Analysis of two strains of Friend murine leukemia viruses differing in ability to induce early splenomegaly: lack of relationship with generation of recombinant mink cell focus-forming viruses.

Friend murine leukemia helper viruses (F-MuLV) 57 and B3 were indistinguishable by genomic structural analyses with RNase T1-resistant oligonucleotide fingerprinting and by antigenic reactivity with a panel of 31 monoclonal antibodies directed against murine leukemia viruses. Nevertheless, F-MuLV 57 and B3 had strikingly different virulences. Approximately 2 months after inoculation, IRW and NFS/N mice inoculated as newborns with F-MuLV 57 had gross splenomegaly caused by erythroid proliferation. In contrast, an equivalent dose of F-MuLV B3 induced spleen or lymph node enlargement 4 to 13 months after inoculation. Although most cases of spleen enlargement in F-MuLV B3-inoculated mice were due to erythroid proliferation, lymphoid or myeloid proliferation was also frequently observed. The replication of both F-MuLV 57 and B3 was equally efficient, and both viruses generated recombinant dual-tropic mink cell focus-forming (MCF) viruses with the same kinetics and efficiency. Moreover, MCF viruses induced by F-MuLV 57 and B3 had the same antigenic patterns. Therefore, the ability of F-MuLV to induce early splenomegaly did not correlate with the generation of recombinant MCF viruses.     

Separate sequences in a murine retroviral envelope protein mediate neuropathogenesis by complementary mechanisms with differing requirements for tumor necrosis factor alpha

The innate immune response, through the induction of proinflammatory cytokines and antiviral factors, plays an important role in protecting the host from pathogens. Several components of the innate response, including tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein 1, interferon-inducible protein 10, and RANTES, are upregulated in the brain following neurovirulent retrovirus infection in humans and in animal models. However, it remains unclear whether this immune response is protective, pathogenic, or both. In the present study, by using TNF-alpha(-/-) mice we analyzed the contribution of TNF-alpha to neurological disease induced by four neurovirulent murine retroviruses, with three of these viruses encoding portions of the same neurovirulent envelope protein. Surprisingly, only one retrovirus (EC) required TNF-alpha for disease induction, and this virus induced less TNF-alpha expression in the brain than did the other retroviruses. Analysis of glial fibrillary acidic protein and F4/80 in EC-infected TNF-alpha(-/-) mice showed normal activation of astrocytes but not of microglia. Thus, TNF-alpha-mediated microglial activation may be important in the pathogenic process initiated by EC infection. In contrast, TNF-alpha was not required for pathogenesis of the closely related BE virus and the BE virus induced disease in TNF-alpha(-/-) mice by a different mechanism that did not require microglial activation. These results provide new insights into the multifactorial mechanisms involved in retrovirus-induced neurodegeneration and may also have analogies to other types of neurodegeneration.     

N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34-94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.     

Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment

Upon entry into mammalian cells, the intracellular pathogen Brucella abortus resides within a membrane-bound compartment, the Brucella -containing vacuole (BCV), the maturation of which is controlled by the bacterium to generate a replicative organelle derived from the endoplasmic reticulum (ER). Prior to reaching the ER, Brucella is believed to ensure its intracellular survival by inhibiting fusion of the intermediate BCV with late endosomes and lysosomes, although such BCVs are acidic and accumulate the lysosomal-associated membrane protein (LAMP-1). Here, we have further examined the nature of intermediate BCVs using confocal microscopy and live cell imaging. We show that BCVs rapidly acquire several late endocytic markers, including the guanosine triphosphatase Rab7 and its effector Rab-interacting lysosomal protein (RILP), and are accessible to fluid-phase markers either delivered to the whole endocytic pathway or preloaded to lysosomes, indicating that BCVs interact with late endosomes and lysosomes. Consistently, intermediate BCVs are acidic and display proteolytic activity up to 12 h post-infection. Expression of dominant-negative Rab7 or overexpression of RILP significantly impaired the ability of bacteria to convert their vacuole into an ER-derived organelle and replicate, indicating that BCV maturation requires interactions with functional late endosomal/lysosomal compartments. In cells expressing dominant-negative Rab7[T22N], BCVs remained acidic, yet displayed decreased fusion with lysosomes. Taken together, these results demonstrate that BCVs traffic along the endocytic pathway and fuse with lysosomes, and such fusion events are required for further maturation of BCVs into an ER-derived replicative organelle.     

Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages.

Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.     

The B12/23 restriction is critically dependent on recombination signal nonamer and spacer sequences

Ag receptor variable region gene assembly is initiated through the formation of a synaptic complex which minimally includes the recombination-activating gene (RAG) 1/2 proteins and a pair of recombination signals (RSs) flanking the recombining gene segments. RSs are composed of conserved heptamer and nonamer sequences flanking relatively nonconserved spacers of 12 or 23 bp. RSs regulate variable region gene assembly within the context of the 12/23 rule which mandates that recombination only occurs between RSs of dissimilar spacer length. RSs can exert additional constraints on variable region gene assembly beyond imposing spacer length requirements. At a minimum this restriction, termed B12/23, is imposed on the Vbeta to DJbeta rearrangement step by the 5' Dbeta RS and is enforced at or before the DNA cleavage step of the V(D)J recombination reaction. In this study, the components of the 5' Dbeta RS required for enforcing the B12/23 rule are assessed on chromosomal substrates in vivo in the context of normal murine thymocyte development and on extrachromosomal substrates induced to undergo recombination in nonlymphoid cell lines. These analyses reveal that the integrity of the nonamer sequence as well as the highly conserved spacer nucleotides of the 5' Dbeta1 RS are critical for enforcing the B12/23 restriction. These findings have important implications for understanding the B12/23 restriction and the manner in which RS synaptic complexes are assembled in vivo.     

Settlement inhibition of fouling invertebrate larvae by metabolites of the marine bacterium halomonas marina within a polyurethane coating

The marine bacterium, Halomonas marina (ATCC 27129), was shown to inhibit settlement and development of the sessile invertebrates Balanus amphitrite and Bugula neritina. Different bacterial treatments were employed to investigate this interaction. Filmed bacteria and liquid suspensions of whole cells, lysed cells and culture filtrate all reduced settlement of B. amphitrite. Polyurethane coatings containing whole cells were partially inhibitory while lysed cells caused complete inhibition of B. amphitrite larval settlement. In contrast, culture filtrate in a polyurethane matrix stimulated settlement of B. amphitrite larvae. Whole cells, culture filtrate, and lysed cells embedded in a polyurethane coating also controlled B. neritina settlement and maturation.     

Use of a focal infectivity assay for testing susceptibility of HIV to antiviral agents

A highly sensitive and quantitative focal immunoassay has been developed for detecting the human immunodeficiency virus (HIV). The assay can be used to measure cell-free virus or the production of HIV by virus-infected cells. Both laboratory-adapted strains of HIV and patient isolates can be studied with this assay. In this communication, we demonstrate the utility of this assay for measuring the effects of anti-HIV agents on viral isolates. We show that the anti-viral effects of such diverse agents as azidothymidine, interferon-alpha, immunotoxins, soluble CD4 and antibody can be accurately quantified. This assay may be used in the discovery and evaluation of new anti-HIV therapies or may be adapted for use in testing the sensitivity of patient isolates to standard therapeutic agents.     

Characterization and localization of myosin in the brush border of intestinal epithelial cells

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.