An antiviral disulfide compound blocks interaction between arenavirus Z protein and cellular promyelocytic leukemia protein.

The promyelocytic leukemia protein (PML) forms nuclear bodies (NB) that can be redistributed by virus infection. In particular, lymphocytic choriomeningitis virus (LCMV) influences disruption of PML NB through the interaction of PML with the arenaviral Z protein. In a previous report, we have shown that the disulfide compound NSC20625 has antiviral and virucidal properties against arenaviruses, inducing unfolding and oligomerization of Z without affecting cellular RING-containing proteins such as the PML. Here, we further studied the effect of the zinc-finger-reactive disulfide NSC20625 on PML-Z interaction. In HepG2 cells infected with LCMV or transiently transfected with Z protein constructs, treatment with NSC20625 restored PML distribution from a diffuse-cytoplasmic pattern to punctate, discrete NB which appeared identical to NB found in control, uninfected cells. Similar results were obtained in cells transfected with a construct expressing a Z mutant in zinc-binding site 2 of the RING domain, confirming that this Z-PML interaction requires the integrity of only one zinc-binding site. Altogether, these results show that the compound NSC20625 suppressed Z-mediated PML NB disruption and may be used as a tool for designing novel antiviral strategies against arenavirus infection.

LCMV-mediated hepatitis in rhesus macaques: WE but not ARM strain activates hepatocytes and induces liver regeneration.

Lymphocytic chorimeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), causative agent of Lassa hemorrhagic fever (LHF), belong to the Old World group of the family Arenaviridae. Both viruses have extensive strain diversity and significant variations in lethality and pathogenicity for man and experimental animals. We have shown that the LHF-like infection of rhesus macaques with the WE strain of LCMV affects liver functions, induces hepatocyte proliferation, and causes a rise in IL-6 and soluble TNF receptors (sTNFR) concomitant with a rise in viremia. The levels of IL-6 and sTNFR can serve as an additional diagnostic tool for liver involvement in pathogenesis of arenavirus infection. Mucosal inoculation of rhesus macaques with LCMV-WE can result in attenuated infection with a transient viremia and liver enzyme abnormalities. The ARM strain of LCMV shares 88% amino acid homology with WE. In contrast to LCMV-WE, ARM strain does not induce manifested disease in monkeys, does not affect liver functions, and does not induce hepatocyte proliferation. Previously we demonstrated that LCMV-ARM infection protected rhesus macaques challenged with LCMV-WE. Here we have shown that the protected animals have no signs of hepatitis and hepatocyte proliferation.

Macaque models for early immunosuppression and T cell loss during acute immunodeficiency virus infections.

The interval of acute infection with immunodeficiency viruses is critically important for determining the long-term rate of disease progression. The steps of initial infection, systemic dissemination, and explosive replication of pathogenic SIV or SHIV in macaques are being mapped to show the mechanisms responsible for remodeling host immunity, for establishing the persistent infection, and for promoting disease progression. Here, we describe recent studies on two ways in which CD4+ T cell populations are depleted during acute infection. Initially, we discuss recent work on the mechanisms for CD4+ T cell-mediated, MHC-unrestricted cytolysis. This mechanism shows how even soluble viral antigens such as the envelope glycoprotein, can prime CD4+ lymphocytes to be both effector and target cells in an unrestricted cytolysis mechanism. The consequence of unrestricted cytolysis is a more rapid destruction of the CD4+ T cell population. Secondly, we discuss the broader issue of T cell hyperactivation during acute infection. Inappropriate activation of this lymphocyte population renders cells susceptible to activation induced cell death and also increases the rate of virus replication. Macaque immunization studies have shown a clear role for extracellular Tat in hyperactivation. These two mechanisms, unrestricted cytolysis and T cell hyperactivation, are components of the acute infection that remodel host immunity and dictate the rate of progression to AIDS.

Mucosal immunization with Salmonella typhimurium expressing Lassa virus nucleocapsid protein cross-protects mice from lethal challenge with lymphocytic choriomeningitis virus.

OBJECTIVES: Lassa fever virus (LAS) is transmitted to man by rodent carriers and is fatal in a third of untreated cases. Our goal is to provide immune protection from Lassa fever by mucosal vaccination. STUDY DESIGN/METHODS: Mice were vaccinated intragastrically with control vectors or with vectors (vaccinia or Salmonella) expressing LAS nucleocapsid protein (NP). Mice were challenged intracranially with a lethal dose of the related arenavirus, lymphocytic choriomeningitis virus (LCMV), as a measure of the vaccine's ability to elicit cross-protection. RESULTS: Salmonella and vaccinia vectors expressing LAS NP each protected a third of the mice from lethal challenge with LCMV. All mice vaccinated with a vector expressing LCMV NP were protected as expected. CONCLUSIONS: The LAS recombinant Salmonella vector is comparable to the LAS recombinant vaccinia vector in its ability to cross-protect mice from lethal challenge. Nucleocapsid protein is an inadequate immunogen on its own, but provides sufficient cross-protection to make it a useful component of a broadly reactive arenavirus vaccine.

Role of the promyelocytic leukemia protein PML in the interferon sensitivity of lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV) induces type I interferon (alpha and beta interferon [IFN-alpha and IFN-beta]) upon infection and yet is sensitive to the addition of type II interferon (gamma interferon [IFN-gamma]) to the culture media. This sensitivity is biologically important because it correlates inversely with the ability of certain LCMV strains to persist in mice (D. Moskophidis, M. Battegay, M. A. Bruendler, E. Laine, I. Gresser, and R. M. Zinkernagel, J. Virol. 68:1951-1955, 1994). The cellular oncoprotein PML is induced by both IFN-alpha/beta and IFN-gamma, and PML binds the LCMV Z protein and becomes redistributed within cells from nucleus to cytoplasm upon LCMV infection. In the present study, increased PML expression results in diminished LCMV replication, implicating PML in the IFN sensitivity of LCMV. Virus production in PML -/- murine embryonic fibroblasts (MEF) exceeds virus production in PML +/+ MEF, and this difference is exacerbated by IFN treatment that upregulates PML expression. IFN-gamma also diminishes LCMV production in PML -/- cells; therefore, viral IFN sensitivity is not entirely due to PML. Both viral mRNA production and viral protein production decrease as PML expression increases. Here we propose that PML reduces LCMV transcription through its interaction with the Z protein.

The lymphocytic choriomeningitis virus RING protein Z associates with eukaryotic initiation factor 4E and selectively represses translation in a RING-dependent manner.

Only a few host cell proteins that associate with arenaviruses have been identified. To date, the arenavirus Z protein associates with the promyelocytic leukemia protein PML and the ribosomal P proteins. The majority of PML is present in nuclear bodies which are translocated to the cytoplasm by infection with the arenavirus, lymphocytic choriomeningitis virus (LCMV). The Z protein is a small zinc-binding RING protein with an unknown function which is required for the viral life cycle. Here, we demonstrate an association between Z and the host cell translation factor, eukaryotic initiation factor 4E (eIF-4E) in infected and transfected cells. Z's association with both ribosomal proteins and this translation factor led us to investigate whether Z could modulate host cell translation. In cell culture, Z selectively represses protein production in an eIF-4E-dependent manner. Specifically, we see reduction in cyclin D1 protein production with no effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cells transfected with Z. Previous reports indicate that cyclin D1 is sensitive to eIF-4E levels, whereas GAPDH is not. Consistent with this, we observe preferential downregulation of cyclin D1 during infection and no effect on GAPDH. Further, no changes in RNA levels were observed for cyclin D1 or GAPDH transcripts. The interaction between eIF-4E and Z may provide a mechanism for slower growth observed in infected cells and a viral strategy for establishing chronic infection.

Murine immune responses to mucosally delivered Salmonella expressing Lassa fever virus nucleoprotein.

Arenaviruses are emerging pathogens known to infect via the mucosa, however no formal attempts to make mucosal vaccines have been undertaken. Here we describe a recombinant aroA attenuated Salmonella typhimurium that expresses the nucleoprotein (NP) gene of Lassa fever virus (LAS). The complete NP gene was cloned downstream of the bacterial groEL promotor and integrated into the aroA locus of S. typhimurium. Lassa NP protein was detected in whole cell extracts from the recombinant Salmonella by immunoblot analysis with serum from Lassa-infected people. Mice were inoculated by intragastric intubation with 5 x 10(9) S. typhimurium and boosted with the same recombinant Salmonella 21 days after the primary inoculation. Both local mucosal IgA and serum immunoglobulins against Lassa NP were observed. Splenic cytotoxic T-lymphocyte responses to LAS NP were detected after the boost and they cross-reacted with target cells infected with the related arenavirus, lymphocytic choriomeningitis virus. Recombinant Salmonella elicits humoral and cell mediated immune responses against Lassa fever virus in mice and should be considered as a potential vaccine strategy in man.

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF.

High major histocompatibility complex-unrestricted lysis of simian immunodeficiency virus envelope-expressing cells predisposes macaques to rapid AIDS progression.

Before the development of virus-specific immune responses, peripheral blood mononuclear cells (PBMC) from uninfected rhesus monkeys and human beings have the capacity to lyse target cells expressing simian immunodeficiency virus (SIV) or human immunodeficiency virus-1 (HIV) envelope (gp130 and gp120) antigens. Lysis by naive effector cells does not require major histocompatibility complex (MHC)-restricted antigen presentation, is equally effective for allogeneic and xenogeneic targets, and is designated MHC-unrestricted (UR) lysis. UR lysis is not sensitive to EGTA and does not require de novo RNA or protein synthesis. Several kinds of envelope-expressing targets, including cells that poorly express MHC class I antigens, can be lysed. CD4(+) effectors are responsible for most of the lytic activity. High lysis is correlated with high expression of HIV or SIV envelope, specifically, the central one-third of the gp130 molecule, and lysis is completely inhibited by a monoclonal antibody against envelope. Our work extends observations of human lymphocytes expressing HIV gp120 to the SIV/rhesus monkey model for AIDS. Additionally, we address the relevance of UR lysis in vivo. A survey of PBMC from 56 uninfected rhesus monkeys indicates that 59% of the individuals had peak UR lytic activity above 15% specific lysis. Eleven of these monkeys were subsequently infected with SIV. Animals with UR lytic activity above 15% specific lysis were predisposed to more rapid disease progression than animals with low UR lytic activity, suggesting a strong correlation between this form of innate immunity and disease progression to AIDS.

Sequence comparison of the large genomic RNA segments of two strains of lymphocytic choriomeningitis virus differing in pathogenic potential for guinea pigs.

Two strains of lymphocytic choriomeningitis virus (LCMV) differ in their ability to cause a lethal disease in outbred guinea pigs: the Armstrong (ARM) strain is not lethal at high doses (10(6) PFU), whereas the WE strain is lethal at less than 10 PFU inoculated intraperitoneally. The high pathogenic potential of LCMV WE has been mapped to the larger (L) of the two genomic RNA segments by genetic reassortment analysis (Riviere, Y., Ahmed, R., Southern, P. J., Buchmeier, M. J. and Oldstone, M. B. A., J. Virol. 55, 704-709, 1985). Here we describe the completed sequence of the LCMV WE L RNA, and its comparison to the L RNA of the non-virulent strain, LCMV ARM. Similar to the L RNA of LCMV ARM, the L RNA of WE is 7.2 kb long and contains two open reading frames (ORFs): the 5" ORF encodes a small RING finger (zinc-binding) protein, p11 Z, and the 3" ORF encodes the putative RNA-dependent RNA polymerase (RdRp or L protein). Comparison of nucleotide sequences for both viruses revealed 84% L RNA homology. At the amino acid level similarity between the two strains is 87% in the Z ORF, and 88% in the RdRp ORF. The most divergent regions are found in the N-terminal parts of the RdRp and Z proteins and are most likely to account for differences in pathogenic potential.

Dissemination of lymphocytic choriomeningitis virus from the gastric mucosa requires G protein-coupled signaling.

The gastric mucosa is an important portal of entry for lymphocytic choriomeningitis virus (LCMV) infections. Within hours after intragastric (i.g.) inoculation, virus appears in the gastric epithelia, then in the mesenteric lymph nodes and spleen, and then in the liver and brain. By 72 h i.g.-inoculated virus is widely disseminated and equivalent to intravenous (i.v.) infection (S. K. Rai, B. K. Micales, M. S. Wu, D. S. Cheung, T. D. Pugh, G. E. Lyons, and M. S. Salvato. Am. J. Pathol. 151:633-639, 1997). Pretreatment of mice with a G protein inhibitor, pertussis toxin (PTx), delays LCMV dissemination after i.g., but not after i.v., inoculation. Delayed infection was confirmed by plaque assays, by reverse transcription-PCR, and by in situ hybridization. The differential PTx effect on i.v. and i.g. infections indicates that dissemination from the gastric mucosa requires signals transduced through heterotrimeric G protein complexes. PTx has no direct effect on LCMV replication, but it modulates integrin expression in part by blocking chemokine signals. LCMV infection of macrophages up-regulates CD11a, and PTx treatment counteracts this. PTx may prevent early LCMV dissemination by inhibiting the G protein-coupled chemotactic response of macrophages infected during the initial exposure, thus blocking systemic virus spread.

Hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation.

Efficient recognition of tumor cells by cytolytic T lymphocytes (CTL) is often dependent on the presentation of cytosolic peptides in the context of MHC class I molecules. This process may be influenced by various molecular chaperones. To analyze this influence, we have utilized B16 melanoma cells, which are not effectively recognized by MHC class I-restricted CTL. This resistance to CTL is apparently due to a very low level of surface MHC expression. We have found that stably transfected clones of B16 which constitutively express the human heat shock protein 72 (Hsp72) exhibit significantly increased levels of MHC class I antigens on their surface. This Hsp72-mediated up-regulation of surface MHC class I antigen represents an increase in the amount of functional MHC-peptide complexes as measured by conformation-dependent antibodies and recognition by MHC class I-restricted CTL. Expression of Hsp72 did not improve the antigen presentation defect in cells lacking the activity of the transporter associated with antigen presentation (TAP). Moreover, mice immunized with Hsp72-expressing B16 cells, but not with control-transfected B16 cells, display significantly increased resistance to a subsequent challenge with live, wild-type B16. Together, our data demonstrate that the immune recognition of tumor cells can be substantially enhanced by the suitable expression of a molecular chaperone.

Two RING finger proteins, the oncoprotein PML and the arenavirus Z protein, colocalize with the nuclear fraction of the ribosomal P proteins.

The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.

An arenavirus RING (zinc-binding) protein binds the oncoprotein promyelocyte leukemia protein (PML) and relocates PML nuclear bodies to the cytoplasm.

The promyelocytic leukemia protein (PML) forms nuclear bodies which are altered in some disease conditions. We report that the cytoplasmic RNA virus lymphocytic choriomeningitis virus (LCMV) influences the distribution of PML bodies. In cells infected with LCMV, the Z protein and PML form large bodies primarily in the cytoplasm. Transient transfection studies indicate that Z alone is sufficient to redistribute PML to the cytoplasm and that PML and Z colocalize. Coimmunoprecipitation studies show specific interaction between PML and Z proteins. A similar result was observed with a Z protein from another arenavirus, Lassa virus, suggesting that this is a general feature of the Arenaviridae. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML and does not need the PML RING or the nuclear localization signal to colocalize. The Z protein acts dominantly to overcome the diffuse phenotype observed in several PML mutants. The interaction between PML and Z may influence certain unique characteristics of arenavirus infection.

The promyelocytic leukemia protein PML has a pro-apoptotic activity mediated through its RING domain.

The promyelocytic leukemia protein PML is known to form nuclear multiprotein complexes which are compromised in several pathogenic conditions including acute promyelocytic leukemia. We show that in cells infected with a single stranded RNA virus, which relocates PML bodies to the cytoplasm, the infected cells are more resistant to serum starvation induced apoptosis than their uninfected counterparts. Antisense PML oligonucleotides increase cell survival under serum deprivation conditions indicating that PML is directly involved in the apoptotic activity. Transient transfection studies have indicated that this pro-apoptotic activity of PML is mediated through the zinc binding region known as the RING finger. Viral attack of PML nuclear bodies appears to allow the virus to deregulate host cell apoptotic machinery in order to establish chronic infection.

Completion of the Lassa fever virus sequence and identification of a RING finger open reading frame at the L RNA 5' End.

Lassa (LAS) fever virus is a highly pathogenic arenavirus with large (L) and small (S) RNA genomic segments. The 5' end of the LAS L segment is described here, thereby completing the sequence of the most virulent arenavirus analyzed to date. In keeping with the ambisense gene structure of the arenaviruses, the LAS L RNA encodes a 250-kDa protein and an 11-kDa protein in opposite senses with respect to each other. The 11-kDa protein, defined previously in arenaviruses lymphocytic choriomeningitis (LCM), Tacaribe (TAC), and Pichinde (PIC), contains a RING type of zinc-binding structure. Expression of the 11-kDa protein in LAS virus-infected cells has been confirmed by binding to peptide-specific antibody.

Timed appearance of lymphocytic choriomeningitis virus after gastric inoculation of mice.

Arenaviruses present an emerging health threat in agrarian areas of Africa and South America; however, the natural routes of arenaviral infections are not clearly understood. Our previous studies with lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, implicate oral and intragastric routes as natural routes of infection. Our studies raised many questions about the primary site of infection and the route of dissemination after gastric infection. In this report, we use in situ hybridization to detect LCMV in various organs at different time points (0 to 96 hours). After gastric inoculation, the gastric mucosa is the initial site of viral infection, followed by infection of the spleen and liver, then ileum and last, lung, kidney, brain, and esophagus. Furthermore, our observations suggest that virus is disseminated lymphatically rather than by a hematogenous route. Infectious center assays using mononuclear cells from stomach, blood, and spleen of mice infected by the gastric route confirmed active infection with LCMV and the presence of mononuclear cells producing infectious virus in these tissues. This is the first identification of gastric epithelia as a primary site of virus infection.

Restoration of MHC class I surface expression and endogenous antigen presentation by a molecular chaperone.

Presentation of cytosolic peptides in the context of major histocompatibility complex (MHC) class I antigen is crucial for immune recognition of virus-infected and malignant cells. This process, which is often defective in cancer cells, involves a series of cellular events which may be facilitated by heat shock proteins (molecular chaperones). To address the influence of chaperone function on the presentation of cytosolic peptides, we have utilized B16 melanoma cells (H-2b). These tumour cells are resistant to lysis by MHC class I-restricted cytotoxic T lymphocytes (CTL), due to a very low level of surface MHC expression. The authors found that stably transfected clones of B16 expressing a heterologous heat shock protein (Hsp65) exhibit significantly increased levels of MHC class I antigens on their surface, and are effectively lysed by alloreactive CTL. These MHC class I molecules can form functional MHC-peptide complexes which are recognized by virus-specific CTL. Moreover, mice immunized with Hsp65-expressing tumour cells, but not with control-transfected tumour cells, display a significantly increased resistance to a subsequent challenge with live, wild-type B16. Together, these results indicate that the suitable expression of a molecular chaperone can overcome a defect in MHC class I expression and antigen presentation, and suggest a novel approach to cancer immunotherapy.

The Lassa fever virus L gene: nucleotide sequence, comparison, and precipitation of a predicted 250 kDa protein with monospecific antiserum.

The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells.

The lymphocytosis-promoting agent pertussis toxin affects virus burden and lymphocyte distribution in the SIV-infected rhesus macaque.

Pertussis toxin from the gram-negative bacterium Bordetella pertussis is an ADP-ribosylase that modifies Gi proteins in mammalian lymphocytes and inhibits their capacity to traffic from blood into lymphoid tissues. We used this compound to induce lymphocytosis in rhesus macaques and to study its effects on SIV infection. Pertussis toxin injected at 25 micrograms/kg induced a transient lymphocytosis that peaked 3-8 days after administration and caused a rapid, transient decrease in the frequency of infectious cells in blood as judged by in vitro virus isolation assays. Lymphocyte subsets were altered during the lymphocytosis interval and sustained changes in CD8+ T cell levels were noted as long as 53 days after pertussis toxin injection. In situ hybridization studies showed that pertussis toxin altered the distribution of viral RNA in lymph nodes during the interval of lymphocytosis, and caused long-term changes with decreased virus replication in some tissue specimens.