Enterovirus A Shows Unique Patterns of Codon Usage Bias in Conventional Versus Unconventional Clade.

Enterovirus A (EV-A) species cause hand, foot and mouth disease (HFMD), threatening the health of young children. Understanding the mutual codon usage pattern of the virus and its host(s) has fundamental and applied values. Here, through examining multiple codon usage parameters, we found that the codon usage bias among EV-A strains varies and is clade-specific. EVA76, EVA89, EVA90, EVA91 and EVA92, the unconventional clade of EV-A strains, show unique codon usage pattern relative to the two conventional clades, including EVA71, CVA16, CVA6 and CVA10, etc. Analyses of Effective Number of Codon (ENC), Correspondence Analysis (COA) and Parity Rule 2 (PR2), etc., revealed that the codon usage patterns of EV-A strains are shaped by mutation pressure and natural selection. Based on the neutrality analysis, we determined the dominant role of natural selection in the formation of the codon usage bias of EV-A. In addition, we have determined the codon usage compatibility of potential hosts for EV-A strains using codon adaptation index (CAI), relative codon deoptimization index (RCDI) and similarity index (SiD) analyses, and found that EV-A showed host-specific codon adaptation patterns in different clades. Finally, we confirmed that the unique codon usage pattern of the unconventional clade affected protein expression level in human cell lines. In conclusion, we identified novel characteristics of codon usage bias in distinct EV-A clades associated with their host range, transmission and pathogenicity.

Rules governing genetic exchanges among viral types from different Enterovirus A clusters.

The species Enterovirus A (EV-A) consists of two conventional clusters and one unconventional cluster. At present, sequence analysis shows no evidence of recombination between conventional and unconventional EV-A types. However, the factors underlying this genetic barrier are unclear. Here, we systematically dissected the genome components linked to these peculiar phenomena, using the viral reverse genetic tools. We reported that viral capsids of the unconventional EV-A types expressed poorly in human cells. The trans-encapsidation outputs across conventional and unconventional EV-A types were also with low efficiency. However, replicons of conventional types bearing exchanged 5'-untranslated region (UTR) or non-structural regions from the unconventional types were replication-competent. Furthermore, we created a viable recombinant EVA71 (conventional type) with its P3 region replaced by that from EVA89 (unconventional type). Thus, our data for the first time reveal the potential for fertile genetic exchanges between conventional and unconventional EV-A types. It also discloses that the mysterious recombination barriers may lie in uncoordinated capsid expression and particle assembly by different EV-A clusters.

Involvement of VCP/UFD1/Nucleolin in the viral entry of Enterovirus A species.

Valosin-containing protein (VCP) plays roles in various cellular activities. Recently, Enterovirus A71 (EVA71) infection was found to hijack the VCP protein. However, the mechanism by which VCP participates in the EVA71 life cycle remains unclear. Using chemical inhibitor, RNA interference and dominant negative mutant, we confirmed that the VCP and its ATPase activity were critical for EVA71 infection. To identify the factors downstream of VCP in enterovirus infection, 31 known VCP-cofactors were screened in the siRNA knockdown experiments. The results showed that UFD1 (ubiquitin recognition factor in ER associated degradation 1), but not NPL4 (NPL4 homolog, ubiquitin recognition factor), played critical roles in infections by EVA71. UFD1 knockdown suppressed the activity of EVA71 pseudovirus (causing single round infection) while it did not affect the viral replication in replicon RNA transfection assays. In addition, knockdown of VCP and UFD1 reduced viral infections by multiple human Enterovirus A serotypes. Mechanistically, we found that knockdown of UFD1 significantly decreased the binding and the subsequent entry of EVA71 to host cells through modulating the levels of nucleolin protein, a coreceptor of EVA71. Together, these data reveal novel roles of VCP and its cofactor UFD1 in the virus entry by EVA71.

The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10.

Coxsackievirus A10 (CVA10) is one of the major etiological agents of hand, foot, and mouth disease. There are no vaccine and antiviral drugs for controlling CVA10 infection. Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals. Here, two infectious clones for the prototype and a Myc-tagged CVA10 were constructed. Viable CVA10 viruses were harvested by transfecting the viral mRNA into human rhabdomyosarcoma (RD) cells. Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally. We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter, respectively. Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293T (HEK293T) cells led to the production of single round infectious particles (SRIPs). Based on CVA10 replicon RNA, SRIPs with either the enterovirus A71 (EVA71) capsid or the CVA10 capsid were generated. Infection by EVA71 SRIPs required SCARB2, while CVA10 SRIPs did not. Finally, we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme (HHRib) before the 5'-untranslated region (UTR). In summary, reverse genetic tools for prototype strain of CVA10, including both the infectious clone and the SRIPs system, were successfully established. These tools will facilitate the basic and translational study of CVA10.