Identification of Amino Acid Residues within Simian Virus 40 Capsid Proteins Vp1, Vp2, and Vp3 That Are Required for Their Interaction and for Viral Infection

Abstract

Interaction of simian virus 40 (SV40) major capsid protein Vp1 with the minor capsid proteins Vp2 and Vp3is an integral aspect of the SV40 architecture. Two Vp3 sequence elements mediate Vp1 pentamer binding in vitro, Vp3 residues 155 to 190, or D1, and Vp3 residues 222 to 234, or D2. Of the two, D1 but not D2 was necessary and sufficient to direct the interaction with Vp1 in vivo. Rational mutagenesis of Vp3 residues (Phe157, Ile158, Pro164, Gly165, Gly166, Leu177, and Leu181) or Vp1 residues (Val243 and Leu245), based on a structural model of the SV40 Vp1 pentamer complexed with Vp3 D1, was carried out to disrupt the interaction between Vp1 and Vp3 and to study the consequences of these mutations for viral viability. Altering these residues to bulky, charged residues blocked the interaction in vitro. When these alterations were introduced into the viral genome, they reduced viral viability. Mutants with alterations in Vp1 Val243, Leu245, or both to glutamate were nearly nonviable, whereas those with Vp3 alterations reduced, but did not eliminate, viability. Our results defined the residues of Vp1 and the minor capsid proteins that are essential for both the interaction of the capsid proteins and viral viability in permissive cells.Simian virus 40 (SV40), of the family Polyomaviridae, is a nonenveloped small DNA tumor virus, about 45 to 50 nm in diameter, whose capsid structure is known at atomic resolution (21, 30). Its capsid is built from pentamers of the major capsid protein Vp1 and the minor capsid proteins Vp2 and Vp3. The two minor capsid proteins are identical except that Vp2 has an amino-terminal segment of 118 residues in addition to the 234 shared carboxy-terminal residues that constitute Vp3. The unique amino terminus of Vp2 is myristoylated, a lipid modification that has been proposed to play a role in entry and uncoating during infection (16,27).Three sequence elements within the shared carboxy-terminal sequence have been identified and are here referred to as amino acids of Vp3. The first of these elements, residues 199 to 206, is the monopartite Vp3 nuclear localization signal (NLS), which functions in the nuclear import of newly synthesized Vp3 and in the nuclear entry of infecting particles (5,25). This NLS interacts with the most abundant cellular NLS receptor complex, the importin ␣2/␤1 heterodimer (25). The second element, residues 222 to 234, or D2, binds DNA (4) and is an in vitro Vp1-interactive determinant (9). Interestingly, Vp3 from the highly related murine polyomavirus (Py) lacks this Vp1-interacting determinant and DNA binding D2 and binds Vp1 via a more internal region (Py Vp3 residues 140 to 181) (1).This internal region is highly conserved among polyomaviruses. Since the crystal structure of this internal region of Py Vp3 bound to the Py Vp1 pentamer is known (3), we have built a homology model of the SV40 Vp3 fragment (Vp3 residues 157 to 184, harboring most of the 01 residues) bound to its Vp1 pentamer (15). Whether these two sequence elements, D1 and D2, function in the in vi...

Minor Capsid Proteins of Simian Virus 40 Are Dispensable for Nucleocapsid Assembly and Cell Entry but Are Required for Nuclear Entry of the Viral Genome

“…The analysis of three Vp3 mutants (F157E-I158E, P164E-G165R-G166E, and P164R-P165E-P166R) and three Vp1 mutants (V243E, L245E, and V243E-L245E) by transfection of the mutant viral genomes into host monkey kidney cells showed that all of the mutants replicate viral DNA and produce capsid proteins normally. However, the mutants are much less infectious than the wild type (41). A plausible interpretation of these data is that the capsids of these particles comprise Vp1 but little or no Vp2/3 owing to compromised Vp1-Vp3 interaction.…”

“…Most mutants are noninfectious (41), so the formation of mutant particles requires cells to be transfected with mutant DNAs, which are then isolated as described previously (27). Briefly, lysates from 5 ϫ 10 6 to 1 ϫ 10 7 cells that had been transfected with mutant DNAs were separated by sedimentation through sucrose gradients, and the presence of particles in the fractions was determined by anti-Vp1 Western blotting using 1/50 of each fraction.…”

“…Based on a structural model, we previously identified residues of SV40 Vp1 and Vp3 that, when mutated, selectively disrupt the interaction of the two proteins (20,41). The analysis of three Vp3 mutants (F157E-I158E, P164E-G165R-G166E, and P164R-P165E-P166R) and three Vp1 mutants (V243E, L245E, and V243E-L245E) by transfection of the mutant viral genomes into host monkey kidney cells showed that all of the mutants replicate viral DNA and produce capsid proteins normally.…”

The Simian Virus 40 Late Viral Protein VP4 Disrupts the Nuclear Envelope for Viral Release

“…Both domains are required for the membrane association and disruption activity found with VP4. In the context of VP2 and VP3, the HD from VP4 serves as the VP1 binding domain that supports virus assembly by binding to the hydrophobic central cavity within VP1 pentamers (2,11,41). The basic amino acid cluster, or the NLS, directs the trafficking of the late proteins to the nucleus (Fig.…”

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

“…Vp2 and Vp3 are required for the transport of the infecting viral DNA to the cell nucleus (5,7). Vp1 is necessary for the packaging of the viral minichromosome and assembly of the capsid and mediates cell attachment and entry (5,6). Thus, the formation of infectious SV40 virions depends on the proper folding of the newly synthesized Vp1 into the functional building block of the capsid, namely, the Vp1 pentamer.…”