Biology subjects

Parthasarathy, H.

Publications and source records attributed to Parthasarathy, H..

4 recordsLinked to original sources

bioRxiv2021

Regulation of eIF2α Phosphorylation by MAPKs Influences Polysome Stability and Protein Translation

Regulation of protein translation occurs primarily at the level of initiation and is mediated by multiple signaling pathways, majorly mechanistic target of rapamycin complex 1 (mTORC1), mitogen-activated protein kinases (MAPKs), and the eukaryotic translation initiation factor eIF2. While mTORC1 and eIF2 influence the polysome stability, MAPKs influence the phosphorylation of the cap-binding protein eIF4E and are known to influence translation of only a small set of mRNAs. Here, we demonstrate that p38 MAPK and ERK1/2 regulate translation through integrated stress response (ISR) pathways. Dual inhibition (dual-Mi) of p38 MAPK and ERK1/2 caused substantial phosphorylation of eIF2 in a synergistic manner, resulting in near-absolute collapse of polysomes. This regulation was independent of Mnk1/2, a well-studied mediator of translation regulation by the MAPKs. Dual-Mi-induced polysome dissociation was far more striking than that caused by sodium arsenite, a strong inducer of ISR. Interestingly, induction of ISR caused increased p38 phosphorylation, and its inhibition resulted in stronger polysome dissociation, indicating the importance of p38 in the translation activities. Thus, our studies demonstrate a major, unidentified role for ERK1/2 and more particularly p38 MAPK in the maintenance of homeostasis of polysome association and translation activities.

biochemistry↗

bioRxiv2021

Suppression of Global Protein Translation in SARS-CoV-2 Infection

Viruses employ distinct strategies to ensure efficient translation of their mRNAs over the host transcripts. SARS-CoV-2 targets host mRNAs and ribosomes to favor its own protein synthesis. However, the modulation of the key signal pathways that control the host protein translation machinery during SARS-CoV-2 infection has not been sufficiently addressed. Here, by employing an early variant and a Delta variant isolate that evolved later in the pandemic demonstrates that SARS-CoV-2 infection results in massive polysome collapse starting from 24 hpi, a hallmark of global translation inhibition. Unexpectedly, eIF2 phosphorylation, commonly targeted by viruses to induce translation arrest, was not involved in the translation arrest, suggesting that SARS-CoV-2 countermeasures by the virus to suppress ISR. eIF4E phosphorylation remained unaltered during the infection, ruling out its involvement in the preferential translation of SARS-CoV-2 transcripts. We find that SARS-CoV-2 infection consistently causes mTORC1 inhibition in a comparable manner across both variants indicating that the virus likely targets mTORC1 pathway to suppress host translation. Interestingly, mTORC1 inhibition by SARS-CoV-2 did not impact the polysomal loading of ribosomal protein transcripts rpS3 and rpL26, suggesting that 5TOP mRNAs are spared from the translation suppression and that ribosomal protein synthesis remains active during the infection. Pharmacological activation of mTORC1 did not significantly impact viral replication, suggesting that mTORC1 inhibition might be selectively restricting the host mRNAs from accessing the translation machinery, facilitating a more robust translation of viral transcripts. This study provides new insights into the molecular interactions by which SARS-CoV-2 variants, despite their different clinical outcomes, converge on a conserved mechanism to manipulate host translation regulatory pathways.

microbiology↗

bioRxiv2021

Inactivation of SARS-CoV-2 by β-propiolactone Causes Aggregation of Viral Particles and Loss of Antigenic Potential

Inactivated viral preparations are important resources in vaccine and antisera industry. Of the many vaccines that are being developed against COVID-19, inactivated whole-virus vaccines are also considered effective. {beta}-propiolactone (BPL) is a widely used chemical inactivator of several viruses. Here, we analyze various concentrations of BPL to effectively inactivate SARS-CoV-2 and their effects on the biochemical properties of the virion particles. BPL at 1:2000 (v/v) concentrations effectively inactivated SARS-CoV-2. However, higher BPL concentrations resulted in the loss of both protein content as well as the antigenic integrity of the structural proteins. Higher concentrations also caused substantial aggregation of the virion particles possibly causing undesirable outcomes including a potential immune escape by infectious virions, and a loss in antigenic potential. We also identify that the viral RNA content in the culture supernatants can be a direct indicator of their antigenic content. Our findings may have important implications in the vaccine and antisera industry during COVID-19 pandemic.

microbiology↗

bioRxiv2021

Development of Equine Immunoglobulin Fragment F(ab')2 with High Neutralizing Capability against SARS-CoV-2

The ongoing pandemic, COVID-19, caused by SARS-CoV-2 has taken the world, and especially the scientific community by storm. While vaccines are being introduced into the market, there is also a pressing need to find potential drugs and therapeutic modules. Remdesivir is one of the antivirals currently being used with a limited window of action. As more drugs are being vetted, passive immunotherapy in the form of neutralizing antibodies can provide immediate action to combat the increasing numbers of COVID-positive cases. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 generate high titers of antibody with a strong virus neutralizing potential. ELISA performed with pooled antisera displayed highest immunoglobulin titer on 42 days post-immunization, at 1:51,200 dilutions. F(ab)2 immunoglobulin fragments generated from the pools also showed very high, antigen-specific affinity at 1:102,400 dilutions. Finally, in vitro virus neutralization assays confirmed that different pools of F(ab)2 fragments could successfully neutralize SARS-CoV-2 with titers well above 25,000, indicating the potential of this strategy in treating severe COVID-19 cases with high titers. The F(ab)2 was able to cross neutralize another SARS-CoV-2 strain, demonstrating its efficacy against the emerging viral variants and the importance of this approach in our efforts of eradication of COVID-19. In conclusion, this study demonstrates that virus-neutralizing antibodies raised in equines can potentially be used as a treatment regimen in the form of effective passive immunotherapy to combat COVID-19.

immunology↗