Effect of rabies virus infection on gene expression in mouse brain

A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6–7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.

Modular organization of the Friend murine leukemia virus envelope protein underlies the mechanism of infection

Retrovirus infection is initiated by receptor-dependent fusion of the envelope to the cell membrane. The modular organization of the envelope protein of C type retroviruses has been exploited to investigate how binding of the surface subunit (SU) to receptor triggers fusion mediated by the transmembrane (TM) subunit. We show that deletion of the receptor-binding domain (RBD) from SU of Friend murine leukemia virus (Fr-MLV) abolishes infection that is restored by supplying RBD as a soluble protein. Infection by this mechanism remains dependent on receptor expression. When membrane attachment of the virus lacking RBD is reestablished by inserting the hormone erythropoietin, infection remains dependent on the RBD/receptor complex. However, infection increases 50-fold to 5 × 105 units/ml on cells that also express the erythropoietin receptor. Soluble RBD from Fr-MLV also restores infection by amphotropic and xenotropic MLVs in which RBD is deleted. These experiments demonstrate that RBD has two functions: mediating virus attachment and activating the fusion mechanism. In addition, they indicate that receptor engagement triggers fusion by promoting a subgroup-independent functional interaction between RBD and the remainder of SU and/or TM.

Immunity to retroviral infection: The Friend virus model

Friend virus infection of adult immunocompetent mice is a well established model for studying genetic resistance to infection by an immunosuppressive retrovirus. This paper reviews both the genetics of immune resistance and the types of immune responses required for recovery from infection. Specific major histocompatibility complex (MHC) class I and II alleles are necessary for recovery, as is a non-MHC gene, Rfv-3, which controls virus-specific antibody responses. In concordance with these genetic requirements are immunological requirements for cytotoxic T lymphocyte, T helper, and antibody responses, each of which provides essential nonoverlapping functions. The complexity of responses necessary for recovery from Friend virus infection has implications for both immunotherapies and vaccines. For example, it is shown that successful passive antibody therapy is dependent on MHC type because of the requirement for T cell responses. For vaccines, successful immunization requires priming of both T cell and B cell responses. In vivo depletion experiments demonstrate different requirements for CD8+ T cells depending on the vaccine used. The implications of these studies for human retroviral diseases are discussed.

The dynamics of hepatitis B virus infection.

We consider a cellular model of infection by the hepatitis B virus and describe how it may be used to account for two important features of the disease, namely (i) the wide variety of manifestations of infection and the age dependence thereof, and (ii) the typically long delay before the development of virus-induced liver cancer (primary hepatocellular carcinoma). The model is based on the assumption that the liver is comprised of both immature and mature hepatocytes, with these two subpopulations of cells responding contrastingly upon infection by the virus.

Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Comparative interleukin (IL-2)/interferon IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SICmac251 virus.

Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals.

A recombinant Mycobacterium bovis bacillus Calmette-Guérin (BCG) vector-based vaccine that secretes the V3 principal neutralizing epitope of human immunodeficiency virus (HIV) could induce immune response to the epitope and prevent the viral infection. By using the Japanese consensus sequence of HIV-1, we successfully constructed chimeric protein secretion vectors by selecting an appropriate insertion site of a carrier protein and established the principal neutralizing determinant (PND)-peptide secretion system in BCG. The recombinant BCG (rBCG)-inoculated guinea pigs were initially screened by delayed-type hypersensitivity (DTH) skin reactions to the PND peptide, followed by passive transfer of the DTH by the systemic route. Further, immunization of mice with the rBCG resulted in induction of cytotoxic T lymphocytes. The guinea pig immune antisera showed elevated titers to the PND peptide and neutralized HIVMN, and administration of serum IgG from the vaccinated guinea pigs was effective in completely blocking the HIV infection in thymus/liver transplanted severe combined immunodeficiency (SCID)/hu or SCID/PBL mice. In addition, the immune serum IgG was shown to neutralize primary field isolates of HIV that match the neutralizing sequence motif by a peripheral blood mononuclear cell-based virus neutralization assay. The data support the idea that the antigen-secreting rBCG system can be used as a tool for development of HIV vaccines.

Broad resistance to plant viruses in transgenic plants conferred by antisense inhibition of a host gene essential in S-adenosylmethionine-dependent transmethylation reactions.

S-Adenosylhomocysteine hydrolase (SAHH) is a key enzyme in transmethylation reactions that use S-adenosylmethionine as the methyl donor. Because of the importance of SAHH in a number of S-adenosylmethionine-dependent transmethylation reactions, particularly the 5' capping of mRNA during viral replication, SAHH has been considered as a target of potential antiviral agents against animal viruses. To test the possibility of engineering a broad type of resistance to plant viruses, we expressed the antisense RNA for tobacco SAHH in transgenic tobacco plants. As expected, transgenic plants constitutively expressing an anti-sense SAHH gene showed resistance to infection by various plant viruses. Among those plants, about half exhibited some level of morphological change (typically stunting). Analysis of the physiological change in those plants showed that they contained excess levels of cytokinin. Because cytokinin has been found to induce acquired resistance, there is also a strong possibility that the observed resistance was induced by cytokinin.

Mouse mammary tumor viruses with functional superantigen genes are selected during in vivo infection.

Mouse mammary tumor virus (MMTV) encodes a superantigen that is important for viral infectivity in vivo. To determine whether superantigen function was required for infection by milk-borne MMTV, we created HYB PRO/Cla transgenic mice. These mice produced a full-length, packaged viral RNA with a frameshift mutation that caused premature termination of the superantigen protein. Young HYB PRO/Cla mice showed no deletion of their cognate V beta 14+ T cells, although they shed virus in their milk. The nontransgenic offspring of the HYB PRO/Cla mice were infected with this virus, since transgene-specific viral transcripts were detected in their mammary glands. Surprisingly, these offspring demonstrated the progressive deletion of V beta 14+ T cells characteristic of exogenous MMTV (C3H) infection. Sequence analysis demonstrated that these newly acquired viruses had reconstituted superantigen open reading frames resulting from recombination between the HYB PRO/Cla and endogenous Mtv-1 proviral RNAs. Thus, there is selection during the infection process for MMTVs with functional superantigen genes.

SPC3, a synthetic peptide derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120, inhibits HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms.

The third variable region (V3 loop) of gp120, the HIV-1 surface envelope glycoprotein, plays a key role in HIV-1 infection and pathogenesis. Recently, we reported that a synthetic multibranched peptide (SPC3) containing eight V3-loop consensus motifs (GPGRAF) inhibited HIV-1 infection in both CD4+ and CD4- susceptible cells. In the present study, we investigated the mechanisms of action of SPC3 in these cell types--i.e., CD4+ lymphocytes and CD4- epithelial cells expressing galactosylceramide (GalCer), an alternative receptor for HIV-1 gp120. We found that SPC3 was a potent inhibitor of HIV-1 infection in CD4+ lymphocytes when added 1 h after initial exposure of the cells to HIV-1, whereas it had no inhibitory effect when present only before and/or during the incubation with HIV-1. These data suggested that SPC3 did not inhibit the binding of HIV-1 to CD4+ lymphocytes but interfered with a post-binding step necessary for virus entry. In agreement with this hypothesis, SPC3 treatment after HIV-1 exposure dramatically reduced the number of infected cells without altering gp120-CD4 interaction or viral gene expression. In contrast, SPC3 blocked HIV-1 entry into CD4-/GalCer+ human colon epithelial cells when present in competition with HIV-1 but had no effect when added after infection. Accordingly, SPC3 was found to inhibit the binding of gp120 to the GalCer receptor. Thus, the data suggest that SPC3 affects HIV-1 infection by two distinct mechanisms: (i) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4-/GalCer+ cells and (ii) post-binding inhibition of HIV-1 entry into CD4+ lymphocytes.

Live and let die: manipulation of host hepatocytes by exoerythrocytic Plasmodium parasites

The generation of rodent Plasmodium strains expressing fluorescent proteins in all life cycle stages has had a big impact on malaria research. With this tool in hand, for the first time it was possible to follow in real time by in vivo microscopy the infection route of Plasmodium sporozoites transmitted to the mammalian host by Anopheles mosquitoes. Recently, this work has been extended to the analysis of both hepatocyte infection by Plasmodium sporozoites, as well as liver merozoite transport into blood vessels. The stunning results of these studies have considerably changed our understanding of hepatocyte invasion and parasite liberation. Here, we describe the most important findings of the last years and in addition, we elaborate on the molecular events during the intracellular development of Plasmodium exoerythrocytic forms that give rise to erythrocyte infecting merozoites.

Induction of inflammatory cytokines and nitric oxide in J774.2 cells and murine macrophages by lipoteichoic acid and related cell wall antigens from Staphylococcus epidermidis

Staphylococcus epidermidis causes infections associated with medical devices including central venous catheters, orthopaedic prosthetic joints and artificial heart valves. This coagulase-negative Staphylococcus produces a conventional cellular lipoteichoic acid (LTA) and also releases a short-glycerophosphate-chain-length form of LTA (previously termed lipid S) into the medium during growth. The relative pro-inflammatory activities of cellular and short-chain-length exocellular LTA were investigated in comparison with peptidoglycan and wall teichoic acid from S. epidermidis and LPS from Escherichia coli O111. The ability of these components to stimulate the production of proinflammatory cytokines [interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α] and nitric oxide was investigated in a murine macrophage-like cell line (J774.2), and in peritoneal and splenic macrophages. On a weight-for-weight basis the short-chain-length exocellular LTA was the most active of the S. epidermidis products, stimulating significant amounts of each of the inflammatory cytokines and nitric oxide, although it was approximately 100-fold less active than LPS from E. coli. By comparison the full-chain-length cellular LTA and peptidoglycan were less active and the wall teichoic acid had no activity. As an exocellular product potentially released from S. epidermidis biofilms, the short-chain-length exocellular LTA may act as the prime mediator of the host inflammatory response to device-related infection by this organism and act as the Gram-positive equivalent of LPS in Gram-negative sepsis. The understanding of the role of short-chain-length exocellular LTA in Gram-positive sepsis may lead to improved treatment strategies. © 2005 SGM.

Interactive Effects of Morphine on HIV Infection: Role in HIV-Associated Neurocognitive Disorder

HIV epidemic continues to be a severe public health problem and concern within USA and across the globe with about 33 million people infected with HIV. The frequency of drug abuse among HIV infected patients is rapidly increasing and is another major issue since injection drug users are at a greater risk of developing HIV associated neurocognitive dysfunctions compared to non-drug users infected with HIV. Brain is a major target for many of the recreational drugs and HIV. Evidences suggest that opiate drug abuse is a risk factor in HIV infection, neural dysfunction and progression to AIDS. The information available on the role of morphine as a cofactor in the neuropathogenesis of HIV is scanty. This review summarizes the results that help in understanding the role of morphine use in HIV infection and neural dysfunction. Studies show that morphine enhances HIV-1 infection by suppressing IL-8, downregulating chemokines with reciprocal upregulation of HIV coreceptors. Morphine also activates MAPK signaling and downregulates cAMP response element-binding protein (CREB). Better understanding on the role of morphine in HIV infection and mechanisms through which morphine mediates its effects may help in devising novel therapeutic strategies against HIV-1 infection in opiate using HIV-infected population.

MECHANISM OF UPREGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS DURING PICORNAVIRUS INFECTION AND ITS ROLE IN THE DEVELOPMENT OF VIRAL REPLICATION ORGANELLES

Picornaviruses are a group of human and animal pathogens capable of inflicting serious public health diseases and economic burdens. Treatments options through vaccines for prevention or antivirals to cure infection are not available for the vast majority of these viruses. These shortcomings, in the development of vaccines or antivirals therapeutic, are linked to the genetic diversity and to an incomplete understanding of the biology of these viruses. Despite the diverse host range, this group of positive-strand RNA viruses shares the same replication mechanisms, including the development of membranous structures (replication organelles) in the cytoplasm of infected cells. The development of these membranous structures, which serve as sites for the replication of the viral RNA genome, has been linked to the hijacking of elements of the cellular membrane metabolism pathways. Here we show that upon picornavirus infection, there is a specific activation of acyl-CoA synthetase enzymes resulting in strong import and accumulation of long chain fatty acids in the cytoplasm of infected cells. We show that the newly imported fatty acids serve as a substrate for the upregulation of phosphatidylcholine synthesis required for the structural development of replication organelles. In this work, we identified that acyl-CoA synthetase long chain 3 (ACSL3) is required for the upregulation of lipids syntheses and the replication of poliovirus. We have shown that the poliovirus protein 2A was required but not sufficient for the activation of import of long chain fatty acids in infected cells. We demonstrated that the fatty acid import is upregulated upon infection by diverse picornaviruses and that such upregulation is not dependent on activation of ER stress response or the autophagy pathways. In this work, we have demonstrated that phosphatidylcholine was required for the structural development of replication organelles. Phosphatidylcholine synthesis was dispensable for the production of infectious particles at high MOI but required at a low MOI for the protection of the replication complexes from the cellular innate immunity mechanisms.