A Critical Observation on the Design and Development of Reported Peptide Inhibitors of DENV NS2B-NS3 Protease in the Last Two Decades

: Dengue is one of the neglected tropical diseases, which remains a reason for concern as cases seem to rise every year. The failure of the only dengue vaccine, Dengvaxia®, has made the problem more severe and humanity has no immediate respite from this global burden. Dengue virus (DENV) NS2B-NS3 protease is an attractive target partly due to its role in polyprotein processing. Also, since it is among the most conserved domains in the viral genome, it could produce a broad scope of opportunities toward antiviral drug discovery in general. This review has made a detailed analysis of each case of the design and development of peptide inhibitors against DENV NS2B-NS3 protease in the last two decades. Also, we have discussed the reasons attributed to their inhibitory activity, and wherever possible, we have highlighted the concerns raised, challenges met, and suggestions to improve the inhibitory activity. Thus, we attempt to take the readers through the designing and development of reported peptide inhibitors and gain insight from these developments, which could further contribute toward strategizing the designing and development of peptide inhibitors of DENV protease with improved properties in the coming future.

A comparative analysis of parechovirus protein structures with other picornaviruses

Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.

DWV Infection in vitro Using Honey Bee Pupal Tissue

The deformed wing virus (DWV) has been best characterized among honey bee viruses; however, very little is known regarding the mechanisms of viral infection and replication due to the lack of immortalized honey bee cell lines. To solve this problem, we established an in vitro system using honey bee pupal tissue to reconstruct DWV binding and entry into the host cell, followed by translation of the RNA genome and polyprotein processing using RNA-dependent RNA polymerase (RdRP) as a marker. Using this system, the P-domain of the virion subunit VP1 was found to be essential for DWV infection, but not for binding and entry into the cell. DWV efficiently infected the head tissue derived from early but not late pupa, suggesting that undifferentiated cells are targeted for viral infection. Furthermore, we found that inhibitors of mammalian picornavirus 3C-protease, rupintrivir and quercetin suppressed RdRP synthesis, indicating that this in vitro system is also useful for screening a compound to control viral infection. Our in vitro system may help to understand the mechanism of DWV infection in host cells.

Analysis of proteolytic processing sites in potyvirus polyproteins revealed differential amino acid preferences of NIa-Pro protease in each of seven cleavage sites

Potyviruses encode a large polyprotein that undergoes proteolytic processing, producing 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb-RdRp, and CP. While P1/HC-Pro and HC-Pro/P3 junctions are cleaved by P1 and HC-Pro, respectively, the remaining seven are processed by NIa-Pro. In this study, we analyzed 135 polyprotein sequences from approved potyvirus species and deduced the consensus amino acid residues at five positions (from −4 to +1, where a protease cleaves between −1 and +1) in each of nine cleavage sites. In general, the newly deduced consensus sequences were consistent with the previous ones. However, seven NIa-Pro cleavage sites showed distinct amino acid preferences despite being processed by the same protease. At position −2, histidine was the dominant amino acid residue in most cleavage sites (57.8–60.7% of analyzed sequences), except for the NIa-Pro/NIb-RdRp junction where it was absent. At position −1, glutamine was highly dominant in most sites (88.2–97.8%), except for the VPg/NIa-Pro junction where glutamic acid was found in all the analyzed proteins (100%). At position +1, serine was the most abundant residue (47.4–86.7%) in five out of seven sites, while alanine (52.6%) and glycine (82.2%) were the most abundant in the P3/6K1 and 6K2/VPg junctions, respectively. These findings suggest that each NIa-Pro cleavage site is finely tuned for differential characteristics of proteolytic reactions. The newly deduced consensus sequences may be useful resources for the development of models and methods to accurately predict potyvirus polyprotein processing sites.

Role of the Matrix-Capsid Cleavage Site Polymorphism S124V of HIV-1 Sub-subtype A2 in Gag Polyprotein Processing

Subtype C and A HIV-1 strains dominate the epidemic in Africa and Asia, while sub-subtype A2 is found at low frequency only in West Africa. To relate Gag processing in vitro with viral fitness, viral protease (PR) enzymatic activity and in vitro Gag processing were evaluated. The rate of sub-subtype A2 Gag polyprotein processing, as production of the p24 protein, was reduced compared to subtype B or C independent of PR subtype, indicating that subtype A2 Gag qualitatively differed from other subtypes. Introduction of subtype B matrix-capsid cleavage site in sub-subtype A2 Gag only partially restored the processing rate. Unique amino acid polymorphism V124S at the matrix-capsid cleavage site, together with other polymorphisms at non-cleavage sites, are differentially influencing the processing of Gag polyproteins. This genetic polymorphisms landscape defining HIV-1 sub-subtypes, subtypes and recombinant forms are determinants of viral fitness and frequency in the HIV-1 infected population.Graphical AbstractHighlightsThe polymorphism at matrix-capsid cleavage site, together with non-cleavage sites polymorphisms, direct the processing rate of the substrate, not the intrinsic activity of the enzyme.The less prevalent and less infectious sub-subtype A2 harbors the matrix-capsid cleavage site polymorphism that we report as a limiting factor for gag processing.Sub-subtype A2 Gag polyprotein processing rate is independent of the PR subtype.

Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

ABSTRACTThe main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued structure-based design of peptidomimetic α-ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease:inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the α-ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against Middle East Respiratory Syndrome coronavirus.

Sensitivity of Alphaviruses to G3BP Deletion Correlates with Efficiency of Replicase Polyprotein Processing

ABSTRACT We present a comprehensive overview of the dependency of several Old World alphaviruses for the host protein G3BP. Based on their replication ability in G3BP-deleted cells, Old World alphaviruses can be categorized into two groups, being either resistant or sensitive to G3BP deletion. We observed that all sensitive viruses have an Arg residue at the P4 position of the cleavage site between the nonstructural protein P1 (nsP1) and nsP2 regions of the replicase precursor polyprotein (1/2 site), while a different residue is found at this site in viruses resistant to G3BP deletion. Swapping this residue between resistant and sensitive viruses also switches the G3BP deletion sensitivity. In the absence of G3BP, chikungunya virus (CHIKV) replication is at the limit of detection. The P4 Arg-to-His substitution partially rescues this defect. The P4 residue of the 1/2 site is known to play a regulatory role during processing at this site, and we found that if processing is blocked, the influence of the P4 residue on the sensitivity to G3BP deletion is abolished. Immunofluorescence experiments with CHIKV replicase with manipulated processing indicate that the synthesis of double-stranded RNA is defective in the absence of G3BP and suggest a role of G3BP during negative-strand RNA synthesis. This study provides a functional link between the host protein G3BP and the P4 residue of the 1/2 site for viral RNA replication of Old World alphaviruses. While this suggests a link between G3BP proteins and viral replicase polyprotein processing, we propose that G3BP proteins do not have a regulatory role during polyprotein processing. IMPORTANCE Old World alphaviruses comprise several medically relevant viruses, including chikungunya virus and Ross River virus. Recurrent outbreaks and the lack of antivirals and vaccines demand ongoing research to fight the emergence of these infectious diseases. In this context, a thorough investigation of virus-host interactions is critical. Here, we highlight the importance of the host protein G3BP for several Old World alphaviruses. Our data strongly suggest that G3BP plays a crucial role for the activity of the viral replicase and, thus, the amplification of the viral RNA genome. To our knowledge, the present work is the first to provide a functional link between the regulation of viral polyprotein processing and RNA replication and a host factor for alphaviruses. Moreover, the results of this study raise several questions about the fundamental regulatory mechanisms that dictate the activity of the viral replicase, thereby paving the way for future studies.

Dengue virus capsid anchor modulates the efficiency of polyprotein processing and assembly of viral particles

The assembly and secretion of flaviviruses are part of an elegantly regulated process. During maturation, the viral polyprotein undergoes several co- and post-translational cleavages mediated by both viral and host proteases. Among these, sequential cleavage at the N and C termini of the hydrophobic capsid anchor (Ca) is crucial in deciding the fate of viral infection. Here, using a refined dengue pseudovirus production system, along with cleavage and furin inhibition assays, immunoblotting and secondary structure prediction analysis, we show that Ca plays a key role in the processing efficiency of dengue virus type 2 (DENV2) structural proteins and viral particle assembly. Replacement of the DENV2 Ca with the homologous regions from West nile or Zika viruses or, alternatively, increasing its length, improved cleavage and hence particle assembly. Further, we showed that substitution of the Ca conserved proline residue (P110) to alanine abolishes pseudovirus production, regardless of the Ca sequence length. Besides providing the results of a biochemical analysis of DENV2 structural polyprotein processing, this study also presents a system for efficient production of dengue pseudoviruses.