Celso Cunha

Malaria Parasites Can Develop Stable Resistance to Artemisinin but Lack Mutations in Candidate Genes atp6 (Encoding the Sarcoplasmic and Endoplasmic Reticulum Ca2+ ATPase), tctp, mdr1, and cg10

ABSTRACT Resistance of Plasmodium falciparum to drugs such as chloroquine and sulfadoxine-pyrimethamine is a major problem in malaria control. Artemisinin (ART) derivatives, particularly in combination with other drugs, are thus increasingly used to treat malaria, reducing the probability that parasites resistant to the components will emerge. Although stable resistance to artemisinin has yet to be reported from laboratory or field studies, its emergence would be disastrous because of the lack of alternative treatments. Here, we report for the first time, to our knowledge, genetically stable and transmissible ART and artesunate (ATN)-resistant malaria parasites. Each of two lines of the rodent malaria parasite Plosmodium chabaudi chabaudi, grown in the presence of increasing concentrations of ART or ATN, showed 15-fold and 6-fold increased resistance to ART and ATN, respectively. Resistance remained stable after cloning, freeze-thawing, after passage in the absence of drug, and transmission through mosquitoes. The nucleotide sequences of the possible genetic modulators of ART resistance (mdr1, cg10, tctp, and atp6) of sensitive and resistant parasites were compared. No mutations in these genes were identified. In addition we investigated whether changes in the copy number of these genes could account for resistance but found that resistant parasites retained the same number of copies as their sensitive progenitors. We believe that this is the first report of a malaria parasite with genetically stable and transmissible resistance to artemisinin or its derivatives.

First Isolation of Borrelia lusitaniae from a Human Patient

ABSTRACT The first human isolate of Borrelia lusitaniae recovered from a Portuguese patient with suspected Lyme borreliosis is described. This isolate, from a chronic skin lesion, is also the first human isolate of Borrelia in Portugal. Different phenotypic and molecular methods are used to characterize it.

Mechanisms and Signals for the Nuclear Import of Proteins

In eukaryotes, the nuclear membrane provides a physical barrier to the passive diffusion of macromolecules from and into the cytoplasm. Nucleocytoplasmic traffic occurs through highly specialized structures known as nuclear pores, and involves the participation of a special class of transport proteins. Active transport across the nuclear pores is an energy-dependent process that relies on the activity of Ran-GTPases both in the nuclear and cytoplasmic compartments. Nuclear import of proteins is an essential step in regulating gene expression and the replication cycle of several viruses. In this review, the key mechanisms, pathways, and models underlying the transport of proteins across nuclear pores are analysed.

Hepatitis delta virus ribonucleoproteins shuttle between the nucleus and the cytoplasm.

Hepatitis delta virus (HDV) infection of individuals infected with hepatitis B virus (HBV) is associated with more severe liver damage and an increased risk of fulminant disease. HDV is a single-stranded RNA virus that encodes a single protein, the delta antigen, which is expressed in two forms, small (S-HDAg) and large (L-HDAg). Here we show that although HDV ribonucleoproteins are mainly detected in the nucleus, they are also present in the cytoplasm of cells infected with HDV or transfected with HDV cDNA. Making use of an heterokaryon assay, we demonstrate that HDV ribonucleoproteins shuttle continuously between the nucleus and the cytoplasm. In the absence of HDV RNA, both forms of the delta antigen are retained in the nucleus, whereas in the absence of the delta antigen, HDV RNA is predominantly detected in the cytoplasm. Coexpression of HDV RNA and S-HDAg (which binds to the viral RNA and contains a nuclear localization signal) results in nuclear accumulation of the viral RNA. This suggests that HDV RNA mediates export of viral particles to the cytoplasm whereas the delta antigen triggers their reimport into the nucleus.

Hepatitis delta virus infects the cells of hepadnavirus‐induced hepatocellular carcinoma in woodchucks

Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus‐induced hepatocellular carcinoma (HCC). Woodchucks (Marmota monax) that were chronically infected with HBV‐related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery the WHV carrier woodchucks were superinfected with WHV‐enveloped HDV (wHDV). Six weeks later the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real‐time polymerase chain reaction assay, and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues. Conclusion: Cells of WHV‐induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Because others previously hypothesized that hepadnavirus‐induced HCCs are resistant to reinfection with a hepadnavirus in vivo, our data suggest that if such a resistance exists it likely occurs via a block at the post‐entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection. (HEPATOLOGY 2012;56:76–85)

Proteome analysis of a human liver carcinoma cell line stably expressing hepatitis delta virus ribonucleoproteins

Hepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identification of host factors involved in pathogenesis. Here, we made use of a HDV cDNA stably transfected cell line, Huh7-D12, in a proteomic approach to identify the changes in the protein expression profiles in human liver cells that arise as a consequence of HDV replication. Total protein extracts from Huh7-D12 cells and of the corresponding non transfected human liver carcinoma cell line, Huh7, were separated by 2-DE. Differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 23 differentially expressed proteins of which 15 were down regulated and 8 up regulated in Huh7-D12 cells. These proteins were found to be involved in different cellular pathways. The down regulation of the histone H1-binding protein and of triosephosphate isomerase was confirmed by Real time PCR, and the up regulation of the La protein and lamin A/C was validated by western blot.

Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles.

ABSTRACT A natural subviral agent of human hepatitis B virus (HBV), hepatitis delta virus (HDV), requires only the envelope proteins from HBV in order to maintain persistent infection. HBV surface antigens (HBsAgs) can be produced either by HBV replication or from integrated HBV DNA regardless of replication. The functional properties of the integrant-generated HBsAgs were examined using two human hepatocellular carcinoma-derived cell lines, Hep3B and PLC/PRF/5, that contain HBV integrants but do not produce HBV virions and have no signs of HBV replication. Both cell lines were able to support HDV replication and assembly/egress of HDV virions. Neither of the cell lines was able to produce substantial amounts of the pre-S1-containing HDV particles. HDV virions assembled in PLC/PRF/5 cells were able to infect primary human hepatocytes, while Hep3B-derived HDV appeared to be noninfectious. These results correlate with the findings that the entire open reading frame (ORF) for the large (L) envelope protein that is essential for infectivity is present on HBV RNAs from PLC/PRF/5 cells, while an L protein ORF that was truncated and fused to inverted precore sequences was found using RNAs from Hep3B cells. This study demonstrates for the first time that at least some of the HBV DNA sequence naturally integrated during infection can produce functional small and large envelope proteins capable of the formation of infectious HDV virions. Our data indicate that in vivo chronic HDV infection can persist in the absence of HBV replication (or when HBV replication is profoundly suppressed) if functional envelope proteins are supplied from HBV integrants. IMPORTANCE The study addresses the unique mechanism of HDV persistence in the absence of ongoing HBV replication, advances our understanding of HDV-HBV interactions, and supports the implementation of treatments directly targeting HDV for HDV/HBV-infected individuals.

Changes in the proteome of Huh7 cells induced by transient expression of hepatitis D virus RNA and antigens

Hepatitis delta virus (HDV) infection of human hepatocytes infected with the hepatitis B virus (HBV) is associated with increased liver damage and risk of fulminant disease. Although considerable progress has been made towards the elucidation of the mechanisms of HDV replication and pathogenesis, little is still known about the host factors involved in the different steps of the replication cycle. Here, we made use of a proteomic approach to analyse the global alterations in protein expression that arise in human hepatocytes separately transfected with each of the HDV components. Huh7 cells were transiently transfected with plasmids that code for the small delta antigen (S-HDAg), large delta antigen (L-HDAg), genomic RNA (gRNA), and antigenomic RNA (agRNA), respectively. Total protein extracts were separated by 2-DE and differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 32 proteins known to be involved in different pathways namely nucleic acid metabolism, protein metabolism, transport, signal transduction, apoptosis, and cell growth. Moreover, the down regulation of hnRNP D, HSP105, and triosephosphate isomerase was further confirmed by Real time PCR.

HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

Hepatitis Delta Virus: A Peculiar Virus

The hepatitis delta virus (HDV) is distributed worldwide and related to the most severe form of viral hepatitis. HDV is a satellite RNA virus dependent on hepatitis B surface antigens to assemble its envelope and thus form new virions and propagate infection. HDV has a small 1.7 Kb genome making it the smallest known human virus. This deceivingly simple virus has unique biological features and many aspects of its life cycle remain elusive. The present review endeavors to gather the available information on HDV epidemiology and clinical features as well as HDV biology.