Establishment of a novel human T-cell leukemia virus type 1 infection model using cell-free virus.

The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.

Release of HSV-1 Cell-Free Virions: Mechanisms, Regulation, and Likely Role in Human-Human Transmission.

Herpes simplex virus type 1, or HSV-1, is a widespread human pathogen that replicates in epithelial cells of the body surface and then establishes latent infection in peripheral neurons. When HSV-1 replicates, viral progeny must be efficiently released to spread infection to new target cells. Viral spread occurs via two major routes. In cell-cell spread, progeny virions are delivered directly to cellular junctions, where they infect adjacent cells. In cell-free release, progeny virions are released into the extracellular milieu, potentially allowing the infection of distant cells. Cell-cell spread of HSV-1 has been well studied and is known to be important for in vivo infection and pathogenesis. In contrast, HSV-1 cell-free release has received less attention, and its significance to viral biology is unclear. Here, I review the mechanisms and regulation of HSV-1 cell-free virion release. Based on knowledge accrued in other herpesviral systems, I argue that HSV-1 cell-free release is likely to be tightly regulated in vivo. Specifically, I hypothesize that this process is generally suppressed as the virus replicates within the body, but activated to high levels at sites of viral reactivation, such as the oral mucosa and skin, in order to promote efficient transmission of HSV-1 to new human hosts.

In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes.

Single-stranded, positive-sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic maturation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions.

Interviral Recombination between Plant, Insect, and Fungal RNA Viruses: Role of the Intracellular Ca2+/Mn2+ Pump.

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

Protocol for Efficient Cell-Free Synthesis of Cowpea Chlorotic Mottle Virus-Like Particles Containing Heterologous RNAs.

We report a protocol for efficient cell-free synthesis of cowpea chlorotic mottle virus (CCMV)-like particles containing a broad range of lengths and sequences of RNA. Our protocol starts with a purified stock of wild-type CCMV (protocols for harvesting and purifying the virus are detailed elsewhere) and features three basic steps: disassembly of the CCMV and purification of the capsid protein (CP) from the viral RNA; coassembly of the purified CP and an RNA of choice; and characterization of the assembly products. We highlight several key factors that increase the yield of the assembly reaction: the CP should be uncleaved and sufficiently free of viral RNA; the length of the RNA should be between about 100 and 4000 nucleotides; and the stoichiometry of CP and RNA should be 6-1 by mass. Additionally, we point out that separating the assembly reaction into multiple steps-by successively lowering the ionic strength and then the pH of the assembly buffers-results in the highest yields of well-formed, nuclease-resistant, CCMV-like particles. Finally, we describe methods for characterizing the assembly products using native agarose gel electrophoresis and negative-stain transmission electron microscopy.

Breast milk from Tanzanian women has divergent effects on cell-free and cell-associated HIV-1 infection in vitro.

Transmission of HIV-1 during breastfeeding is a significant source of new pediatric infections in sub-Saharan Africa. Breast milk from HIV-positive mothers contains both cell-free and cell-associated virus; however, the impact of breast milk on HIV-1 infectivity remains poorly understood. In the present study, breast milk was collected from HIV-positive and HIV-negative Tanzanian women attending antenatal clinics in Dar es Salaam. Milk was analyzed for activity in vitro against both cell-free and cell-associated HIV-1. Potent inhibition of cell-free R5 and X4 HIV-1 occurred in the presence of milk from all donors regardless of HIV-1 serostatus. Inhibition of cell-free HIV-1 infection positively correlated with milk levels of sialyl-Lewis(X) from HIV-positive donors. In contrast, milk from 8 of 16 subjects enhanced infection with cell-associated HIV-1 regardless of donor serostatus. Milk from two of these subjects contained high levels of multiple pro-inflammatory cytokines including TNFα, IL-1ß, IL-6, IL-8, MIP-1α, MIP-1ß, MCP-1 and IP-10, and enhanced cell-associated HIV-1 infection at dilutions as high as 1∶500. These findings indicate that breast milk contains innate factors with divergent activity against cell-free and cell-associated HIV-1 in vitro. Enhancement of cell-associated HIV-1 infection by breast milk may be associated with inflammatory conditions in the mother and may contribute to infant infection during breastfeeding.

Production of infectious poliovirus from synthetic viral genomes.

Making use of the nucleotides sequence of the RNA genome (7,440 nt) of poliovirus, synthetic deoxyoligonucleotides, 60-70 nt in length are synthesized. The oligonucleotides that map to adjacent segments in the genome are designed such that they are of plus- and minus-strand polarity with the overlapping complementary sequences at their termini. The oligonucleotides are assembled by asymmetric PCR, and then, the segments are ligated directly into a plasmid. The segments are assembled stepwise via common unique restriction endonuclease cleavage sites to yield a full-length poliovirus complementary DNA (cDNA), carrying a phage T7 RNA polymerase promoter at the (left) 5' end. Genomic RNA is generated with a phage T7 RNA polymerase. The viral RNA is incubated in a cell-free extract, where it is translated and replicated, resulting in the de novo synthesis of poliovirus (PV). Finally, in vivo and in vitro experiments are carried out to confirm that infectious material isolated from the cell-free extract is indeed infectious PV.All components of the synthetic PV are generated by biochemical means. No virus-related structure or component that may have been generated previously in vivo is used as template or as building block for the viral particles. Our work shows that it is possible to synthesize an infectious agent in test tube by solely following instructions from a written sequence.

Murine FLT3 ligand-derived dendritic cell-mediated early immune responses are critical to controlling cell-free human T cell leukemia virus type 1 infection.

Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. We observed previously that depletion of dendritic cells (DCs) in CD11c-diphtheria toxin receptor transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished cellular immune response compared with mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host-pathogen interaction between DCs and cell-free HTLV-1, we used FLT3 ligand-cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers (CD80, CD86, and MHC class II) on infection; however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3(+) T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of IFN-stimulated genes, most cytokines, and transcription factors, but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.

Depletion of dendritic cells enhances susceptibility to cell-free infection of human T cell leukemia virus type 1 in CD11c-diphtheria toxin receptor transgenic mice.

Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c(+) DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response (IFN-gamma(+)CD8(+) T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis after the initial infection.

Reactivation of latent HIV-1 infection by the periodontopathic bacterium Porphyromonas gingivalis involves histone modification.

Latently infected cells harbor the HIV-1 proviral DNA genome primarily integrated into heterochromatin, allowing the persistence of transcriptionally silent proviruses. Hypoacetylation of histone proteins by histone deacetylases (HDAC) is involved in the maintenance of HIV-1 latency by repressing viral transcription. In addition, periodontal diseases, caused by polymicrobial subgingival bacteria including Porphyromonas gingivalis, are among the most prevalent infections of mankind. Here we demonstrate the effects of P. gingivalis on HIV-1 replication. This activity could be ascribable to the bacterial culture supernatant but not to other bacterial components such as fimbriae or LPS. We found that this HIV-1-inducing activity was recovered in the lower molecular mass (<3 kDa) fraction of the culture supernatant. We also demonstrated that P. gingivalis produces high concentrations of butyric acid, acting as a potent inhibitor of HDACs and causing histone acetylation. Chromatin immunoprecipitation assays revealed that the corepressor complex containing HDAC1 and AP-4 was dissociated from the HIV-1 long terminal repeat promoter upon stimulation with bacterial culture supernatant concomitantly with the association of acetylated histone and RNA polymerase II. We thus found that P. gingivalis could induce HIV-1 reactivation via chromatin modification and that butyric acid, one of the bacterial metabolites, is responsible for this effect. These results suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to the systemic dissemination of the virus.

Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation.

The 5' ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3' end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3' end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed.

HIV-1 persists in breast milk cells despite antiretroviral treatment to prevent mother-to-child transmission.

BACKGROUND: The effects of short-course antiretrovirals given to reduce mother-to-child transmission (MTCT) on temporal patterns of cell-associated HIV-1 RNA and DNA in breast milk are not well defined. METHODS: Women in Kenya received short-course zidovudine (ZDV), single-dose nevirapine (sdNVP), combination ZDV/sdNVP or short-course highly active antiretroviral therapy (HAART). Breast milk samples were collected two to three times weekly for 4-6 weeks. HIV-1 DNA was quantified by real-time PCR. Cell-free and cell-associated RNA levels were quantified by the Gen-Probe HIV-1 viral load assay. RESULTS: Cell-free HIV-1 RNA levels in breast milk were significantly suppressed by sdNVP, ZDV/sdNVP or HAART therapy compared with ZDV between day 3 and week 4 postpartum (P < or = 0.03). Breast milk HIV-1 DNA levels (infected cell levels) were not significantly different between treatment arms at any timepoint during the 4-6-week follow-up. At 3 weeks postpartum, when the difference in cell-free RNA levels was the greatest comparing HAART directly with ZDV (P = 0.0001), median log10 HIV-1 DNA copies per 1 x 10 cells were 2.78, 2.54, 2.69, and 2.31 in the ZDV, sdNVP, ZDV/sdNVP and HAART arms, respectively (P = 0.23). Cell-associated HIV-1 RNA levels were modestly suppressed in HAART versus ZDV/sdNVP during week 3 (3.37 versus 4.02, P = 0.04), as well as over time according to a linear mixed-effects model. CONCLUSION: Cell-free and, to a lesser extent, cell-associated HIV-1 RNA levels in breast milk were suppressed by antiretroviral regimens used to prevent MTCT. However, even with HAART, there was no significant reduction in the reservoir of infected cells, which could contribute to breast milk HIV-1 transmission.

Molecular mechanisms by which human immunodeficiency virus type 1 integrase stimulates the early steps of reverse transcription.

Reverse transcriptase (RT) and integrase (IN) are two essential enzymes that play a critical role in synthesis and integration of the retroviral cDNA, respectively. For human immunodeficiency virus type 1 (HIV-1), RT and IN physically interact and certain mutations and deletions of IN result in viruses defective in early steps of reverse transcription. However, the mechanism by which IN affects reverse transcription is not understood. We used a cell-free reverse transcription assay with different primers and compositions of deoxynucleoside triphosphates to differentially monitor the effect of IN on the initiation and elongation modes of reverse transcription. During the initiation mode, addition of IN stimulated RT-catalyzed reverse transcription by fourfold. The stimulation was specific to IN and could not be detected when the full-length IN was replaced with truncated IN derivatives. The IN-stimulated initiation was also restricted to the template-primer complex formed using tRNA(3)(Lys) or short RNA oligonucleotides as the primer and not those formed using DNA oligonucleotides as the primer. Addition of IN also produced a threefold stimulation during the elongation mode, which was not primer dependent. The stimulation of both initiation and elongation by IN was retained in the presence of an RT trap. Furthermore, IN had no effect on steps at or before template-primer annealing, including packaging of viral genomic RNA and tRNA(3)(Lys). Taken together, our results showed that IN acts at early steps of reverse transcription by increasing the processivity of RT and suppressing the formation of the pause products.

Lysosomes: fusion and function.

Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Live-cell imaging has shown that fusion with lysosomes occurs by both transient and full fusion events, and yeast genetics and mammalian cell-free systems have identified much of the protein machinery that coordinates these fusion events. Many pathogens that hijack the endocytic pathways to enter cells have evolved mechanisms to avoid being degraded by the lysosome. However, the function of lysosomes is not restricted to protein degradation: they also fuse with the plasma membrane during cell injury, as well as having more specialized secretory functions in some cell types.

Cellulose acetate 1,2-benzenedicarboxylate inhibits infection by cell-free and cell-associated primary HIV-1 isolates.

Cellulose acetate 1,2-benzenedicarboxylate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was previously shown to have potent inhibitory activity against infection by human immunodeficiency virus type 1 (HIV-1) T cell line-adapted (TCLA) strains. In the present study, we determined the inhibitory activity of CAP against infection by cell-free and cell-associated primary HIV-1 isolates with distinct genotypes and biotypes in cervical explants, peripheral blood mononuclear cells (PBMCs), monocytederived macrophages (MDMs), and CEMx174 5.25M7 cells. CAP blocked infection by cell-free and cell-associated HIV-1 in cervical explants. It inhibited infection by cell-free primary HIV-1 isolates (clades A to G and group O) in PBMCs, MDMs, and CEMx174 5.25M7 cells and blocked transmissions of the cell-associated primary HIV-1 isolates from dendritic cells (DCs) to PBMCs, from MDMs to PBMCs, and from PBMCs to CEMx174 5.25M7 cells. The inhibitory activity of CAP on infection by the cell-free and cell-associated primary HIV-1 isolates is independent of viral subtypes and coreceptor usage. These data suggest that CAP is a good microbicide candidate that can be further developed for preventing sexual transmission of HIV-1.

Proteinase-activated receptor-2 induction by neuroinflammation prevents neuronal death during HIV infection.

Proteinase-activated receptors (PARs), a newly discovered subgroup of G-protein coupled receptors, are widely expressed by neural cells, but their roles in the nervous system remain uncertain. In this study, we report that PAR-2 was up-regulated on neurons in conjunction with neuroinflammation in brain tissue from patients with HIV-1-associated dementia. The inflammatory cytokines TNF-alpha and IL-1beta were also increased in HIV-1-associated dementia brains compared with patients without dementia (p < 0.05), but these same cytokines induced PAR-2 expression on neurons. Enhanced PAR-2 expression and subsequent activation prevented neuronal cell death and induction of the tumor suppressor, p53, caused by the HIV-encoded protein, Tat (p < 0.01). Intrastriatal implantation of a PAR-2 peptide agonist also inhibited Tat-induced neurotoxicity in a mouse model of HIV neuropathogenesis (p < 0.05). Moreover, PAR-2 null animals showed more severe neuroinflammation and neuronal loss caused by Tat neurotoxicity (p < 0.05). TNF-alpha protected wild-type neurons from Tat-related neurotoxicity, but in PAR-2-deficient neurons, the same concentrations of TNF-alpha were cytotoxic (p < 0.001). Thus, neuroinflammation can exert protective effects by which it induces PAR-2 expression with the ensuing abrogation of neuronal death.

Enzymatic characterization of membrane-associated hepatitis C virus NS3-4A heterocomplex serine protease activity expressed in human cells.

The hepatitis C virus (HCV) nonstructural (NS)3-NS4A serine protease heterocomplex is a prime target for development of novel HCV therapies, due to its essential role in maturation of the viral polyprotein. While the mode of substrate/inhibitor recognition of the HCV NS3/NS4A serine protease has been extensively studied in vitro, important molecular aspects of the mechanism of action for this membrane-bound multifunctional enzyme remain unresolved in vivo. In particular, what influence does membrane association exert on the specificity and catalysis of NS3-4A protease? To carry out this study, we developed a specific and sensitive protease assay using a unique internally quenched fluorogenic substrate (IQFS). Our IQFS enables for the first time the direct, specific detection of NS3-4A protease activity within membrane fractions isolated from human cells expressing NS3-4A and the determination of its steady-state kinetic parameters, which were found to be K(m) = 51 +/- 3 microM and k(cat) = 0.39 min(-1). We also show that our fluorescence-based bioassay can be used to evaluate specifically the potency and mode of action of NS3-4A directed inhibitors, such as in the case of a known NS3-4A substrate-analogue inhibitor (K(i) = 22 nM). Our results indicate that the membrane anchoring of NS3 by NS4A does not affect the substrate/inhibitor recognition by the NS3-4A protease domain. Further investigation may reveal whether membrane association could be important for regulating other enzymatic activities associated with NS3 (e.g., helicase and/or ATPase) and/or regulating the recently proposed cross-talk between the protease and helicase activities.

Comparing capsid assembly of primate lentiviruses and hepatitis B virus using cell-free systems.

Many viruses that assemble their capsids in the eukaryotic cytoplasm require a threshold concentration of capsid protein to achieve capsid assembly. Strategies for achieving this include maintaining high levels of capsid protein synthesis and targeting to specific sites to raise the effective concentration of capsid polypeptides. To understand how different viruses achieve the threshold capsid protein concentration required for assembly, we used cell-free systems to compare capsid assembly of hepatitis B virus (HBV) and three primate lentiviruses. Capsid formation of these diverse viruses in a common eukaryotic extract was dependent on capsid protein concentration. HBV capsid assembly was also dependent on the presence of intact membrane surfaces. Surprisingly, not all of the primate lentiviral capsid proteins examined required myristoylation and intact membranes for assembly, even though all contain a myristoylation signal. These findings reveal significant diversity in how different capsid proteins assemble in the same cellular extract.

A soluble chemokine-binding protein from vaccinia virus reduces virus virulence and the inflammatory response to infection.

Many poxviruses express a secreted protein that binds CC chemokines with high affinity and has been called viral CC chemokine inhibitor (vCCI). This protein is unrelated to any known cellular protein, yet can compete with host cellular CC chemokine receptors to modulate host inflammatory and immune responses. Although several strains of vaccinia virus (VV) express a vCCI, the best characterized VV strains Western Reserve and Copenhagen do not. In this study, we have expressed the vCCI from VV strain Lister in a recombinant Western Reserve virus (v Delta B8R-35K) and characterized its binding properties in vitro and its effect on virulence in vivo relative to wild-type virus (v Delta B8R) or a revertant virus (v Delta B8R-R) where Lister 35-kDa had been removed. Cells infected with v Delta B8R-35K secreted a 35-kDa protein that bound the CC chemokine macrophage-inflammatory protein 1 alpha. Expression of vCCI attenuated the virus in a murine intranasal model, characterized by reduced mortality and weight loss, decreased virus replication and spread, and a reduced recruitment of inflammatory cells into the lungs of VV-infected mice. The CC chemokines macrophage-inflammatory protein 1 alpha, eotaxin, and macrophage chemotactic protein 1 were detected in bronchoalveolar lavage fluids from v Delta B8R-infected mice; however, bronchoalveolar lavage fluids from v Delta B8R-35K-infected mice had lower levels of chemokines and a reduced chemotactic activity for murine leukocytes in vitro. These observations suggest that vCCI plays an important role in regulating leukocyte trafficking to the lungs during VV infection by binding to CC chemokines and blocking their chemotactic activities.