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Papers by Stephen Chiweshe

The discovery of a class of small ribonucleic acid (RNA) molecules known as microRNAs (miRNAs) ha... more The discovery of a class of small ribonucleic acid (RNA) molecules known as microRNAs (miRNAs) has led to extensive interest in their biological relevance and role. The first miRNA was discovered in Caenorhabditis elegans in 1993. Since then studies have shown that miRNAs represent a fundamental mechanism of gene expression regulation, regulating thousands of genes at the post-transcriptional level. Given that viruses are highly adept at exploiting cellular processes, it is perhaps unsurprising they have evolved miRNAs of their own. The majority of known viral miRNAs are expressed by herpes viruses underscoring viii Stephen M Chiweshe CRAC New approaches and technologies are required to elucidate the functions of these miRNAs. The initial goal of this project is to establish and optimise the CRAC technique. This will be performed in HEK293 cells transfected with a plasmid expressing a cluster of HCMV miRNAs termed miR-US25. The long-term goal of the project will be to use CRAC technology to identify miRNA targets in infected cells.

Recent advances in microRNA target identification have greatly increased the number of putative t... more Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effec...

Friends and family, I owe you massively. To my beautiful family, Bridget, Esther and Tinaye, love... more Friends and family, I owe you massively. To my beautiful family, Bridget, Esther and Tinaye, love you lots, thank you, thank you and thank you for putting up with me. You fought an amazing battle, together we have succeeded. Victor Masaka Chiweshe sonny, this is for youmuch love. Mum, dad and mum, here is what you made. Masaka Chiweshe OG and Esther T Chiweshe, one word, INSPIRATIONALthank you Moyondizvo, thank you Soko Jakopo. Bro Tendai and family, Hugh and Munyaradzi, together we have achieved. The Ranjisis, Kuudzadombos, Kuris, Muguwes and Sayis, in one way or another you played a part, an invaluable part it was, thank you very much. Dr Geoffrey Mainda, the writing up was just about manageable knowing you are suffering as much if not more, thanks for the companionship. As always, from 2005 to date, partly or fully, I am here because you guys fought some battles for me, Mrs.

Open biology, Nov 1, 2017

Successful generation of virions from infected cells is a complex process requiring orchestrated ... more Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral ther...

PLoS Pathogens, 2013

Recent advances in microRNA target identification have greatly increased the number of putative t... more Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions.

PLOS Neglected Tropical Diseases

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health an... more Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG di... more The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as direct inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with a specific suppression of CpGs within the IE1 major immediate early tr...

Journal of …, 2005

The innate immune response, and in particular the alpha/beta interferon (IFN-/ß) system, plays a ... more The innate immune response, and in particular the alpha/beta interferon (IFN-/ß) system, plays a critical role in the control of viral infections. Interferons and ß exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While ...

The discovery of a class of small ribonucleic acid (RNA) molecules known as microRNAs (miRNAs) ha... more The discovery of a class of small ribonucleic acid (RNA) molecules known as microRNAs (miRNAs) has led to extensive interest in their biological relevance and role. The first miRNA was discovered in Caenorhabditis elegans in 1993. Since then studies have shown that miRNAs represent a fundamental mechanism of gene expression regulation, regulating thousands of genes at the post-transcriptional level. Given that viruses are highly adept at exploiting cellular processes, it is perhaps unsurprising they have evolved miRNAs of their own. The majority of known viral miRNAs are expressed by herpes viruses underscoring viii Stephen M Chiweshe CRAC New approaches and technologies are required to elucidate the functions of these miRNAs. The initial goal of this project is to establish and optimise the CRAC technique. This will be performed in HEK293 cells transfected with a plasmid expressing a cluster of HCMV miRNAs termed miR-US25. The long-term goal of the project will be to use CRAC technology to identify miRNA targets in infected cells.

Recent advances in microRNA target identification have greatly increased the number of putative t... more Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effec...

Friends and family, I owe you massively. To my beautiful family, Bridget, Esther and Tinaye, love... more Friends and family, I owe you massively. To my beautiful family, Bridget, Esther and Tinaye, love you lots, thank you, thank you and thank you for putting up with me. You fought an amazing battle, together we have succeeded. Victor Masaka Chiweshe sonny, this is for youmuch love. Mum, dad and mum, here is what you made. Masaka Chiweshe OG and Esther T Chiweshe, one word, INSPIRATIONALthank you Moyondizvo, thank you Soko Jakopo. Bro Tendai and family, Hugh and Munyaradzi, together we have achieved. The Ranjisis, Kuudzadombos, Kuris, Muguwes and Sayis, in one way or another you played a part, an invaluable part it was, thank you very much. Dr Geoffrey Mainda, the writing up was just about manageable knowing you are suffering as much if not more, thanks for the companionship. As always, from 2005 to date, partly or fully, I am here because you guys fought some battles for me, Mrs.

Open biology, Nov 1, 2017

Successful generation of virions from infected cells is a complex process requiring orchestrated ... more Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral ther...

PLoS Pathogens, 2013

Recent advances in microRNA target identification have greatly increased the number of putative t... more Recent advances in microRNA target identification have greatly increased the number of putative targets of viral microRNAs. However, it is still unclear whether all targets identified are biologically relevant. Here, we use a combined approach of RISC immunoprecipitation and focused siRNA screening to identify targets of HCMV encoded human cytomegalovirus that play an important role in the biology of the virus. Using both a laboratory and clinical strain of human cytomegalovirus, we identify over 200 putative targets of human cytomegalovirus microRNAs following infection of fibroblast cells. By comparing RISC-IP profiles of miRNA knockout viruses, we have resolved specific interactions between human cytomegalovirus miRNAs and the top candidate target transcripts and validated regulation by western blot analysis and luciferase assay. Crucially we demonstrate that miRNA target genes play important roles in the biology of human cytomegalovirus as siRNA knockdown results in marked effects on virus replication. The most striking phenotype followed knockdown of the top target ATP6V0C, which is required for endosomal acidification. siRNA knockdown of ATP6V0C resulted in almost complete loss of infectious virus production, suggesting that an HCMV microRNA targets a crucial cellular factor required for virus replication. This study greatly increases the number of identified targets of human cytomegalovirus microRNAs and demonstrates the effective use of combined miRNA target identification and focused siRNA screening for identifying novel host virus interactions.

PLOS Neglected Tropical Diseases

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health an... more Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis.

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG di... more The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as direct inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with a specific suppression of CpGs within the IE1 major immediate early tr...

Journal of …, 2005

The innate immune response, and in particular the alpha/beta interferon (IFN-/ß) system, plays a ... more The innate immune response, and in particular the alpha/beta interferon (IFN-/ß) system, plays a critical role in the control of viral infections. Interferons and ß exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While ...