Biological Characterization of Conserved Residues within the Cytoplasmic Tail of the Pichinde Arenaviral Glycoprotein Subunit 2 (GP2).

Several mammarenaviruses can cause deadly hemorrhagic fever infections in humans, with limited preventative and therapeutic measures available. Arenavirus cell entry is mediated by the viral glycoprotein (GP) complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The GP2 cytoplasmic tail (CT) is relatively conserved among arenaviruses and is known to interact with the SSP to regulate GP processing and membrane fusion, but its biological role in the context of an infectious virus has not been fully characterized. Using a Pichinde virus (PICV) GP expression vector and a PICV reverse genetics system, we systematically characterized the functional roles of 12 conserved residues within the GP2 CT in GP processing, trafficking, assembly, and fusion, as well as in viral replication. Except for P478A and K505A R508A, alanine substitutions at conserved residues abolished GP processing and membrane fusion in plasmid-transfected cells. Six invariant H and C residues and W503 are essential for viral replication, as evidenced by the fact that their mutant viruses could not be rescued. Both P480A and R482A mutant viruses were rescued, grew similarly to wild-type (WT) virus, and produced evidently processed GP1 and GP2 subunits in virus-infected cells, despite the fact that the same mutations abolished GP processing and membrane fusion in a plasmid-based protein expression system, illustrating the importance of using an infectious-virus system for analyzing viral glycoprotein function. In summary, our results demonstrate an essential biological role of the GP2 CT in arenavirus replication and suggest it as a potential novel target for developing antivirals and/or attenuated viral vaccine candidates.IMPORTANCE Several arenaviruses, such as Lassa virus (LASV), can cause severe and lethal hemorrhagic fever diseases with high mortality and morbidity, for which no FDA-approved vaccines or therapeutics are available. Viral entry is mediated by the arenavirus GP complex, which consists of the stable signal peptide (SSP), the receptor-binding subunit GP1, and the transmembrane subunit GP2. The cytoplasmic tail (CT) of GP2 is highly conserved among arenaviruses, but its functional role in viral replication is not completely understood. Using a reverse genetics system of a prototypic arenavirus, Pichinde virus (PICV), we show that the GP2 CT contains certain conserved residues that are essential for virus replication, implicating it as a potentially good target for developing antivirals and live-attenuated viral vaccines against deadly arenavirus pathogens.

Assays to Assess Arenaviral Glycoprotein Function.

Arenaviruses, such as Lassa virus (LASV) and Pichindé virus (PICV), are enveloped viruses with a bi-segmented ambisense RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein precursor complex (GPC). GPC is processed by signal peptidase in the endoplasmic reticulum into the stable signal peptide (SSP) and GP1/GP2, which is further cleaved by the Golgi-resident subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) into the cellular receptor-recognition subunit GP1 and the transmembrane subunit GP2, which helps promote the membrane fusion reaction to allow virus entry into the cell. This article describes assays to assess PICV GPC expression, proteolytic processing, fusion function, and GPC-mediated virus-like particle (VLP) entry into cells under tissue-culture conditions.

Roles of Arenavirus Z Protein in Mediating Virion Budding, Viral Transcription-Inhibition and Interferon-Beta Suppression.

The smallest arenaviral protein is the zinc-finger protein (Z) that belongs to the RING finger protein family. Z serves as a main component required for virus budding from the membrane of the infected cells through self-oligomerization, a process that can be aided by the viral nucleoprotein (NP) to form the viral matrix of progeny virus particles. Z has also been shown to be essential for mediating viral transcriptional repression activity by locking the L polymerase onto the viral promoter in a catalytically inactive state, thus limiting viral replication. The Z protein has also recently been shown to inhibit the type I interferon-induction pathway by directly binding to the intracellular pathogen-sensor proteins RIG-I and MDA5, and thus inhibiting their normal functions. This chapter describes several assays used to examine the important roles of the arenaviral Z protein in mediating virus budding (i.e., either Z self-budding or NP-Z budding activities), viral transcriptional inhibition in a viral minigenome (MG) assay, and type I IFN suppression in an IFN-ß promoter-mediated luciferase reporter assay.

Narrowed TCR repertoire and viral escape as a consequence of heterologous immunity.

Why some virus-specific CD8 TCR repertoires are diverse and others restricted or "oligoclonal" has been unknown. We show here that oligoclonality and extreme clonal dominance can be a consequence of T cell cross-reactivity. Lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV) encode NP(205-212) epitopes that induce different but highly cross-reactive diverse TCR repertoires. Homologous viral challenge of immune mice only slightly skewed the repertoire and enriched for predictable TCR motifs. However, heterologous viral challenge resulted in a narrow oligoclonal repertoire with dominant clones with unpredictable TCR sequences. This shift in clonal dominance varied with the private, i.e., unique, specificity of the host's TCR repertoire and was simulated using affinity-based computer models. The skewing differences in TCR repertoire following homologous versus heterologous challenge were observed within the same private immune system in mice adoptively reconstituted with memory CD8 T cell pools from the same donor. Conditions driving oligoclonality resulted in an LCMV epitope escape variant in vivo resembling the natural Lassa virus sequence. Thus, T cell oligoclonality, including extremes in clonal dominance, may be a consequence of heterologous immunity and lead to viral escape. This has implications for the design of peptide-based vaccines, which might unintentionally prime for skewed TCR responses to cross-reactive epitopes.

5' termini of Pichinde arenavirus S RNAs and mRNAs contain nontemplated nucleotides.

Primer extension of Pichinde arenavirus purified virion RNA suggests that genomes have at least a single nontemplated base at the 5' end which is a G in all cDNA clones having one such single base. On the other hand, the predominant products of primer extension on total virus-infected-cell RNA are at positions -1 and -2. The primer extension product at position -2 is not represented in virion RNA, and neither of these products is proportionally represented in mRNA. mRNA is predominantly 3 or 4 bases longer than genomes and antigenomes, but primer extension products as long as 7 bases were observed. The sequence of nontemplated bases reported here is unambiguous with respect to the 5'-terminal base and supports the view that there is a sequence preference for a G at the 5' termini of mRNAs. Assessment of our sequence data in the context of the sequences of Tacaribe and lymphocytic choriomeningitis viruses suggests that the mechanism of initiation of arenavirus transcription is fundamentally different from that of members of the families Orthomyxoviridae and Bunyaviridae.