Biology subjects

Mahalingam, M.

Publications and source records attributed to Mahalingam, M..

3 recordsLinked to original sources

bioRxiv2021

Engineered Bacteriophage T4 Nanoparticle as a Potential Targeted Activator of HIV-1 Latency in CD4+ Human T cells

A major barrier for HIV-1 eradication is the latent virus reservoir containing stably integrated and silent proviruses in CD4+ T-cells. Targeted reactivation and removal of this latent reservoir is a potential strategy for HIV-1 cure but remains a major challenge. Here, we investigated whether CD4-targeted bacteriophage T4 capsid nanoparticles that mimic HIV envelope can reactivate HIV-1 latency. The nanoparticles were arrayed with CD4-binding CD4-DARPin, or HIV-1 gp140 envelope trimer. When exposed to J-Lat T-cell model of HIV-1 latency or primary T-lymphocytes from human PBMCs, these nanoparticles activated CD4+ T-cells without causing global T-cell activation, which led to activation of HIV-1 proviral transcription, viral protein production and release. Intriguingly, the observed T-cell activation and HIV-1 latency reversal do not involve the classic PKC or NFAT pathways and did not lead to cytokine storm. These studies indicate that engineered non-infectious bacteriophages can be exploited for HIV-1 cure and targeted T-cell therapies.

immunology↗

bioRxiv2021

A genetic shift in an escaped transmitted/founder virus guides combinatorial vaccine design against HIV-1

A productive HIV-1 infection in humans is often established by transmission and propagation of a single transmitted/founder (T/F) virus, which then evolves into a complex mixture of variants during the lifetime of infection. An effective HIV-1 vaccine should elicit broad immune responses in order to block the entry of diverse T/F viruses. Currently, no such vaccine exists. An in-depth study of escape variants emerging under host immune pressure during very early stages of infection might provide insights into such a HIV-1 vaccine design. Here, in a rare longitudinal study involving HIV-1 infected individuals just days after infection in the absence of antiretroviral therapy, we discovered a remarkable genetic shift that resulted in near complete disappearance of the original T/F virus and appearance of a variant with H173Y mutation in the variable V2 domain of the HIV-1 envelope protein. This coincided with the disappearance of the first wave of strictly H173-specific antibodies and emergence of a second wave of Y173-specific antibodies with increased breadth. Structural analyses indicated conformational dynamism of the envelope protein which likely allowed selection of escape variants with a conformational switch in the V2 domain from an -helix (H173) to a {beta}-strand (Y173) and induction of broadly reactive antibody responses. This differential breadth due to a single mutational change was also recapitulated in a mouse model. Rationally designed combinatorial libraries containing 54 conformational variants of V2 domain around position 173 further demonstrated increased breadth of antibody responses elicited to diverse HIV-1 envelope proteins. These results offer new insights into designing broadly effective HIV-1 vaccines.

microbiology↗

bioRxiv2021

A viral genome packaging ring-ATPase is a flexibly coordinated pentamer

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, by direct counting using single-molecule fluorescence, we have determined that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

biophysics↗