Knut Ivan Kristiansen

Increased Expression of Toll-Like Receptor 2 on Monocytes in HIV Infection: Possible Roles in Inflammation and Viral Replication

BACKGROUND Toll-like receptors (TLRs) are key pattern-recognition receptors of the innate immune system, but their role in human immunodeficiency virus (HIV) infection is largely unknown. METHODS In the present study, we examined the expression of TLR2 and TLR4 on monocytes from 48 HIV-infected patients and 21 healthy control subjects by flow cytometry. RESULTS We found that freshly isolated monocytes from HIV-infected patients displayed enhanced expression of TLR2 but not TLR4, that TLR2 expression on the surface of monocytes was significantly increased upon stimulation of HIV type 1 envelope protein gp120, and that TLR2 stimulation in HIV-infected patients induced increased viral replication and TNF- alpha response. CONCLUSION Our findings suggest potential roles for TLR2 in chronic immune activation and viral replication in HIV infection.

Predicting non-coding RNA genes in Escherichia coli with boosted genetic programming

Several methods exist for predicting non-coding RNA (ncRNA) genes in Escherichia coli (E.coli). In addition to about sixty known ncRNA genes excluding tRNAs and rRNAs, various methods have predicted more than thousand ncRNA genes, but only 95 of these candidates were confirmed by more than one study. Here, we introduce a new method that uses automatic discovery of sequence patterns to predict ncRNA genes. The method predicts 135 novel candidates. In addition, the method predicts 152 genes that overlap with predictions in the literature. We test sixteen predictions experimentally, and show that twelve of these are actual ncRNA transcripts. Six of the twelve verified candidates were novel predictions. The relatively high confirmation rate indicates that many of the untested novel predictions are also ncRNAs, and we therefore speculate that E.coli contains more ncRNA genes than previously estimated.

A new protein superfamily includes two novel 3-methyladenine DNA glycosylases from Bacillus cereus, AlkC and AlkD

Soil bacteria are heavily exposed to environmental methylating agents such as methylchloride and may have special requirements for repair of alkylation damage on DNA. We have used functional complementation of an Escherichia coli tag alkA mutant to screen for 3‐methyladenine DNA glycosylase genes in genomic libraries of the soil bacterium Bacillus cereus. Three genes were recovered: alkC, alkD and alkE. The amino acid sequence of AlkE is homologous to the E. coli AlkA sequence. AlkC and AlkD represent novel proteins without sequence similarity to any protein of known function. However, iterative and indirect sequence similarity searches revealed that AlkC and AlkD are distant homologues of each other within a new protein superfamily that is ubiquitous in the prokaryotic kingdom. Homologues of AlkC and AlkD were also identified in the amoebas Entamoeba histolytica and Dictyostelium discoideum, but no other eukaryotic counterparts of the superfamily were found. The alkC and alkD genes were expressed in E. coli and the proteins were purified to homogeneity. Both proteins were found to be specific for removal of N‐alkylated bases, and showed no activity on oxidized or deaminated base lesions in DNA. B. cereus AlkC and AlkD thus define novel families of alkylbase DNA glycosylases within a new protein superfamily.

Single Transmembrane Peptide DinQ Modulates Membrane-Dependent Activities

The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated. The +44 dinQ transcript translates into a toxic single transmembrane peptide localized in the inner membrane. AgrB regulates dinQ RNA by RNA interference to counteract DinQ toxicity. Thus the dinQ-agr locus shows the classical features of a type I TA system and has many similarities to the tisB-istR locus. DinQ overexpression depolarizes the cell membrane and decreases the intracellular ATP concentration, demonstrating that DinQ can modulate membrane-dependent processes. Augmented DinQ strongly inhibits marker transfer by Hfr conjugation, indicating a role in recombination. Furthermore, DinQ affects transformation of nucleoid morphology in response to UV damage. We hypothesize that DinQ is a transmembrane peptide that modulates membrane-dependent activities such as nucleoid compaction and recombination.

Modulatory effect of mannose-binding lectin on cytokine responses: possible roles in HIV infection

Background  Mannose‐binding lectin (MBL) is a soluble receptor of the innate immune system, probably contributing to antimicrobial defence. The possible role of MBL in HIV infection is unclear.

Prevalence of reverse transcriptase and protease mutations associated with antiretroviral drug resistance among drug-naïve HIV-1 infected pregnant women in Kagera and Kilimanjaro regions, Tanzania

BackgroundAccess to antiretroviral drugs for HIV-1 infection has increased in sub-Saharan Africa (SSA) during the past few years. Mutations in the HIV-1 genome are often associated with treatment failure as indicated by viral replication and elevated levels of virus in the blood. Mutations conferring resistance to antiretroviral drugs are based on comparing gene sequences with corresponding consensus sequences of HIV-1 subtype B that represents only 10% of the AIDS pandemic. The HIV pandemic in SSA is characterized by high viral genetic diversity. Before antiretroviral drugs become more widely available, it is important to characterize baseline naturally occurring genetic mutations and polymorphisms associated with antiretroviral drug resistance among circulating HIV-1 subtypes.MethodsThe prevalence of mutations associated with antiretroviral drug resistance in protease (PR) and reverse transcriptase (RT) regions among antiretroviral treatment-naïve HIV-1 infected pregnant women was investigated in Bukoba (Kagera) and Moshi (Kilimanjaro) municipalities, Tanzania, between September and December 2005. The HIV-1 pol gene was amplified using primers recognizing conserved viral sequences and sequenced employing BigDye chemistry from 100 HIV-1 seropositive treatment-naïve pregnant women and 61 HIV-1 seropositive women who had received a single dose of Nevirapine (sdNVP). Positions 1–350 of the RT and 1–99 of the PR genes were analyzed for mutations based on the Stanford University HIV Drug Resistance Database.ResultsHIV-1 subtypes A, C, D, CRF10_CD and Unique Recombinant Forms (URF) were detected. Primary mutations associated with NRTI and NNRTI resistance were detected among 3% and 4% of treatment-naïve strains, respectively. Primary mutations associated with NRTI and NNRTI resistance were detected in 1.6% and 11.5% of women who had received sdNVP, respectively. None of the primary mutations associated with PI resistance was found. Polymorphisms detected in RT and PR sequences were mainly mutations that are found in the consensus sequences of non-B subtypesConclusionBased on the WHO HIV Drug Resistance Research Network Threshold of less than 5%, the baseline prevalence of primary mutations among treatment-naïve HIV-1 infected pregnant women in Kagera and Kilimanjaro regions was low. The significance of HIV-1 subtype B polymorphic positions with respect to antiretroviral resistance identified among the prevalent HIV-1 subtypes is unknown. More studies addressing the correlation between polymorphic mutations, antiretroviral resistance and clinical outcome are warranted in regions where non-B subtypes are prevalent.

Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions, Tanzania.

A strategy to prevent the spread of HIV-1 worldwide is complicated by the high genetic diversity of the virus. To gain a better understanding of the HIV-1 genetic diversity in Tanzania, a molecular epidemiological investigation was conducted in Kagera and Kilimanjaro regions. While several studies have addressed HIV-1 subtypes in Tanzania, this is the first study to describe the virus subtypes circulating in Kagera. The Kagera region is the epicenter of the HIV-1 epidemic in Africa, and it was therefore of interest to compare the prevalence of HIV subtypes in this region and Kilimanjaro. Blood samples were obtained from 246 HIV-1-infected pregnant women attending antenatal clinics. Plasma HIV-1 RNA was extracted, amplified, and sequenced in the env C2V3 and/or pol regions from 209 samples. Based on the analysis of env C2V3 and pol sequences, 47.4% had concordant subtypes, 19.1% were discordant indicating recombination, and for 33.5% sequences were obtained for only one region. The distribution HIV-1 subtypes based on the phylogenetic analysis of paired env C2V3/ pol sequences in Kagera region was A/A (27.8%), C/C (29.6%), D/D (16.7%), and unique recombinant forms (25.9%), and in Kilimanjaro region was A/A (32.9%), C/C (25.9%), D/D (10.6%), CRF10_CD (1.2%), and unique recombinant forms (29.4%). The env C2V3 subsubtype A2 and env C2V3/pol CRF10_CD were also observed indicating that these recombinants are circulating in Tanzania. The high diversity of HIV-1 subtypes and the high prevalence of recombinants demonstrated in this study necessitate expanded and continuous monitoring of the epidemic in Tanzania. The trend may have implications for current national control strategies against the HIV-1 epidemic.

Overexpression of the LexA-regulated tisAB RNA in E. coli inhibits SOS functions; implications for regulation of the SOS response

The DNA damage induced SOS response in Escherichia coli is initiated by cleavage of the LexA repressor through activation of RecA. Here we demonstrate that overexpression of the SOS-inducible tisAB gene inhibits several SOS functions in vivo. Wild-type E. coli overexpressing tisAB showed the same UV sensitivity as a lexA mutant carrying a noncleavable version of the LexA protein unable to induce the SOS response. Immunoblotting confirmed that tisAB overexpression leads to higher levels of LexA repressor and northern experiments demonstrated delayed and reduced induction of recA mRNA. In addition, induction of prophage λ and UV-induced filamentation was inhibited by tisAB overexpression. The tisAB gene contains antisense sequences to the SOS-inducible dinD gene (16 nt) and the uxaA gene (20 nt), the latter encoding a dehydratase essential for galacturonate catabolism. Cleavage of uxaA mRNA at the antisense sequence was dependent on tisAB RNA expression. We showed that overexpression of tisAB is less able to confer UV sensitivity to the uxaA dinD double mutant as compared to wild-type, indicating that the dinD and uxaA transcripts modulate the anti-SOS response of tisAB. These data shed new light on the complexity of SOS regulation in which the uxaA gene could link sugar metabolism to the SOS response via antisense regulation of the tisAB gene.

Genotypic Analysis of Human Immunodeficiency Virus Type 1 env V3 Loop Sequences: Bioinformatics Prediction of Coreceptor Usage Among 28 Infected Mother–Infant Pairs in a Drug-Naive Population

We sought to predict virus coreceptor utilization using a simple bioinformatics method based on genotypic analysis of human immunodeficiency virus types 1 (HIV-1) env V3 loop sequences of 28 infected but drug-naive women during pregnancy and their infected infants and to better understand coreceptor usage in vertical transmission dynamics. The HIV-1 env V3 loop was sequenced from plasma samples and analyzed for viral coreceptor usage and subtype in a cohort of HIV-1-infected pregnant women. Predicted maternal frequencies of the X4, R5X4, and R5 genotypes were 7%, 11%, and 82%, respectively. Antenatal plasma viral load was higher, with a mean log(10) (SD) of 4.8 (1.6) and 3.6 (1.2) for women with the X4 and R5 genotypes, respectively, p = 0.078. Amino acid substitution from the conserved V3 loop crown motif GPGQ to GPGR and lymphadenopathy were associated with the X4 genotype, p = 0.031 and 0.043, respectively. The maternal viral coreceptor genotype was generally preserved in vertical transmission and was predictive of the newborns viral genotype. Infants born to mothers with X4 genotypes were more likely to have lower birth weights relative to those born to mothers with the R5 genotype, with a mean weight (SD) of 2870 (±332) and 3069 (±300) g, respectively. These data show that at least in HIV-1 subtype C, R5 coreceptor usage is the most predominant genotype, which is generally preserved following vertical transmission and is associated with the V3 GPGQ crown motif. Therefore, antiretroviral-naive pregnant women and their infants can benefit from ARV combination therapies that include R5 entry inhibitors following prediction of their coreceptor genotype using simple bioinformatics methods.

Evolution of human immunodeficiency virus type 1 serotypes in northern Tanzania: a retrospective study.

The HIV‐1 epidemic in Tanzania is characterized by the circulation of heterogeneous virus subtypes. A retrospective study was conducted to determine the changing pattern of circulating HIV‐1 subtypes in northern Tanzania. A peptide‐binding enzyme immunoassay (PEIA) was employed to analyse 305 HIV‐1 positive serum and plasma samples collected between 1985 and 2005. Samples were serotyped using synthetic peptides representing HIV‐1 genotypes A–E derived from consensus gp120 V3 sequences. Plasma samples collected in 2005 were V3 genotyped for comparison with PEIA results. In 1985, serotypes A and D were co‐circulating while serotype C was first detected in 1990. In 2001 and 2005, serotype C was the most prevalent, serotype A was stable, and serotype D was declining. PEIA is relatively rapid and simple to perform compared to molecular approaches, and is a valuable epidemiological tool in regions with limited resources. HIV‐1 classification into serotypes based on antigenic V3 diversity may be a useful screening tool for the identification of HIV‐1 variants with regard to diagnosis, treatment, disease progression and candidate vaccine development.