and tifuvirtide (T 1249). The development of entry inhibitors has been rapid, with an increasing number entering clinical trials. Moreover, some entry inhibitors are also being evaluated as candidate microbicides to prevent mucosal transmission of HIV. The integrase enzyme facilitates the integration of viral DNA into the host cell genome. The uniqueness and specificity of this reaction makes integrase an attractive drug target. However, integrase inhibitors have been slow to reach clinical development, although recent contenders, including L 870810, show promise. Inhibitors that target viral maturation via a unique mode of action, such as PA 457, also have potential. In addition, recent advances in our understanding of cellular pathways involved in the life cycle of HIV have also identified novel targets that may have potential for future antiretroviral intervention, including interactions between the cellular proteins APOBEC3G and TSG101, and the viral proteins Vif and p6, respectively. In summary, a number of antiretroviral agents in development make HIV entry, integration and maturation emerging drug targets. A multifaceted approach to ART, using combinations of inhibitors that target different steps of the viral life cycle, has the best potential for long-term control of HIV infection. Furthermore, the development of microbicides targeting HIV holds promise for reducing HIV transmission events.

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is a rapidly emerging pathogen with potentially serious consequences for public health. Here we describe conditions that result not only in the efficient expression of the SARS-CoV spike (S) protein on the surface of cells, but in its incorporation into lentiviral particles that can be used to transduce cells in an S glycoprotein-dependent manner. We found that although some primate cell lines, including Vero E6, 293T and Huh-7 cells, could be efficiently transduced by SARS-CoV S glycoprotein pseudoviruses, other cells lines were either resistant or very poorly permissive to virus entry. Infection by pseudovirions could be inhibited by several lysosomotropic agents, suggesting a requirement for acidification of endosomes for efficient S-mediated viral entry. In addition, we were able to develop a cell-cell fusion assay that could be used to monitor S glycoprotein-dependent membrane fusion. Although proteolysis did not enhance the infectivity of cell-free pseudovirions, trypsin activation is required for cell-cell fusion. Additionally, there was no apparent pH requirement for S glycoprotein-mediated cell-cell fusion. Together, these studies describe important tools that can be used to study SARS-CoV S glycoprotein structure and function, including approaches that can be used to identify inhibitors of the entry of SARS-CoV into target cells.

Equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release.

A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.

A comparative study of the human T-cell leukemia virus type 2 integrase expressed in and purified from Escherichia coli and Pichia pastoris.

The human T-cell leukemia virus type-2 (HTLV-2) integrase (IN) catalyzes the insertion of the viral genome into the host chromosome. HTLV-2 IN was expressed as an N-terminal hexa-histidine tagged protein in the methylotrophic yeast Pichia pastoris and as a C-terminal hexa-histidine fusion in Escherichia coli. Maximal IN expression was observed at 48h post-induction for the yeast system and 2h post-induction for E. coli. Effective purification strategies were developed using non-ionic and zwitterionic detergents for initial protein extraction, followed by a one-step nickel-chelating chromatography purification. IN from both sources was routinely greater than 90% pure with yields exceeding 1.5mg of purified IN per liter of culture for P. pastoris. The relative pI was defined for both INs, pH 5.0-5.4, by 2D-gel electrophoresis. Specific activities for IN purified from E. coli and P. pastoris were calculated from in vitro 3(') processing assays and were comparable. In vitro IN assays were also performed to optimize reaction buffer pH and metal concentrations for both 3(') processing and strand transfer assays. Strand transfer was optimal from pH 6.2-6.8, more than 1.5 pH units below the optimal 3(') processing pH of 8.3. IN from both sources showed no enhancement in activity with MnCl(2) concentrations greater than 5mM. The specific activity of P. pastoris purified IN was 0.35 product (pmol)/h/microg IN, and E. coli produced IN was 0.48 product (pmol)/h/microg IN.