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Fulltext Protein-protein interactions between A. aegypti midgut and dengue virus 2: two-hybrid screens using the midgut cDNA library

Tham HW, Balasubramaniam VR, Chew MF, Ahmad H, Hassan SS

J Infect Dev Ctries, 2015 Dec 30;9(12):1338-49.
PMID: 26719940 DOI: 10.3855/jidc.6422

INTRODUCTION: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection.
METHODOLOGY: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.
RESULTS: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays.
CONCLUSIONS: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.
Fulltext CPB1 of Aedes aegypti interacts with DENV2 E protein and regulates intracellular viral accumulation and release from midgut cells

Tham HW, Balasubramaniam VR, Tejo BA, Ahmad H, Hassan SS

Viruses, 2014 Dec;6(12):5028-46.
PMID: 25521592 DOI: 10.3390/v6125028

Aedes aegypti is a principal vector responsible for the transmission of dengue viruses (DENV). To date, vector control remains the key option for dengue disease management. To develop new vector control strategies, a more comprehensive understanding of the biological interactions between DENV and Ae. aegypti is required. In this study, a cDNA library derived from the midgut of female adult Ae. aegypti was used in yeast two-hybrid (Y2H) screenings against DENV2 envelope (E) protein. Among the many interacting proteins identified, carboxypeptidase B1 (CPB1) was selected, and its biological interaction with E protein in Ae. aegypti primary midgut cells was further validated. Our double immunofluorescent assay showed that CPB1-E interaction occurred in the endoplasmic reticulum (ER) of the Ae. aegypti primary midgut cells. Overexpression of CPB1 in mosquito cells resulted in intracellular DENV2 genomic RNA or virus particle accumulation, with a lower amount of virus release. Therefore, we postulated that in Ae. aegypti midgut cells, CPB1 binds to the E protein deposited on the ER intraluminal membranes and inhibits DENV2 RNA encapsulation, thus inhibiting budding from the ER, and may interfere with immature virus transportation to the trans-Golgi network.
Fulltext Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF

Balasubramaniam VR, Hong Wai T, Ario Tejo B, Omar AR, Syed Hassan S

PLoS One, 2013;8(9):e72429.
PMID: 24073193 DOI: 10.1371/journal.pone.0072429

We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.
Fulltext Cellular transcripts of chicken brain tissues in response to H5N1 and Newcastle disease virus infection

Balasubramaniam VR, Wai TH, Omar AR, Othman I, Hassan SS

Virol J, 2012;9:53.
PMID: 22361110 DOI: 10.1186/1743-422X-9-53

Highly-pathogenic avian influenza (HPAI) H5N1 and Newcastle disease (ND) viruses are the two most important poultry viruses in the world, with the ability to cause classic central nervous system dysfunction in poultry and migratory birds. To elucidate the mechanisms of neurovirulence caused by these viruses, a preliminary study was design to analyze host's cellular responses during infections of these viruses.
Fulltext Cellular transcripts regulated during infections with Highly Pathogenic H5N1 Avian Influenza virus in 3 host systems

Balasubramaniam VR, Hassan SS, Omar AR, Mohamed M, Noor SM, Mohamed R, et al.

Virol J, 2011;8:196.
PMID: 21529348 DOI: 10.1186/1743-422X-8-196

Highly pathogenic Avian Influenza (HPAI) virus is able to infect many hosts and the virus replicates in high levels in the respiratory tract inducing severe lung lesions. The pathogenesis of the disease is actually the outcome of the infection as determined by complex host-virus interactions involving the functional kinetics of large numbers of participating genes. Understanding the genes and proteins involved in host cellular responses are therefore, critical for the elucidation of the mechanisms of infection.
Chemotherapeutic Role of Polyphenols Present in Ocimum sanctum

Khatoon S, Kalam N, Balasubramaniam VR, Shaikh MF, Ansari MT

Anticancer Agents Med Chem, 2022;22(20):3325-3342.
PMID: 35578854 DOI: 10.2174/1871520622666220516142839

Ocimum sanctum is a sacred herb of India and is commonly known as 'Tulsi' or 'Holy Basil' in regional languages of the country. Various parts of O. sanctum are recognised to have remarkable therapeutic efficacy, and are therefore used in Indian traditional medicine system, Ayurveda. Scientific studies have shown that O. sanctum has a range of pharmacological activities. The presence of a substantial amount of polyphenols in O. sanctum could be the reason for its excellent bioactivity. Polyphenols are used to prevent or treat oncologic diseases due to their anti-cancer effects, which are related to activation of apoptotic signaling, cell cycle arrest, binding ability with membrane receptors, and potential effects on immunomodulation and epigenetic mechanisms. The poor bioavailability of polyphenols restricts their clinical use. The application of nanonization has been implemented to improve their bioavailability, penetrability, and prolong their anticancer action. The present review analyses the recent preclinical studies related to the chemo-preventive and therapeutic potential of polyphenols present in O. sanctum. Moreover, the current article also examines in-depth the biochemical and molecular mechanisms involved in the antineoplastic actions of the considered polyphenols.
Targeting glucose metabolism with dichloroacetate (DCA) reduces zika virus replication in brain cortical progenitors at different stages of maturation

Gilbert-Jaramillo J, Komarasamy TV, Balasubramaniam VR, Heather LC, James WS

Antiviral Res, 2024 Aug;228:105933.
PMID: 38851593 DOI: 10.1016/j.antiviral.2024.105933

The underlying threat of new Zika virus (ZIKV) outbreaks remains, as no vaccines or therapies have yet been developed. In vitro research has shown that glycolysis is a key factor to enable sustained ZIKV replication in neuroprogenitors. However, neither in vivo nor clinical investigation of glycolytic modulators as potential therapeutics for ZIKV-related fetal abnormalities has been conducted. Accordingly, we tested the therapeutic potential of metabolic modulators in relevant in vitro systems comprising two pools of neuroprogenitors (NPCs), which resemble early and late stages of pregnancy. Effective doses of metabolic modulators [3.0 μM] dimethyl fumarate (DMF), [3.2 mM] dichloroacetate (DCA), and [6.3 μM] VER-246608 were determined for these cells by their effect on lactate release, pyruvate dehydrogenase (PDH) activity and cell survival. The drugs were used in a 24h pre-treatment and kept throughout ZIKV infection of NPCs. Drug effects and ZIKV replication were assessed at 24- and 56-h post-infection. In early NPCs treated with DMF, DCA and VER-246608, there was a significant reduction in the extracellular release of ZIKV potentially by PDH-mediated increased mitochondrial oxidation of glucose. Out of the three drugs, only DCA was observed to reduce viral replication in late NPCs treated with DCA. Altogether, our findings suggest that reduction of anaerobic glycolysis could be of therapeutic potential against ZIKV-related fetal abnormalities and that clinical translation should consider the use of specific glycolytic modulators over different trimesters.
Fulltext Detection and genetic characterization of canine astroviruses in pet dogs in Guangxi, China

Zhou H, Liu L, Li R, Qin Y, Fang Q, Balasubramaniam VR, et al.

Virol J, 2017 08 17;14(1):156.
PMID: 28814340 DOI: 10.1186/s12985-017-0823-4

BACKGROUND: Astroviruses (AstVs) have been reported to infect and cause gastroenteritis in most animal species. Human AstVs were regarded the causative agent of viral diarrhea in children. In dogs, little is known about the epidemiology and clinical significance of AstV infection.
FINDINGS: In this study, we collected and tested 253 rectal swabs from pet dogs; of which 64 samples (25.3%) tested positive for AstVs with diarrhea and 15 more samples (5.9%) also was identified as AstVs, however without any clinical signs. Phylogenetic analysis of 39 partial ORF1b sequences from these samples revealed that they are similar to AstVs, which can be subdivided into three lineages. Interestingly, out of the 39 isolates sequenced, 16 isolates are shown to be in the Mamastrovirus 5/canine astrovirus (CAstV) lineage and the remaining 23 isolates displayed higher similarities with known porcine astrovirus (PoAstV) 5 and 2. Further, analysis of 13 capsid sequences from these isolates showed that they are closely clustered with Chinese or Italy CAstV isolates.
CONCLUSIONS: The findings indicate that CAstVs commonly circulate in pet dogs, and our sequencing results have shown the genomic diversity of CAstVs leading to increasing number of clusters.