Rous sarcoma virus RNA stability requires an open reading frame in the gag gene and sequences downstream of the gag-pol junction.

The intracellular accumulation of the unspliced RNA of Rous sarcoma virus was decreased when translation was prematurely terminated by the introduction of nonsense codons within its 5' proximal gene, the gag gene. Subcellular fractionation of transfected cells suggested that nonsense codon-mediated instability occurred in the cytoplasm. Analysis of constructs containing an in-frame deletion in the nucleocapsid domain of gag, which prevents interaction between the Gag protein and viral RNA, showed that an open reading frame extending to approximately 30 nucleotides from the natural gag termination codon was needed for RNA stability. Sequences at the gag-pol junction necessary for ribosomal frameshifting were not required for RNA stability; however, sequences located 100 to 200 nucleotides downstream of the natural gag termination codon were found to be necessary for stable RNA. The stability of RNAs lacking this downstream sequence was not markedly affected by premature termination codons. We propose that this downstream RNA sequence may interact with ribosomes translating gag to stabilize the RNA.

Role of endogenous murine leukemia virus in immunologically triggered lymphoreticular tumors. I. Development and use of oncogenic cellfree preparations serially passaged in vivo.

Cellfree extracts (CFEs) prepared from (BALB/cJ X A/J)F1 (CAF1) and (BALB/cJ X C57BL/6J)F1 (CB6F1) mice in which a graft-versus-host reaction (GVHR) has been induced are known to be oncogenic, but only after a protracted latent period (mean, 16 mo). Serial passage of such CFEs in successive generations of syngeneic mice inoculated at birth led to the development of two separate oncogenic preparations, the CA serioes in CAF, mice and the CB series in CB6F, mice, in which the mean latent period was reduced to 6 and 12 months, respectively. Both oncogenic preparations contained infectious B-tropic murine leukemia virus (MuLV) and particles with the ultrastructural characteristics of MuLV. No other kind of virus particle was seen. When these preparations were injected into infant syngeneic mice, B-tropic MuLV could be detected in the reticular tissues as early as 2 weeks thereafter. The virus persisted in the reticular tissues and was present in the lymphoreticular tumors that subsequently developed. However, if the same preparation was injected into young adult recipients, there may have been transient MuLV replication, but the virus subsequently disappeared from the reticular tissues and no lymphoreticular tumors developed. Previous experiments showed that MuLV was present in CFEs prepared from CAF, animals with the GVHR but absent in those of normal control mice. Since the lymphoreticular tumors arising in mice with the GVHR were the same as those induced by the CA and CB MuLV preparations, it was concluded that tumorigenesis in mice with the GVHR was caused by endogenous B-tropic MuLV activated by the immunologic disturbance.

Mycobacteria-containing phagosomes associate less annexins I, VI, VII and XI, but not II, concomitantly with a diminished phagolysosomal fusion.

We have studied the intracellular localization of annexins I,II, VI, VII, and XI in cells containing latex beads or Mycobacterium avium at different times after ingestion in order to establish whether a correlation existed between the association of annexins to phagosomes and phagolysosomal fusion, since the intracellular survival of mycobacteria is linked to an impairment of phagosome maturation. We demonstrate an important decrease in the levels of association of annexins I, VI, VII and XI, but not II to phagosomes containing either live or killed mycobacteria compared with phagosomes containing inert latex particles. The reduced association of annexins observed was detected only on M. avium-containing phagosomes and not in other cell membrane nor in cytosolic fractions from infected cells, and was apparent from 8 hours through to 4 days after phagocytosis. These findings add elements to the present knowledge of the phagosomal modifications that accompany the survival of intracellular pathogens, suggesting that annexins I, VI, VII, and XI play a secondary role in phagosomal fusion events while annexin II does not seem to be related to the mechanism of regulation of endolysosomal fusion.

Quantitation of HIV-1 proviral DNA in cells from cerebrospinal fluid.

The number of proviral copies in the cerebrospinal fluid of 13 patients in various stages of HIV-1 infection was determined using the polymerase chain reaction. HIV-1 proviral DNA was detected at a median value of 1 provirus per 300 cells with a range from 1 per 20 to 1 per 2,400 cells and a mean value of 1 provirus per 570 cells. Interestingly, this indicates a higher proportion of infected cells in cerebrospinal fluid than is reported to be the case for blood mononuclear cells. However, a clear correlation of these findings to the CDC stage or to neurologic complications was not obvious.

Cellular antibiotic pharmacology.

Many perinatal pathogens are able to survive and in some cases replicate intracellularly. With the exception of viruses and toxoplasma, these pathogens principally infect phagocytic cells of the reticuloendothelial system. Such intracellular organisms, by evading the effects of antibiotics that act only extracellularly, may respond poorly to conventional therapy. Of currently available antibiotics, rifampin, chloramphenicol and trimethoprim are the most active intracellularly. Other antibiotics are either taken up by cells but appear to be inactive intracellularly (lincomycin) or are excluded from cells (penicillins, cephalosporins, aminoglycosides). The clinical role of antibiotics that are active intracellularly is not clear; anecdotal human experience and limited controlled animal experience suggests that they may be useful in the treatment of some infections. Because of the decreased microbicidal activity of newborn phagocytes, intracellular activity of antibiotics may be of greater importance than in older patients. Further study is needed to answer these questions. Methods of enhancing intracellular activity of antibiotics are available should this property prove to be desirable.

Subcellular localization of the 65-kDa heat shock protein in mycobacteria by immunoblotting and immunogold ultracytochemistry.

The 65-kDa heat shock protein (hsp65) is an immunodominant antigen in mycobacterial infections and also the key etiologic factor in mycobacteria-induced autoimmune arthritis. Because the subcellular distribution of hsp65 in the mycobacteria may be relevant to understand its immunoreactivity, we have investigated the presence of hsp65 in the envelope and cytoplasmic compartments of the bacilli. Anti-hsp65 antibodies were used in western blottings to investigate the presence of hsp65 in cell fractions (membrane, envelope and cytosol) of Mycobacterium avium and M. smegmatis, and also to label hsp65 in situ by the immunogold method on thin-sectioned mycobacteria, including the non-cultivable M. leprae, that were studied by transmission electron microscopy. All of the three subcellular mycobacterial fractions showed significant labelling by anti-hsp65 antibodies. Immunogold ultracytochemistry revealed the presence of hsp65 in both the cytoplasm and the envelope of mycobacteria. The data indicate that hsp65 molecules are commonly present not only in the cytoplasm but also in the envelope of mycobacteria. The latter topography of hsp65 may contribute to the strong immunogenicity of hsp65 since it may correspond to export hsp65 molecules captured before being secreted into the extracellular milieu, thus making hsp65 a mycobacterial antigen readily available for presentation to the immune system of infected hosts.

Intracellular localization of recombinant vaccinia virus produced HIV antigens and their use for confirmation of HIV seropositivity.

Recombinant vaccinia strains vC5 and vE234L expressing the rp50 and rgp160/rgp120 recombinant proteins were used in immunoblot and immunofluorescence assays. No false reactions were found, although 30 sera giving false reactivity with gag or env encoded proteins in a commercial immunoblot assay were included into the test panel. We recommend the recombinant protein-based assay for confirmation and discrimination of HIV seropositivity.

Heterologous expression of a gene encoding a 35 kDa protein of Mycobacterium avium paratuberculosis in Escherichia coli.

The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total protein of E. coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response.

[Early stages in the replication of aphthous fever virus. Kinetics of partial degradation and sites for the establishment of penetrating residual virus]. / Etapas tempranas en la replicación del virus de la fiebre aftosa. (VFA). Cinética de la degradación parcial y sitios de estabilización del virus penetrante residual.

At input multiplicities between 5-10 UFP/cell FMDV O1 Caseros adsorb in BHK21 Clon 13 cells at a rate of 5-20% depending whether cells are in suspension or in monolayers. At least four washings with medium are required to eliminate non specifically adsorbed virions. The remaining attached virus appears to be in contact with "specific" receptors of the cytoplasmic membrane. After penetration, 80% of virus became degraded to slow molecular weight material which is detected at first in the subcellular fraction and is gradually excreted out of the cells. The degradation process occurs from 0 to 15 minutes and it is demonstrated by a parallel decrease in infectivity and stability of purified labelled FMDV O1 Caseros. The 20% remaining virus is found firmly attached to a subcellular fraction precipitable at 15,000 xg. Parental residual infectivity remains remarkably stable for 90 min at which time newly synthetized RNA appears. Fraction of 15,000 xg from infected cultures was obtained at 60 min PI and incubated in vitro at 37 C. The infectivity remains unaltered for a period of 120 min even if the preparation is treated with 0.2% of triton x 100. Fraction of 15,000 xg from infected and control cultures obtained at different time PI were seeded on a linear sucrose gradient. A sharp peak of acid phosphatase associated with the lower mobility bands indicates the presence of intact lysosome structures. A shoulder of the enzyme activity is detected between the two main protein bands. Associated with the region rich in unbroken lysosomes, a very sharp peak of infectivity and/or 3H uridine or 32P labelled incoming virus can be detected. A different pattern of the residual incoming infectivity is found associated with the more rapid sedimentation protein band. 3H uridine incorporation at the beginning of viral RNA synthesis initiation shows that an important amount of newly synthesized viral RNA is found in the lower mobility band of the 15,000 xg fraction. Some incorporation is also detected in the faster band. The results presented here suggest that some functional activity is associated with the residual virus present at the beginning of infection in the 15,000 xg fraction. It can also be accepted that this virus penetrates by pinocytosis of specific receptors at the cytoplasmic membrane.

[Abortive influenza virus infection of Ehrlich cells. Virion and subcellular fraction proteins]. / Abortivnaia infektsiia virusa grippa v kletkakh Erlikha. Belki virionov i subkletochnykh fraktsii.

Ehrlich ascitic carcinoma cells infected with the WSN strain of influenza virus produced noninfectious virus particles containing no M protein. Polypeptides P1, NP, and NS were synthesized in the infected ascites cells to the same concentration was in permissive cells (chick fibroblasts and MDCK cells); the synthesis of HA and M proteins was markedly disturbed, however. In the permissive cells, M protein was found in the fraction of the perinuclear martial and plasma membranes whereas in the ascitic cells M protein was detected only in perinuclear material but not in plasma membrane. Thus, nonpermissiveness of the ascitic cells appears to be due to disturbance of both synthesis and transport of M protein to the plasma membrane.

Salmonella induced IL-23 and IL-1beta allow for IL-12 production by monocytes and Mphi1 through induction of IFN-gamma in CD56 NK/NK-like T cells.

BACKGROUND: The type-1 cytokine pathway plays a pivotal role in immunity against intracellular bacterial pathogens such as Salmonellae and Mycobacteria. Bacterial stimulation of pattern recognition receptors on monocytes, macrophages and dendritic cells initiates this pathway, and results in the production of cytokines that activate lymphocytes to produce interferon (IFN)-gamma. Interleukin (IL)-12 and IL-23 are thought to be the key cytokines required for initiating a type-1 cytokine immune response to Mycobacteria and Salmonellae. The relative contribution of IL-23 and IL-12 to this process is uncertain. METHODOLOGY/PRINCIPAL FINDINGS: We show that various TLR agonists induce the production of IL-23 but not IL-12 in freshly isolated human monocytes and cultured human macrophages. In addition, type 1 pro-inflammatory macrophages (Mphi1) differentiated in the presence of GM-CSF and infected with live Salmonella produce IL-23, IL-1beta and IL-18, but not IL-12. Supernatants of Salmonella-infected Mphi1 contained more IL-18 and IL-1beta as compared with supernatants of Mphi1 stimulated with isolated TLR agonists, and induced IFN-gamma production in human CD56(+) cells in an IL-23 and IL-1beta-dependent but IL-12-independent manner. In addition, IL-23 together with IL-18 or IL-1beta led to the production of GM-CSF in CD56(+) cells. Both IFN-gamma and GM-CSF enhanced IL-23 production by monocytes in response to TLR agonists, as well as induced IL-12 production. CONCLUSIONS/SIGNIFICANCE: The findings implicate a positive feedback loop in which IL-23 can enhance its release via induction of IFN-gamma and GM-CSF. The IL-23 induced cytokines allow for the subsequent production of IL-12 and amplify the IFN-gamma production in the type-1 cytokine pathway.

Intracellular trafficking of Parachlamydia acanthamoebae.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that naturally infects free-living amoebae. It is a potential agent of pneumonia that resists destruction by human macrophages. However, the strategy used by this Chlamydia-like organism in order to resist to macrophage destruction is unknown. We analysed the intracellular trafficking of P. acanthamoebae within monocyte-derived macrophages. Infected cells were immunolabelled for the bacteria and for various intracellular compartments by using specific antibodies. We analysed the bacteria colocalization with the different subcellular compartments by using epifluorescence and confocal microscopy. Bacterial replication took place 4-6 h post infection within acidic vacuoles. At that time, P. acanthamoebae colocalized with Lamp-1, a membrane marker of late endosomal and lysosomal compartments. A transient accumulation of EEA1 15 min post infection, and of rab7 and the mannose 6-phosphate receptor 30 min post infection confirmed that P. acanthamoebae traffic through the endocytic pathway. The acquisition of Lamp-1 was not different after infection with living and heat-inactivated bacteria. However, 24.5% and 79.5% of living and heat-inactivated P. acanthamoebae, respectively, colocalized with the vacuolar proton ATPase. Moreover, P. acanthamoebae did not colocalized with cathepsin D, a lysosomal hydrolase, suggesting that P. acanthamoebae interferes with maturation of its vacuole. Thus, P. acanthamoebae survives to destruction by human macrophages probably by controlling the vacuole biogenesis.

Mode of penetration and intracellular localization of incoming parental foot and mouth disease virus (FMDV) in BHK cells.

The process of penetration and subsequent early stages of replication of Foot and Mouth Disease Virus (FMDV) in BHK21 cell cultures have been studied in order to obtain further data about the infectious cycle of this virus. Results suggest that FMDV penetrates BHK21 cells by way of pinocytic vesicles. Studies of lysosomal (LF) and supernatant (SF) fractions of homogenized suspension of infected cells were carried out to learn the percentage of possible non-specific absorption of infectious virus particles. Furthermore, analysis of intracellular virus shows that LF-associated virus is not completely degraded by the enzymatic process expressed by cellular proteases and RNAses and can be potentially useful for genetic expression. Data suggest that: i) only a part of LF-associated virus replicates; ii) the remaining LF-associated virus may be a reservoir of potentially active virus.

Suppression of endocytosis in neutrophils by influenza A virus in vitro.

The effect of influenza A virus on the endocytic pathway in polymorphonuclear leukocytes (PMNLs) and the relationship of altered endocytic activity to virus-induced inhibition of other PMNL functions were examined with virus that caused decreased phagosomelysosome fusion and bacterial killing (depressing virus [DV]) and virus that did not (non-DV). Binding of both viruses to PMNL surface receptors was similar, but uptake of DV into PMNLs was decreased compared with that of non-DV. Both viruses were associated with the PMNL plasma membrane and were in endosomes. DV caused less stimulation of pinocytosis than did non-DV. The rate of exocytosis of fluoresceinated-dextran (FL-dextran) from cells stimulated with DV was significantly less than for non-DV. When PMNLs were pretreated with buffer, DV, or non-DV and then exposed to FL-dextran and N-formylmethionylleucylphenylalanine, the pinocytosis of FL-dextran was significantly less in cells pretreated with DV as compared with non-DV or buffer.

Expression of the 16K cistron of tobacco rattle virus in protoplasts.

An antiserum was raised against a synthetic peptide corresponding to the 18 C-terminal amino acids of a putative 16K protein encoded by the 3'-terminal open reading frame of tobacco rattle virus (TRV) RNA-1. This antiserum was used to demonstrate expression of the 16K cistron in vivo. TRV-infected tobacco protoplasts accumulated similar amounts of 16K protein and viral coat protein but in tobacco plants only the coat protein was detectable. Time course experiments revealed that in protoplasts the accumulation of 16K protein lagged somewhat behind that of coat protein. The 16K protein was incorporated in a high-molecular-weight cellular component that was resistant to treatment with nonionic detergents.

Viral RNA synthesis in tomato spotted wilt virus-infected Nicotiana rustica plants.

The synthesis of viral RNA species in tomato spotted wilt virus-infected Nicotiana rustica plants was followed in terms of time and relative abundance. Systemic symptoms were visible after 4 days postinoculation (p.i.), but viral (v) and viral-complementary (vc) strands of all three genomic RNA segments [large (L) RNA, medium (M) RNA and small (S) RNA] were detected from 2 days p.i. In addition, two subgenomic mRNAs, derived from S RNA, were detected. For the L RNA segment no subgenomic mRNAs were detected, suggesting that this segment is expressed via the synthesis of a genome-sized vc mRNA. A possible M-specific subgenomic mRNA was detected, showing a similar time course of appearance as the subgenomic mRNAs derived from the S RNA segment. Analysis of cytoplasmic RNA fractions revealed that both v and vc strands of all three genomic segments associate with the nucleocapsid protein into nucleocapsid structures, the vcRNA species being present in lower amounts. Intact, enveloped virus particles contained only the v strand of the L RNA segment and, surprisingly, both v and vc strands of the M and S RNA segment, though in different ratios.