The Influence of Envelope C-Terminus Amino Acid Composition on the Ratio of Cell-Free to Cell-Cell Transmission for Bovine Foamy Virus

Abstract

Foamy viruses (FVs) have extensive cell tropism in vitro, special replication features, and no clinical pathogenicity in naturally or experimentally infected animals, which distinguish them from orthoretroviruses. Among FVs, bovine foamy virus (BFV) has undetectable or extremely low levels of cell-free transmission in the supernatants of infected cells and mainly spreads by cell-to-cell transmission, which deters its use as a gene transfer vector. Here, using an in vitro virus evolution system, we successfully isolated high-titer cell-free BFV strains from the original cell-to-cell transmissible BFV3026 strain and further constructed an infectious cell-free BFV clone called pBS-BFV-Z1. Following sequence alignment with a cell-associated clone pBS-BFV-B, we identified a number of changes in the genome of pBS-BFV-Z1. Extensive mutagenesis analysis revealed that the C-terminus of envelope protein, especially the K898 residue, controls BFV cell-free transmission by enhancing cell-free virus entry but not the virus release capacity. Taken together, our data show the genetic determinants that regulate cell-to-cell and cell-free transmission of BFV.

Bovine Foamy Virus: Shared and Unique Molecular Features In Vitro and In Vivo

“…The Gag-Env interaction is very important for the budding and release of FV virions. Yet, the interaction of Gag and Env in BFV-B and BFV-Z1 was almost the same, which suggests that the contribution of Gag-Z1 to enhanced cell-free transmission is not through promoting interaction with Env [25].…”

“…BFV as a well-established infection model in life-stock animals (cattle and sheep) [13,14] BFV as the only known FV in the general human food chain (beef and dairy products) [15,16] BFV Riems as the only FV passaged exclusively on primary and homologous host cells [17,18] Integrase domain: disrupted HH-CC zinc finger and unique sequence insertion into the extreme C-terminus [19] Detailed understanding of gene expression and transactivation of a non-simian FV [20,21] RNA Pol III miRNAs, unique precursor structure and their functions [14,22,23] Extremely tight cell association and identification of residues critical for this phenotype [24][25][26] Detailed understanding of new restriction factors against FVs [27] Broad tissue tropism and gene expression in BFV-infected calves [5,28] Although a so-called prototype (and/or primate) FV exists in the literature, conserved, prototypic features, besides those basic characteristics that led to the establishment of an independent subfamily of FVs, are currently only partially known. Here, an unbiased comparison of distant FVs and their replication strategies might be worth trying to discriminate basic from deduced, secondary features.…”

“…Unlike Orthoretroviruses, but similar to the other FVs, BFV Gag is not myristoylated and it cannot produce cell-free Gag-only virus-like particles [24,25]. Similar to hepatitis B virus (HBV), BFV particle budding and release are instead dependent on the co-expression of the cognate viral envelope (Env) protein [24,25], which suggests that Env provides a critical membrane-targeting function inherently lacking in BFV Gag. In the case of BFV, this occurs at the plasma membrane rather than the endoplasmic reticulum (ER), due to a lack of a functional ER retrieval signal (ERRS) [68].…”

“…This 14-residue deletion is located in the central and non-conserved region of FV Gag, which strongly contributes to the size differences of simian versus non-simian FVs [69]. This deletion led to a smaller Gag-Z1 that enhanced cell-free infectivity by four-to five-fold [25]. At the same site in Gag in some high titer (HT) cell-free BFV-Riems variants, insertions, and duplications occurred.…”

“…This lack, or only low level, of cell-free transmission appears to be independent of whether the BFV isolates have been exclusively propagated in primary bovine cells (the BFV Riems isolate) or whether immortalized bovine (MDBK cells) and hamster and canine cell lines, like BHK-21 and Cf2Th, have been used for virus propagation. BFV is an excellent model for studying virus adaption to cell-free transmission and identifying the principles of viral transmission by in vitro selection and evolution analyses, since the BFV particle budding machinery is similar to that of other FVs [24,25].…”