Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles

Abstract

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

Triatoma virus structural polyprotein expression, processing and assembly into virus-like particles

“…6d) and an icosahedral morphology indistinguishable from that of previously described native TrV empty capsids (31.1±1.8 nm of diameter; Fig. 6e) (Agirre et al, 2011). Average yields of particles purified from cells infected with BV_P1 or coinfected with BV_P1 and BV_NS were of y65 mg/10 27 cells.…”

“…6b). The 35.5 kDa product might correspond to PP3, a non-canonical TrV polypeptide previously detected in native TrV empty particles (Agirre et al, 2011).…”

“…3c). It is noteworthy that the anti-VP1 serum recognized two protein products in extracts from cells coinfected with BV_NS and BV_P1; a major species corresponding to a polypeptide of y29 kDa, similar to the mature VP1 polypeptide found in natural TrV capsids (Agirre et al, 2011;Czibener et al, 2000), and a minor one, comigrating with the intact P1 product detected in cells infected with BV_P1 alone, thus likely corresponding to the unprocessed form of the P1 protein ( Fig. 3c).…”

“…To overcome this experimental limitation we have generated two recombinant baculoviruses (rBVs) independently expressing the TrV ORFs. Data presented here show that the proteolytic processing of the P1 polyprotein is strictly dependent upon coexpression of the NS polyprotein, and that coexpression of both TrV cistrons leads to the assembly of virus-like particles (VLPs) akin to natural TrV empty capsids (Agirre et al, 2011). We also demonstrate that the unprocessed form of the P1 polypeptide assembles into quasi-spherical structures larger than VLPs produced in NS/P1-coexpressing cells.…”

“…VP0 (37.3 kDa), VP1 (29.7 kDa) and VP2 (28.4 kDa). VP0 is further processed releasing the VP3 (31.8 kDa) and VP4 (5.5 kDa) polypeptides (Agirre et al, 2011;Czibener et al, 2000).…”