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.

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.

Whole body positron emission tomography imaging of activated lymphoid tissues during acute simian-human immunodeficiency virus 89.6PD infection in rhesus macaques.

Mechanisms of acute retroviral pathogenesis have been examined during primary infection of rhesus macaques with simian-human immunodeficiency virus 89.6PD (SHIV(89.6PD)). During acute infection, between initial exposure and establishment of antigen-specific immune responses that stabilize the virus burden, rapid immune system changes influence the viral set-point and dictate subsequent steps in disease progression. In a previous study, we described specific patterns of lymphocyte activation during acute SHIV(89.6PD) infection. We now extend these studies to describe lymphoid tissue activation, using whole body positron emission tomography (PET) and the radioactive tracer 2-[(18)F]fluorodeoxyglucose (FDG). Within a few days after primary infection by intravenous, intrarectal, or intravaginal routes, PET-FDG imaging revealed a distinct pattern of lymphoid tissue activation centered on axillary, cervical, and mediastinum lymph nodes. Increased tissue FDG uptake preceded fulminant virus replication at these sites, suggesting that a diffusible factor of host or viral origin was responsible for lymphoid tissue changes. These data show that activation of lymphoid tissues in the upper body is an early response to virus infection and that diffusible mediators of activation might be important targets for vaccine or therapeutic intervention strategies.

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.

Lymphocyte activation during acute simian/human immunodeficiency virus SHIV(89.6PD) infection in macaques.

Host-virus interactions control disease progression in human immunodeficiency virus-infected human beings and in nonhuman primates infected with simian or simian/human immunodeficiency viruses (SHIV). These interactions evolve rapidly during acute infection and are key to the mechanisms of viral persistence and AIDS. SHIV(89.6PD) infection in rhesus macaques can deplete CD4(+) T cells from the peripheral blood, spleen, and lymph nodes within 2 weeks after exposure and is a model for virulent, acute infection. Lymphocytes isolated from blood and tissues during the interval of acute SHIV(89.6PD) infection have lost the capacity to proliferate in response to phytohemagglutinin (PHA). T-cell unresponsiveness to mitogen occurred within 1 week after mucosal inoculation yet prior to massive CD4(+) T-cell depletion and extensive virus dissemination. The lack of mitogen response was due to apoptosis in vitro, and increased activation marker expression on circulating T cells in vivo coincided with the appearance of PHA-induced apoptosis in vitro. Inappropriately high immune stimulation associated with rapid loss of mature CD4(+) T cells suggested that activation-induced cell death is a mechanism for helper T-cell depletion in the brief period before widespread virus dissemination. Elevated levels of lymphocyte activation likely enhance SHIV(89.6PD) replication, thus increasing the loss of CD4(+) T cells and diminishing the levels of virus-specific immunity that remain after acute infection. The level of surviving immunity may dictate the capacity to control virus replication and disease progression. We describe this level of immune competence as the host set point to show its pivotal role in AIDS pathogenesis.

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.

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.

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.

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.

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.