Home » Alphavirus nsP2 interacts with host pathways for viral minus-strand synthesis and replication complex stability. by Junbo Mai
Alphavirus nsP2 interacts with host pathways for viral minus-strand synthesis and replication complex stability. Junbo Mai

Alphavirus nsP2 interacts with host pathways for viral minus-strand synthesis and replication complex stability.

Junbo Mai

Published
ISBN : 9781109657920
NOOKstudy eTextbook
192 pages
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 About the Book 

Alphavirus nsP2 has been described to play a critical role in regulating host responses. To investigate the function of P23 proteins in host-cell interaction, SIN2V, a Sindbis virus (SIN) mutant unable to cleave P23 to nsP2 and nsP3, was constructedMoreAlphavirus nsP2 has been described to play a critical role in regulating host responses. To investigate the function of P23 proteins in host-cell interaction, SIN2V, a Sindbis virus (SIN) mutant unable to cleave P23 to nsP2 and nsP3, was constructed and found to bear two phenotypes: continuous minus strand synthesis and unstable RTC activity (Gorchakov et al., 2008). This finding allowed me to test the hypothesis that only mature nsPs, not P23 can activate and control host responses. SIN2V was employed to probe the fate of SIN2V minus strands, the conditions needed to rescue its phenotypes and the mechanism of RTC inactivation. It was found that unlike wt SIN, SIN2V RTC containing P23 was not able to switch between 26S mRNA synthesis and 49S plus strand synthesis and did not alter the ratios of 49S:26S plus-strand RNA during infection. Ethidium bromide staining of the RTC double-stranded (RF) RNA cores and 3H-uridine radiolabeling of the RFs were used to quantify RTC levels and replication activity. It was shown that wt SIN accumulated ∼ 27,000 RFs per infected BHK21 cell. SIN2V RFs were accumulated to 112% of wt SIN levels although SIN2V theoretically produced ∼260% of wildtype minus strand amounts. This meant that those RTC complexes in excess of wt levels must have been degraded. The SIN2V minus strands made early or late functioned as authentic templates and their template activity was similarly susceptible to inactivation. Alphavirus nsP proteins introduced through superinfection with Semliki Forest virus rescued the P23 defective function needed to induce minus strand synthesis cessation. The loss of SIN2V RTC activity was not due to the physical degradation, but rather to the proteasome action. Inhibition of unbiquitin activating enzyme E1 activity also rescued SIN2V RTC activity, suggesting that the mature nsP2 and possibly nsP3 proteins block this ubiquitin-proteasome pathway. Taken together, these findings demonstrated that mature nsP2 and possibly nsP3 but not P23 activates a host response to shut off minus strand synthesis and blocks a host response to protect RTC activity.