Decreased human immunodeficiency virus type 1 plasma viremia during antiretroviral therapy reflects downregulation of viral replication in lymphoid tissue.

Although several immunologic and virologic markers measured in peripheral blood are useful for predicting accelerated progression of human immunodeficiency virus (HIV) disease, their validity for evaluating the response to antiretroviral therapy and their ability to accurately reflect changes in lymphoid organs remain unclear. In the present study, changes in certain virologic markers have been analyzed in peripheral blood and lymphoid tissue during antiretroviral therapy. Sixteen HIV-infected individuals who were receiving antiretroviral therapy with zidovudine for > or = 6 months were randomly assigned either to continue on zidovudine alone or to add didanosine for 8 weeks. Lymph node biopsies were performed at baseline and after 8 weeks. Viral burden (i.e., HIV DNA copies per 10(6) mononuclear cells) and virus replication in mononuclear cells isolated from peripheral blood and lymph node and plasma viremia were determined by semiquantitative polymerase chain reaction assays. Virologic and immunologic markers remained unchanged in peripheral blood and lymph node of patients who continued on zidovudine alone. In contrast, a decrease in virus replication in lymph nodes was observed in four of six patients who added didanosine to their regimen, and this was associated with a decrease in plasma viremia. These results indicate that decreases in plasma viremia detected during antiretroviral therapy reflect downregulation of virus replication in lymphoid tissue.

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.

Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs.

Do RNA viruses require genome cyclisation for replication?

Complementary sequences at the 5′ and 3′ ends of the dengue virus RNA genome are essential for viral replication, and are believed to cyclise the genome through long-range base pairing in cis. Although consistent with evidence in the literature, this view neglects possible biologically active multimeric forms that are equally consistent with the data. Here, we propose alternative multimeric structures, and suggest that multigenome noncovalent concatemers are more likely to exist under cellular conditions than single cyclised monomers. Concatemers provide a plausible mechanism for the dengue virus to overcome the single-stranded (+)-sense RNA virus dilemma, and can potentially assist genome transport from the virus-induced vesicles into the cytosol.

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

Limited dengue virus replication in field-collected Aedes aegypti mosquitoes infected with Wolbachia

Introduction Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus. Methodology/Principal Findings Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes. Conclusions/Significance Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.

The Interplay Between KSHV-encoded Viral Cyclin and CDK-inhibitors p27Kip1 and p21Cip1 -implications for Viral Lymphomagenesis

Cell division, which leads to the birth of two daughter cells, is essential for the growth and development of all organisms. The reproduction occurs in a series of events separated in time, designated as the cell cycle. The cell cycle progression is controlled by the activity of cyclin-dependent kinases (CDK). CDKs pair with cyclins to become catalytically active and phosphorylate a broad range of substrates required for cell cycle progression. In addition to cyclins, CDKs are regulated by inhibitory and activating phosphorylation events, binding to CDK-inhibitory proteins (CKI), and also by subcellular localization. The control of the CDK activity is crucial in preventing unscheduled progression of the cell cycle with mistakes having potentially hazardous consequences, such as uncontrolled proliferation of the cells, a hallmark of cancer. The mammalian cell cycle is a target of several DNA tumor viruses that can deregulate the host s cell cycle with their viral oncoproteins. A human herpesvirus called Kaposi s sarcoma herpesvirus (KSHV) is implicated in the cause of Kaposi s sarcoma (KS) and lymphoproliferative diseases such as primary effusion lymphomas (PEL). KSHV has pirated several cell cycle regulatory genes that it uses to manipulate its host cell and to induce proliferation. Among these gene products is a cellular cyclin D homologue, called viral cyclin (v-cyclin) that can activate cellular CDKs leading to the phosphorylation of multiple target proteins. Intriguingly, PELs that are naturally infected with KSHV consistently express high levels of CDK inhibitor protein p27Kip1 and still proliferate actively. The aim of this study was to investigate v-cyclin complexes and their activity in PELs, and search for an explanation why CKIs, such as p27Kip1 and p21Cip1 are unable to inhibit cell proliferation in this type of lymphoma. In this study, we found that v-cyclin binds to p27Kip1 in PELs, and confirmed this novel interaction also in the overexpression models. We observed that p27Kip1 associated with v-cyclin was also phosphorylated by a v-cyclin-associated kinase and identified cellular CDK6 as the major kinase partner of v-cyclin responsible for this phosphorylation. Analysis of the p27Kip1 residues targeted by v-cyclin-CDK6 revealed that serine 10 (S10) is the major phosphorylation site during the latent phase of the KSHV replication cycle. This phosphorylation led to the relocalization of p27Kip1 to the cytoplasm, where it is unable to inhibit nuclear cyclin-CDK complexes. In the lytic phase of the viral replication cycle, the preferred phosphorylation site on p27Kip1 by v-cyclin-CDK6 changed to threonine 187 (T187). T187 phosphorylation has been shown to lead to ubiquitin-mediated degradation of p27Kip1 and downregulation of p27Kip1 was also observed here. v-cyclin was detected also in complex with p21Cip1, both in overexpression models and in PELs. Phosphorylation of p21Cip1 on serine 130 (S130) site by v-cyclin-CDK6 functionally inactivated p21Cip1 and led to the circumvention of G1 arrest induced by p21Cip1. Moreover, p21Cip1 phosphorylated by v-cyclin-associated kinase showed reduced binding to CDK2, which provides a plausible explanation why p21Cip1 is unable to inhibit cell cycle progression upon v-cyclin expression. Our findings clarify the mechanisms on how v-cyclin evades the inhibition of cell cycle inhibitors and suggests an explanation to the uncontrolled proliferation of KSHV-infected cells.

Controlling transduction and replication of oncolytic adenoviruses

Despite progress in conventional cancer treatment regimes, metastatic disease essentially remains incurable and new treatment alternatives are needed. Virotherapy is a relatively novel approach in cancer treatment. It harnesses the natural ability of oncolytic viruses to kill the cells they proliferate in and to spread to neighboring cells, thereby amplifying the therapeutic effect of the initial input dose. The use of replicating, oncolytic viruses for cancer treatment necessitates introduction of various genetic modifications to the viral genome, thereby restraining replication exclusively to tumor cells and eventually obtaining selective eradication of the tumor without side effects to healthy tissue. Furthermore, various modifications can be applied to the viral capsid in hope of gaining effective transduction of target tissue. In other words, the entry of viruses into tumor tissue can be augmented by allowing the virus to utilize non-native receptors for entry. Genetic capsid modifications may also help to avoid some major hurdles in systemic delivery that ultimately lead to the rapid clearance of the virus from the blood and virus induced toxicity. In addition to genetic modifications that alter the phenotype of the virus, some pharmacologic agents may be utilized to enhance the virus entry to target site. Liver kupffer cells (KC) are responsible for the majority of viral clearance after systemic viral delivery and they play a major role in adenovirus induced acute toxicity. The therapeutic window could possibly be widened by transiently depleting KCs, allowing smaller viral input doses and diminishing KC related toxicity. The transductional efficacy of various capsid modified viruses was analyzed in vitro and in vivo in murine orthotopic breast cancer model. The effect of capsid modifications on the oncolytic efficacy, i.e. the ability of the viruses to kill cancer cells, was evaluated in vitro and in vivo in murine cancer models. We concluded that capsid modifications result in transductional enhancement, and that enhanced transduction translates into more potent oncolysis in vitro and in vivo. When KC depleting agents were used in vivo prior to viral injections, enhanced tumor transduction was seen, but this effect was not translated into enhanced antitumor activity. Transcriptional regulation of replicative oncolytic viruses is a prerequisite for virotherapy. Tumor or tissue specific promoters can be used to control the transcription of adenoviral early genes to gain cancer specific viral replication. Specific deletions in viral regions essential for virus replication in normal cells can further increase the safety by allowing viral genome replication in cancer cells featuring specific mutations. Genetically modified viruses were shown to be able to kill putative cancer stem cells that are thought to be responsible for post treatment relapses and metastasis. Further, pharmacologic intervention reduced viral replication and thereby might offer an additional safety switch in case viral replication related side effects are encountered.

Interactions of 3' terminal and 5' terminal regions of physalis mottle virus genomic RNA with its replication complex

Physalis mottle virus (PhMV) belongs to the tymogroup of positive-strand RNA viruses with a genome size of 6 kb. Crude membrane preparations from PhMV-infected Nicotiana glutinosa plants catalyzed the synthesis of PhMV genomic RNA from endogenously bound template. Addition of exogenous genomic RNA enhanced the synthesis which was specifically inhibited by the addition of sense and antisense transcripts corresponding to 3' terminal 242 nucleotides as well as the 5' terminal 458 nucleotides of PhMV genomic RNA while yeast tRNA or ribosomal RNA failed to inhibit the synthesis. This specific inhibition suggested that the 5' and 3' non-coding regions of PhMV RNA might play an important role in viral replication.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3 ` Untranslated Region and Enhances Hepatitis C Virus Replication

HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3' untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3' UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3' UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3' UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3' untranslated region (UTR) of HCV RNA. At the 3' UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

Genome-wide mRNA expression correlates of viral control in CD4+ T-cells from HIV-1-infected individuals.

There is great interindividual variability in HIV-1 viral setpoint after seroconversion, some of which is known to be due to genetic differences among infected individuals. Here, our focus is on determining, genome-wide, the contribution of variable gene expression to viral control, and to relate it to genomic DNA polymorphism. RNA was extracted from purified CD4+ T-cells from 137 HIV-1 seroconverters, 16 elite controllers, and 3 healthy blood donors. Expression levels of more than 48,000 mRNA transcripts were assessed by the Human-6 v3 Expression BeadChips (Illumina). Genome-wide SNP data was generated from genomic DNA using the HumanHap550 Genotyping BeadChip (Illumina). We observed two distinct profiles with 260 genes differentially expressed depending on HIV-1 viral load. There was significant upregulation of expression of interferon stimulated genes with increasing viral load, including genes of the intrinsic antiretroviral defense. Upon successful antiretroviral treatment, the transcriptome profile of previously viremic individuals reverted to a pattern comparable to that of elite controllers and of uninfected individuals. Genome-wide evaluation of cis-acting SNPs identified genetic variants modulating expression of 190 genes. Those were compared to the genes whose expression was found associated with viral load: expression of one interferon stimulated gene, OAS1, was found to be regulated by a SNP (rs3177979, p = 4.9E-12); however, we could not detect an independent association of the SNP with viral setpoint. Thus, this study represents an attempt to integrate genome-wide SNP signals with genome-wide expression profiles in the search for biological correlates of HIV-1 control. It underscores the paradox of the association between increasing levels of viral load and greater expression of antiviral defense pathways. It also shows that elite controllers do not have a fully distinctive mRNA expression pattern in CD4+ T cells. Overall, changes in global RNA expression reflect responses to viral replication rather than a mechanism that might explain viral control.

In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system

Porcine circovirus type 2 (PCV2) nucleic acid and/or antigens are consistently observed in cells of monocytic morphology in lesions of pigs affected by post-weaning multisystemic wasting syndrome (PMWS). In this study, PCV2 antigen was detected in the cytoplasm of monocytes, pulmonary macrophages (PMs) and monocyte-derived macrophages exposed to the virus in vitro, by immunofluorescence analysis (IFA) and the phenotype of these cells confirmed by detection of monocytic cell surface markers using flow cytometry. Viral antigen was not observed in lymphocytic cells. Replication of the virus in PMs was investigated further by comparison to that observed in the continuous pig kidney cell line (PK15A) using quantitative virus titration, quantitative PCR and by the detection of double stranded DNA intermediates of viral replication by Southern blotting analyses. Although increases in viral DNA and levels of infectious virus progeny and the presence of replicative intermediates, indicative of viral replication, were observed in PK15A cells, no such changes were observed in PMs in spite of the fact that infectious virus, viral antigen and viral DNA persisted in the cells for at least the duration of the experiment. These results suggest that in vivo, monocytic cells may not represent the primary target for PCV2 replication. (C) 2003 Elsevier B.V. All rights reserved.

Immune system's role in viral encephalitis.

Viral infections can be a major thread for the central nervous system (CNS), therefore, the immune system must be able to mount a highly proportionate immune response, not too weak, which would allow the virus to proliferate, but not too strong either, to avoid collateral damages. Here, we aim at reviewing the immunological mechanisms involved in the host defense in viral CNS infections. First, we review the specificities of the innate as well as the adaptive immune responses in the CNS, using several examples of various viral encephalitis. Then, we focus on three different modes of interactions between viruses and immune responses, namely human Herpes virus-1 encephalitis with the defect in innate immune response which favors this disease; JC virus-caused progressive multifocal leukoencephalopathy and the crucial role of adaptive immune response in this example; and finally, HIV infection with the accompanying low grade chronic inflammation in the CNS in some patients, which may be an explanation for the presence of cognitive disorders, even in some well-treated HIV-infected patients. We also emphasize that, although the immune response is generally associated with viral replication control and limited cellular death, an exaggerated inflammatory reaction can lead to tissue damage and can be detrimental for the host, a feature of the immune reconstitution inflammatory syndrome (IRIS). We will briefly address the indication of steroids in this situation.