The New World Alphavirus Protein Interactomes

Alphavirus Structure and Life Cycle

The alphavirus genome is divided into two reading frames. The first reading frame encodes the nonstructural polyprotein (nsP), which is further processed into four nonstructural proteins, nsP1-nsP4. The nonstructural proteins form a replication complex that first replicates the viral RNA in a negative-sense, and then transitions it back to the positive-sense RNA, which is incorporated into virion. The second reading frame is controlled by the 26S promoter on the negative-stranded RNA and encodes structural polyproteins. The structural polyprotein consists of the capsid protein and the envelope glycoproteins E1 and E2. nsP3, capsid and E2 proteins have attracted much attention in recent years.

Figure 1. Alphavirus genome and protein coding
(Source: Carey BD, et al. 2019)

Different species of alphaviruses bind to host protein receptors via fusion or viral E2 protein-mediated receptor binding and enter the host cell plasma membrane. Subsequent internalization is mediated by clathrin-mediated endocytosis. In this endocytosis, intracellular vesicles become more acidic during maturation, and the low pH environment leads to dissociation of the E1/E2 dimer, resulting in the insertion of E1 into the endosomal membrane and subsequent fusion of the viral and cellular membranes, releasing the viral nucleocapsid into the cytoplasm of the host. Genomic RNA (gRNA) is immediately translated into two nonstructural precursors, P123 or P1234. p1234 is produced by read-through of the opal termination codon (UGA) between nsP3 and nsP4, followed by cis or trans cleavage of P123 and nsP4, and cis cleavage of nsP1 and P23 only. P123-nsP4 and nsP1-P23-nsP4 utilize host proteins to synthesize negative-stranded viral RNA intermediates through the formation of early-replication complexes. All four mature nonstructural proteins are finally produced between P23, and these late replication complexes are responsible for the synthesis of positive-sense genomic (49S) and subgenomic (26S) RNAs.

The second open reading frame occupies about 1/3 of the alphavirus genome and is controlled by a subgenomic promoter on the negative strand of the viral genome. This ORF encodes the capsid proteins, E3, E2, 6K, and E1 proteins, which are primarily structural proteins that are translated into polyprotein precursors, where the capsid proteins cleave themselves by protein hydrolysis and subsequently encapsidating the gRNAs into the nucleocapsid. This process exposes an ER localization signal on pE2 (the precursor of E3 and E2) that directs the remaining multimeric proteins to the ER, where the proteins enter the secretory pathway for export to the plasma membrane. Later in this pathway, furin-mediated splitting of pE2 forms E2 and E3 proteins, and trimers of E1 and E2 proteins are transported to the plasma membrane.

Figure 2. Alphavirus Life Cycle
(Source: Kim AS, et al. 2023)

Capsid

The capsid proteins are divided into two separate structural domains, the N- terminal and the C-terminal. The C-terminal is highly conserved in all alphavirus capsid proteins, but the N-terminal domain is highly variable. The capsid proteins have multiple functions in the viral life cycle. The C-terminal domain of the capsid has protease activity and is responsible for releasing the capsid from the translated polypeptide and exposing the ER-localized sequences on the remaining polypeptide so that the glycoprotein can be further processed in the ER. The N-terminal domain is highly positively charged and is responsible for binding viral RNA during assembly. Capsid proteins also play an important role in cellular pathogenicity. The capsid proteins of VEEV and EEEV can lead to host transcriptional turn-off, resulting in cytopathic effects. Further studies have shown that host transcriptional turn-off in VEEV-infected cells is due to the presence of nuclear localization signals (NLS) and nuclear export sequences (NES).

The role of VEEV capsid proteins in host transcriptional turn-off is an important mechanism for evading the innate immune response. In the absence of RanGTP, NES in the N-terminal domain of the capsid interacts with chromosome maintenance 1 (CRM1) (also known as exportin 1). The presence of NLS allows the capsid to bind to the host proteins importin α (Impα) and importin β (Impβ), thereby facilitating the entry of the capsid into the nucleus.

Table 2. Known host protein interactions and inhibitors for capsid

(Source: Carey BD, et al. 2019)