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.

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.

Biochemical and immunological evidence that the 11 kDa zinc-binding protein of lymphocytic choriomeningitis virus is a structural component of the virus.

The completed sequence of the arenavirus, lymphocytic choriomeningitis virus, revealed a new gene encoding a small protein with a single zinc-binding domain. The cDNA for this gene has been expressed in E. coli to produce fusion protein that has been used to raise antisera. The antisera facilitated the positive identification of the p11 'Z' gene product as a structural component of the virion. A related arenavirus, Tacaribe, has a comparable p11 gene product. The abundance of the p11 Z protein relative to other virion components has been determined by metabolic labeling. Triton X-114 extraction and dimethyl suberimidate-HCl crosslinking indicate that the p11 Z protein is a hydrophobic protein associated with the nucleocapsid of the virion core.

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.

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.

The completed sequence of lymphocytic choriomeningitis virus reveals a unique RNA structure and a gene for a zinc finger protein.

The arenavirus, lymphocytic choriomeningitis virus (LCMV) has a single-stranded RNA genome composed of a large (L) and a small (S) RNA segment. The completed sequence of LCMV, presented here, reveals a formerly unknown gene (Z) on the L genomic segment. This gene is encoded in the positive or message-sense of the viral genomic RNA, whereas the adjacent gene (L) is in the genome-complementary, or negative sense. The ambisense polarity of the genes on the L RNA reiterates the polarity of genes on the small (S) genomic segment. The Z gene encodes a 10-kDa protein containing a single zinc-finger sequence (Cys2His2). A small RNA representing the message sense of the Z gene is found in infected cells and within virions. In contrast to the known LCMV proteins having structural or enzymatic functions, the predicted Z gene product is most likely to be an RNA-binding protein with a regulatory role. The encapsidation of a message sense Z RNA suggests a role for this gene immediately following virus penetration. The L/Z intergenic region is rich in cytidylic acid (C) and presents an unusual RNA structure. All cDNA clones of the intergenic region differ from each other within a certain poly(C) stretch and lack a 30-base region present in the direct RNA sequence. Finally, the completed sequence establishes that the L RNA 5' end is complementary to its 3' end. The L RNA termini, similar to the S RNA termini, have a small but potentially important asymmetry of sequence. LCMV is the first arenavirus to be completely sequenced.

Translation of tobacco necrosis virus and its satellite in a cell-free wheat germ system.

Tobacco necrosis virus (TNV) and its satellite virus (STNV) each contain a single-stranded RNA genome, of about 1.4 X 10(6) and 0.4 X 10(6) daltons, respectively, which is very active in stimulating amino acid incorporation in a wheat germ cell-free system. With STNV RNA the predominant incorporation product of 22,000 daltons coelectrophoreses with viral coat protein and crossreacts with antibody to viral coat protein. A similar result is obtained with TNV RNA, the only major translation product being a 30,000-dalton protein which corresponds to the coat protein by gel sizing, serological tests, and tryptic peptide analysis. Other products appearing in smaller amounts are about 63,000, 43,000, and 26,000 daltons and smaller. The possible nature of these products is discussed, as well as the unusual feature of a large, presumably multigenic, viral RNA yielding the coat protein as the predominant translation product in a eukaryotic system. Much less STNV RNA than TNV RNA produces maximal translation. Cotranslation of both RNAs in vitro indicates that STNV RNA has a translational advantage over TNV RNA. The fact that these RNAs lack 5'-terminal capping and 3'-terminal poly(A) is discussed.

Neutralizing epitopes of lymphocytic choriomeningitis virus are conformational and require both glycosylation and disulfide bonds for expression.

Lymphocytic choriomeningitis virus (Armstrong strain) bears two overlapping epitopes, GP-1A (A) and GP-1D (D), recognized by neutralizing antibodies on the major surface glycoprotein GP-1. Both are discontinuous conformational epitopes that require prior formation of disulfide bridges and addition of N-linked oligosaccharides. Using monoclonal antibodies specific for each of these epitopes, as well as for conformation-independent epitopes, we have investigated the requirements for biosynthesis and folding of the epitopes. The carbohydrate residues themselves do not appear to comprise critical informational components of these epitopes, but are required for proper folding of the nascent glycopeptide chain within the rough endoplasmic reticulum. These epitopes differ in their resistance to denaturation; epitope D is retained when denatured with SDS under nonreducing conditions, whereas epitope A is lost. Monoclonal antibodies to epitope A cross-react with several strains of LCMV. However, epitope D is detected in only a subset of isolates derived from the Armstrong strain of LCMV. By RNA sequence analysis, we have mapped a single amino acid change distinguishing those virions containing epitope D. Acquisition of binding activity of the epitope D-specific monoclonal correlates with a Thr----Ala or Thr----Lys mutation at amino acid 173 of the GP-1 molecule and concomitant disruption of a consensus N-linked glycosylation site.

Tracking of dye-labeled lymphocytes in rhesus macaques.

Lymphocytes were isolated from rhesus monkeys and marked with a fluorescent lipophilic dye to monitor their distribution in vivo. Dye-labeled cells were either monitored by blood draws over a three-month period, or identified within peripheral organs upon autopsy. Lymphocyte labeling conditions were optimized. Dye-labeled lymphocytes could be detected in the circulation for at least 100 days by flow cytometry and fluorescence microscopy. Activated lymphocytes were removed from the circulation more rapidly than lymphocytes that had not been activated.

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.

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.

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.

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.

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.

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.

Pertussis toxin induces lymphocytosis in rhesus macaques.

Lymph nodes and other solid tissues of the immune system are the principal sites for antigen presentation and lymphocyte activation. Lymphocytes in peripheral blood recognize the high endothelial venules within lymphoid tissues and cross from blood to tissue by the process of extravasation. Pertussis toxin is known to block extravasation and cause lymphocytosis in murine models but has not been studied extensively in nonhuman primates. We used intravenous injection of soluble pertussis toxin to induce a transient lymphocytosis in rhesus monkeys. The increase in total white blood cells was proportionally greater for lymphocytes than for polymorphonuclear cells and the CD4+ lymphocyte subpopulation increased more than the CD8+ cell population. The presence of immature polymorphonuclear cells suggested some activation of bone marrow. Clinical chemistry studies revealed an effect of pertussis toxin on liver function. Pertussis toxin is a powerful immunomodulatory agent that can disrupt and reorganize solid lymphoid tissues.

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.

Cellular immune responses in rhesus macaques infected rectally with low dose simian immunodeficiency virus.

Monkeys infected rectally with low dose simian immunodeficiency virus (SIV) were resistant to high dose challenge with SIV. Peripheral blood mononuclear cells (PBMC) from two of four challenged monkeys were unable to support SIV replication in vitro unless cultures were depleted of CD8+ lymphocytes. Monkeys that had survived high dose rectal infection with SIV also suppressed virus replication in cultured PBMC. PBMC from uninfected monkeys supported virus replication in both unfractionated and CD8-depleted cultures. Virus-suppressive activity of PBMC may be an important correlate of protective immunity in AIDS.

Murine infection with lymphocytic choriomeningitis virus following gastric inoculation.

Laboratory studies of arenaviruses have been limited to parenteral routes of infection; however, recent epidemiological studies implicate virus ingestion as a natural route of infection. Accordingly, we developed a model for oral and gastric infection with lymphocytic choriomeningitis virus to enable studies of mucosal transmission and vaccination by this additional route.