Comparison of Biochemical Properties of HIV-1 and HIV-2 Capsid Proteins.

Timely disassembly of viral core composed of self-assembled capsid (CA) in infected host cells is crucial for retroviral replication. Extensive in vitro studies to date on the self-assembly/disassembly mechanism of human immunodeficiency virus type 1 (HIV-1) CA have revealed its core structure and amino acid residues essential for CA-CA intermolecular interaction. However, little is known about in vitro properties of HIV-2 CA. In this study, we comparatively analyzed the polymerization properties of bacterially expressed HIV-1 and HIV-2 CA proteins. Interestingly, a much higher concentration of NaCl was required for HIV-2 CA to self-assemble than that for HIV-1 CA, but once the polymerization started, the reaction proceeded more rapidly than that observed for HIV-1 CA. Analysis of a chimeric protein revealed that N-terminal domain (NTD) is responsible for this unique property of HIV-2 CA. To further study the molecular basis for different in vitro properties of HIV-1 and HIV-2 CA proteins, we determined thermal stabilities of HIV-1 and HIV-2 CA NTD proteins at several NaCl concentrations by fluorescent-based thermal shift assays. Experimental data obtained showed that HIV-2 CA NTD was structurally more stable than HIV-1 CA NTD. Taken together, our results imply that distinct in vitro polymerization abilities of the two CA proteins are related to their structural instability/stability, which is one of the decisive factors for viral replication potential. In addition, our assay system described here may be potentially useful for searching for anti-CA antivirals against HIV-1 and HIV-2.

The intracellular pathogen Orientia tsutsugamushi responsible for scrub typhus induces lipid droplet formation in mouse fibroblasts.

Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L-929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogen-infection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets.

Decontamination of mycoplasma-contaminated Orientia tsutsugamushi strains by repeating passages through cell cultures with antibiotics.

BACKGROUND: Mycoplasmas-contamination of Orientia tsutsugamushi, one of the obligated intracellular bacteria, is a very serious problem in in vitro studies using cell cultures because mycoplasmas have significant influence on the results of scientific studies. Only a recommended decontamination method is to passage the contaminated O. tsutsugamushi strains through mice to eliminate only mycoplasmas under influence of their immunity. However, this method sometimes does not work especially for low virulent strains of O. tsutsugamushi which are difficult to propagate in mice. In this study, we tried to eliminate mycoplasmas contaminants from both high virulent and low virulent strains of the contaminated O. tsutsugamushi by repeating passage through cell cultures with antibiotics in vitro. RESULTS: We cultured a contaminated, high virulent strain of O. tsutsugamushi using a mouse lung fibroblasts cell line, L-929 cell in the culture medium containing lincomycin at various concentrations and repeated passages about every seven days. At the passage 5 only with 10 µg/ml of lincomycin, we did not detect mycoplasmas by two PCR based methods whereas O. tsutsugamushi continued good growth. During following four passages without lincomycin, mycoplasmas did not recover. These results suggested that mycoplasmas were completely eliminated from the high virulent strain of O. tsutsugamushi. Furthermore, by the same procedures with 10 µg/ml of lincomycin, we also eliminated mycoplasmas from a contaminated, low virulent strain of O. tsutsugamushi. Our additional assay showed that 50 µg/ml of lyncomycin did not inhibit the growth of O. tsutsugamushi, although MICs of many mycoplasmas contaminants were less than 6 µg/ml as shown previously. CONCLUSION: Our results showed an alternative method to eliminate mycoplasmas from the contaminated O. tsutsugamushi strains in place of in vivo passage through mice. Especially this notable method works for the decontamination not only from the high virulent strain also from the low virulent strain of O. tsutsugamushi. For further elimination, lincomycin at the limit concentration, which does not inhibit the growth of O. tsutsugamushi, can possibly eliminate most mycoplasmas from contaminated O. tsutsugamushi strains.

Tropism and pathogenicity of rickettsiae.

Rickettsiae are obligate intracellular parasitic bacteria that cause febrile exanthematous illnesses such as Rocky Mountain spotted fever, Mediterranean spotted fever, epidemic, and murine typhus, etc. Although the vector ranges of each Rickettsia species are rather restricted; i.e., ticks belonging to Arachnida and lice and fleas belonging to Insecta usually act as vectors for spotted fever group (SFG) and typhus group (TG) rickettsiae, respectively, it would be interesting to elucidate the mechanisms controlling the vector tropism of rickettsiae. This review discusses the factors determining the vector tropism of rickettsiae. In brief, the vector tropism of rickettsiae species is basically consistent with their tropism toward cultured tick and insect cells. The mechanisms responsible for rickettsiae pathogenicity are also described. Recently, genomic analyses of rickettsiae have revealed that they possess several genes that are homologous to those affecting the pathogenicity of other bacteria. Analyses comparing the genomes of pathogenic and non-pathogenic strains of rickettsiae have detected many factors that are related to rickettsial pathogenicity. It is also known that a reduction in the rickettsial genome has occurred during the course of its evolution. Interestingly, Rickettsia species with small genomes, such as Rickettsia prowazekii, are more pathogenic to humans than those with larger genomes. This review also examines the growth kinetics of pathogenic and non-pathogenic species of SFG rickettsiae (SFGR) in mammalian cells. The growth of non-pathogenic species is restricted in these cells, which is mediated, at least in part, by autophagy. The superinfection of non-pathogenic rickettsiae-infected cells with pathogenic rickettsiae results in an elevated yield of the non-pathogenic rickettsiae and the growth of the pathogenic rickettsiae. Autophagy is restricted in these cells. These results are discussed in this review.

Restriction of the growth of a nonpathogenic spotted fever group rickettsia.

The growth kinetics of pathogenic and nonpathogenic rickettsiae were compared to elucidate the mechanism responsible for the pathogenicity of rickettsiae. Vero and HeLa cells derived from mammals were inoculated with a nonpathogenic species of spotted fever group rickettsia, Rickettsia montanensis, before being infected with the pathogenic species Rickettsia japonica. The mammalian cells became persistently infected with R. montanensis and produced low levels of rickettsiae. On the other hand, superinfection of the R. montanensis-infected cells with R. japonica resulted in increased yields of R. montanensis accompanied by R. japonica growth. Both rickettsiae also grew well in the R. japonica-infected cells subjected to superinfection with R. montanensis. Western blotting with an antibody to the autophagy-related protein LC3B found that autophagy was induced in the cells infected with R. montanensis alone. On the contrary, autophagy was restricted in the cells that were co-infected with R. japonica. Electron microscopy of the cells infected with R. montanensis alone demonstrated rickettsia particles being digested in intracytoplasmic vacuoles. Conversely, many freely growing rickettsiae were detected in the co-infected cells.

Different interaction between HIV-1 Vif and its cellular target proteins APOBEC3G/APOBEC3F.

We examined a series of site-directed point mutants of human immunodeficiency virus type 1 (HIV-1) Vif for their interaction with cellular anti-viral factors APOBEC3G/APOBEC3F. Mutant viruses that display growth-defect in H9 cells did not counteract effectively APOBEC3G and/or APOBEC3F without exception, as monitored by single-cycle infectivity assays. While growth-defective mutants of Vif C-terminal region were unable to suppress APOBEC3G/APOBEC3F, some N-terminal region mutants did neutralize one of APOBEC3G/APOBEC3F. These data have suggested that members of APOBEC3 family other than APOBEC3G/APOBEC3F are not important for anti-HIV-1 activity. Furthermore, APOPEC3G/APOBEC3F were found to differently associate with Vif in virions as analyzed by equilibrium density centrifugation. Taken together, these results indicated that interaction of HIV-1 Vif and APOBEC3G is distinct from that between Vif and APOBEC3F.

Status of APOBEC3G/F in cells and progeny virions modulated by Vif determines HIV-1 infectivity.

We examined various HIV-1 Vif mutants for interaction with APOBEC3 proteins (A3G/A3F). All replication-defective proviral mutants were found to carry A3G/A3F in virions, and of these, a replication-defective mutant with Vif that binds to A3G in cells but not in virions was noted. Furthermore, a mutant Vif protein that suppresses A3F activity but does not exclude A3F from virions was identified. We also showed that incorporation of Vif into virions is dependent on its interaction with A3G/A3F. Taken together, we concluded that functional binding of Vif to A3G/A3F in cells and/or virions is critical for viral infectivity.

Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner.

Rickettsia conorii, an obligate intracellular tick-borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin- and caveolin-dependent endocytic events likely contribute to the internalization process.

Generation and characterization of APOBEC3G-positive 293T cells for HIV-1 Vif study.

We have established a number of 293T cell lines that express a human anti HIV-1 factor APOBEC3G. Out of seven cell clones examined, four were readily demonstrated to express APOBEC3G by immunoblotting analysis. In particular, two clones (A3G-C1 and -C4) were found to produce a much higher level of functional APOBEC3G relative to that by pooled cell clones. The transfection efficiency of all these cell clones were similar to that of the parental cells, producing a comparable level of virions upon transfection of wild type and vif-minus proviral DNA clones. Furthermore, the expression level of APOBEC3G in the best cell line (A3G-C1) was far much higher than those of an APOBEC3G-positive lymphocyte cell line and peripheral blood mononuclear cells. We finally monitored the incorporation of APOBEC3G into virions produced in A3G-C1. APOBEC3G was easily detected in progeny viral particles upon transfection of vif-minus proviral clone but not of wild type. These results indicated that our new A3G-C1 cell line is eminently useful for various studies on the interaction of human APOBEC3G and HIV-1 Vif.

A protein from a parasitic microorganism, Rickettsia prowazekii, can cleave the signal sequences of proteins targeting mitochondria.

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the alpha and beta subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with beta-MPP but barely contained any of the glycine-rich loop region characteristic of alpha-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast beta-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.

Generation of HIV-1 derivatives that productively infect macaque monkey lymphoid cells.

The narrow host range of human immunodeficiency virus type 1 (HIV-1) is caused in part by innate cellular factors such as apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) and TRIM5alpha, which restrict virus replication in monkey cells. Variant HIV-1 molecular clones containing both a 21-nucleotide simian immunodeficiency virus (SIV) Gag CA element, corresponding to the HIV-1 cyclophilin A-binding site, and the entire SIV vif gene were constructed. Long-term passage in a cynomolgus monkey lymphoid cell line resulted in the acquisition of two nonsynonymous changes in env, which conferred improved replication properties. A proviral molecular clone, derived from infected cells and designated NL-DT5R, was used to generate virus stocks capable of establishing spreading infections in the cynomolgus monkey T cell line and CD8-depleted peripheral blood mononuclear cells from five of five pig-tailed macaques and one of three rhesus monkeys. NL-DT5R, which genetically is >93% HIV-1, provides the opportunity, not possible with currently available SIV/HIV chimeric viruses, to analyze the function of multiple HIV-1 genes in a broad range of nonhuman primate species.

Effects of lysine to arginine mutations in HIV-1 Vif on its expression and viral infectivity.

We previously demonstrated that the expression in cells of human immunodeficiency virus type 1 (HIV-1) Vif is maintained at low level by proteasome-degradation. We examined the contribution of 16 lysines present in Vif (NL432 clone), which is composed of 192 amino acids (aa), to its expression within cells and to viral infectivity for non-permissive cells. To this end, various lysine-arginine mutations were introduced into wild-type (wt) Vif, and the mutational effects were monitored by transfection experiments. When all the lysines were changed to arginines, the mutant Vif was expressed in cells at much higher level than wt and was much more stable. Both N-terminal (aa nos. 34 and 36) and C-terminal (aa nos. 179 and 181) lysines were found to be almost sufficient for wt property. Different from this observation, one of the lysines at aa nos. 22 and 26 was demonstrated to be essential for the virus to grow in non-permissive cells. Our results showed that there is no clear co-relationship between the expression level of HIV-1 Vif and viral infectivity.

Morphological study on biologically distinct vpx/vpr mutants of HIV-2.

We have previously shown that human immunodeficiency virus type 2 (HIV-2) without functional vpx and vpr genes is severely defective for viral growth in lymphocytic cells, and suggested that the virions produced in the absence of Vpx and Vpr are critically damaged. To examine the nature of replication-defect for the vpx/vpr double mutant, we quantitatively and morphologically studied the virions produced in cells transfected or infected with wild type clone, single (vpx and vpr mutants) or the double mutant. While no significant difference in virion production was found for various virus clones in transfected cells, a major growth retardation in infected cells was readily observed for the vpx and vpx/vpr mutants. In particular, no viral growth was detected for the double mutant. By contrast to the very distinct growth characteristics of the three mutant clones, no appreciable difference in virion morphology was noted. These results indicated that Vpx and Vpr of HIV-2 may cooperatively contribute to virion infectivity without affecting virion morphogenesis.

Construction of gag-chimeric viruses between HIV-1 and SIVmac that are capable of productive multi-cycle infection.

Forty-nine recombinant viral clones between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus from the rhesus monkey (SIVmac), which carry chimeric gag (capsid/p2 region) genes in the background of the HIV-1 genome, were constructed to establish an HIV-1/monkey infection model system for human AIDS. Upon transfection, all the recombinants generated progeny virions at a level comparable to the parental HIV-1 clone and no major abnormalities were found in the virions, as examined by Western blot analysis. In infection experiments, 18 recombinants grew in human lymphocytic cells and six of these clones propagated as well as the parental virus, as monitored by virion associated-reverse transcriptase production. By contrast, none of the recombinants grew at a detectable level in monkey lymphocytic cells. The defective replication site(s) in human cells for non-infectious recombinants was mapped to the step before and/or during reverse transcription. Our results described here showed that HIV-1 type chimeric viruses between HIV-1 and SIVmac, which are capable of spreading productive infection, are readily constructed throughout the capsid/p2 region. In addition, it is suggested that there may be a viral determinant(s), other than Gag, responsible for the species-specific tropism of HIV-1 and which is associated with viral DNA synthesis.

The major outer membrane protein rOmpB of spotted fever group rickettsiae functions in the rickettsial adherence to and invasion of Vero cells.

The role of one of the major outer membrane proteins, rOmpB, of spotted fever group rickettsiae was examined. Antibodies generated against native rOmpB inhibited plaque formation by Rickettsia japonica in Vero cells when applied at the time of inoculation of the rickettsiae. However, antibodies to heat-denatured rOmpB did not. Moreover, the soluble recombinant rOmpB also inhibited plaque formation to some extent. Thus it seems that rOmpB functions at least in the adherence of rickettsiae to host cells. To obtain direct evidence of its function in the adherence to and invasion of Vero cells, we generated Escherichia coli transformed by the vector pET-22b(+) inserted with the ompB open reading frame of R. japonica. The recombinant bacteria expressed a 165-kDa protein consistent with the precursor of rOmpB. The protein reacted with monoclonal antibodies to heat-labile epitopes of rOmpB. Immunofluorescence of the recombinant bacteria demonstrated surface expression of the protein. It was shown by light microscopy and transmission and scanning electron microscopy that the bacteria adhered to and invaded Vero cells. Thus, although the recombinant precursor rOmpB was not processed on the outer membrane of E. coli, it functions during these steps. The manner of entry was similar to that of rickettsiae although at a slower rate.

Comparative study on the structure and cytopathogenic activity of HIV Vpr/Vpx proteins.

The three-dimensional (3-D) structure of human immunodeficiency virus type 2 (HIV-2) Vpr/Vpx was predicted by homology modeling based on the NMR structure of human immunodeficiency virus type 1 (HIV-1) Vpr. The three proteins similarly have three major amphipathic alpha-helices. In contrast to HIV-1 Vpr, Vpr/Vpx of HIV-2 have a long N-terminal loop and clustered prolines in the second half of the C-terminal loop. HIV-2 Vpx uniquely contains a long region between the second and third major helices, and bears several glycines in the first half of the C-terminal loop. Instead of the glycines, there is a group of hydrophilic amino acids and arginines in the corresponding regions of the two Vprs. To compare the cytopathogenic potentials of HIV-1 Vpr and HIV-2 Vpr/Vpx, we examined the production of luciferase as a marker of cell damage. We further analyzed the characteristics of cells transduced with vpr/vpx genes driven by an inducible promoter. The results obtained clearly show that structurally similar, but distinct, HIV Vpr/Vpx proteins are detrimental to target cells.

Establishment of a biological assay system for human retroviral protease activity.

In order to obtain indicator cell lines that are exquisitely susceptible to human T-lymphotropic virus type 1 (HTLV-1), luciferase gene driven by HTLV-1 long terminal repeat (LTR) was transfected into lymphocytic H9 cells with neo gene, and cell lines were selected by G418. A cell line (H9/K30luc) was found to produce an extremely high level of luciferase only when co-cultured with HTLV-1 producer MT-2 cells. Both in the absence and presence of a reverse transcriptase (RT) inhibitor azidothymidine, H9/K30luc cells generated similarly high luciferase activity upon co-cultivation with MT-2 cells. To develop an equivalent system for human immunodeficiency virus type 1 (HIV-1), H9/NL432 cells, which are stably infected with HIV-1 and producing a low level of the virus-like MT-2 cells for HTLV-1, were generated. Together with the indicator cell line H9/H1luc for HIV-1 already reported, antiviral effects of some agents on HTLV-1 and HIV-1 could be readily and sensitively evaluated by similar methods. In fact, by using our system, an HIV-1 protease inhibitor, saquinavir, was demonstrated to be highly effective against HIV-1 but not against HTLV-1.

Unique characteristics of HIV-1 Vif expression.

We examined the steady-state expression in cells of four accessory proteins of human immunodeficiency virus type 1 (HIV-1). For this purpose, a series of single gene expression vectors for these viral proteins were constructed and were monitored for their production by transfection. Among them, the expression level of Vif was found to be lowest in both the absence and presence of APOBEC3G. In addition, we noticed the presence of its truncated form, which was not observed for the other accessory proteins. When a subgenomic vector was used for transfection, authentic and several small forms of Vif were produced. By mutational analysis, these forms were demonstrated to be mutant Vif proteins translated from M8, M16 and M29. When a full-length molecular clone was used, the smaller versions of Vif were hardly observed. Functional analysis of these mutant Vif proteins showed that they are incapable of modulating viral infectivity. The results described above, i.e. the low steady-state expression and the presence of truncated forms, represent the unique characteristics of HIV-1 Vif.