Evidence that SV40 VP1-DNA interactions contribute to the assembly of 40-nm spherical viral particles

The simian virus 40 (SV40) particle is mainly composed of the major capsid protein termed VP1. VP1 self-assembles into virus-like particles (VLPs) of approximately 40 nm in diameter when over-expressed in bacteria or in insect cells, but purified VP1 does not form such a structure under physiological conditions, and thus, the mechanism of VP1 assembly is not well understood. Using a highly purified VP1 assembly/disassembly system in vitro, here we provide evidence that DNA is a factor that contributes to VP1 assembly into 40-nm spherical particles. At pH 5, for example, VP1 preferentially assembles into 40-nm particles in the presence of DNA, whereas VP1 assembles into tubular structures in the absence of DNA. Electron microscopic observations revealed that the concentration of DNA and its length are important for the formation of 40-nm particles. In addition, sucrose gradient sedimentation analysis and DNase I-sensitivity assays indicated that DNA of up to 2,000 bp is packaged into the 40-nm particles under the conditions examined. We propose that DNA may facilitate the formation of 40-nm spherical particles by acting as a scaffold that increases the local concentration of VP1 and/or by acting as an allosteric effector that alters the structure of VP1.     

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

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.     

Size and position of truncations in the carboxy-terminal region of major capsid protein VP1 of hamster polyomavirus expressed in yeast determine its assembly capacity

Summary. The hamster polyomavirus major capsid protein VP1 was modified in its carboxy-terminal region by consecutive truncations and single amino acid exchanges. The ability of yeast-expressed VP1 variants to form virus-like particles (VLPs) strongly depended on the size and position of the truncation. VP1 variants lacking 21, 69, and 79 amino acid (aa) residues in their carboxy-terminal region efficiently formed VLPs similar to those formed by the unmodified VP1 (diameter 40–45 nm). In contrast, VP1 derivatives with carboxy-terminal truncations of 35 to 56 aa residues failed to form VLPs. VP1 mutants with a single A336G aa exchange or internal deletions of aa 335 to aa 346 and aa 335 to aa 363 resulted in the formation of VLPs of a smaller size (diameter 20 nm). These data indicate that certain parts of the carboxy-terminal region of VP1 are not essential for pentamer–pentamer interactions in the capsid, at least in the yeast expression system used.     

The structure of infectious bovine rhinotracheitis virus

Summary The structure of infectious bovine rhinotracheitis virus was explored with the negative contrast technique. The virus was propagated in bovine testicle tissue culture cells. The essential components of the virus particles were: (1) the core, (2) the capsid, and (3) the envelope. The core measured 945 Å. The capsid consisted of 162 capsomeres and had an average diameter of 1085 Å. The capsomeres were arrayed in a highly regular pattern characteristic of 532 axial symmetry. The envelope varied in size but had an average diameter of 2000 Å.This work was supported in part by United States Public Health Service Research Grant AI -03820 from the National Institute of Allergy and Infectious Diseases.     

Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation.

Human polyomavirus is a naked capsid virus containing a closed circular double-stranded DNA genome. The mechanism of DNA encapsidation for the viral progeny formation is not fully understood. In this study, DNA encapsidation domain of the major capsid protein, VP1, of the human polyomavirus JCV was investigated. When the first 12 amino acids were deleted, the E. coli expressed VP1 (Delta N12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of Delta N12VP1 did not package exogenous DNA in vitro, which is in contrast to that of the full-length VP1 protein. These findings suggest that the N-terminal of the first 12 amino acids of VP1 were responsible for DNA encapsidation. The importance of amino acids in the DNA encapsidation domain was determined further using site-directed mutagenesis. All of the positively charged amino acids at the N-terminal region of VP1 were essential for DNA encapsidation. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation.     

Characterization of the poliovirus 147S particle: new insights into poliovirus uncoating

A Sabin 1 strain poliovirus (PV) mutant, S1(2Y-1I), carrying a Tyr at amino acid position VP2(142) and an Ile at position VP1(160), can establish persistent infections in HEp-2c cells. This mutant forms atypical 147S particles upon interaction at 0 degrees C with either cells expressing PV receptor (PVR) CD155, or PVR-IgG2a, a chimeric molecule consisting of an extracellular moiety of PVR and the hinge and Fc portion of a mouse IgG2a. Upon interaction with PVR at 37 degrees C, S1(2Y-1I), similar to the parental strain, forms both 135S A particles and 80S empty capsids. At 0 degrees C, surprisingly, at a concentration equal to or greater than 5 nM, PVR-IgG2a induced both the extrusion of VP4 from the capsid of S1(2Y-1I) and the formation of 80S particles. The same transitions were observed at 0 degrees C with the parental strain Sabin 1 at 40 nM PVR-IgG2a. Thus, the formation of 80S particles and VP4 extrusion, considered as one of the steps of PV uncoating, can be temperature-independent at high PVR concentration. This implies that structural changes of the PV capsid occurred following adsorption at low temperature.     

Genetic characterization of a novel calicivirus from a chicken

We describe the identification and genetic characterization of a novel enteric calicivirus, detected by transmission electron microscopy and RT-PCR in two clinically normal chickens and in a chicken with runting and stunting syndrome from different flocks in southern Germany. Positive findings were confirmed by sequencing. The complete nucleotide sequence and genome organization of one strain (Bavaria/04V0021) was determined. The genome of the Bavaria virus is 7,908 nt long and contains two coding open reading frames. Phylogenetic analysis of the deduced partial 2C helicase/NTPase, 3C cysteine protease, RNA-dependent RNA polymerase and complete VP1 capsid protein amino acid sequences showed that the virus is genetically related to but distinct from sapoviruses and lagoviruses. Morphologically, the Bavaria virus particles are 37-42 nm in diameter and exhibit characteristic cup-shaped surface depressions.     

Molecular Interactions between the HSV-1 Capsid Proteins as Measured by the Yeast Two-Hybrid System

HSV-1 B capsids are composed of seven major proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26. VP indicates that the capsid protein is also a component of the infectious virion. Capsid proteins 21, 22a, and VP24 are specified by a single open reading frame (UL26) that encodes 635 amino acids. An objective of the work in our laboratory is to identify and map interactions among and between capsid proteins. In the present studies we employed the yeast GAL4 two-hybrid system developed by Fields and his colleagues (Nature240, 245–246 (1989)) for this purpose. DNA corresponding to the capsid open reading frames was derived as a PCR product and fused to sequences of the GAL4 activation and DNA binding domains. Using this system each of the capsid proteins has been tested for interactions with all of the other capsid proteins. Three interactions have been identified: a relatively strong self-interaction between 22a molecules (residues 307–635 of UL26), bimolecular interactions between 22a and VP5, and another between VP19C and VP23. The interactions were detected by the expression of β-galactosidase enzyme activity, and yielded 289, 86, and 63 units of enzyme activity, respectively. For the 22a self-interaction, elimination of residues 611–635 resulted in an approximately twofold decrease in enzyme activity. The C-terminal 25 amino acids of 22a were also essential for the bimolecular interaction between 22a and VP5.     

Characterization of structural proteins of Solenopsis invicta virus 1

Purification of Solenopsis invicta virus 1 (SINV-1) from its host, S. invicta, and subsequent examination by electron microscopy revealed a homogeneous fraction of spherical particles with a diameter of 30-35 nm. Quantitative PCR with SINV-1-specific oligonucleotide primers verified that this fraction contained high copy numbers of the SINV-1 genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SINV-1 purified fraction revealed three major and one minor protein bands. The protein bands were labeled VP1 (40.8+/-1.4 kDa), VP2 (35.7+/-2.8 kDa), VP3 (25.2+/-1.8 kDa), and VP4 (22.2+/-2.5 kDa) based on mass. N-terminal sequence was acquired successfully for VP1, VP2, and VP3, but not VP4, and delineated each capsid protein within the 3'-proximal open reading frame of SINV-1. Positional organization of the viral proteins within the SINV-1 structural polyprotein was consistent with dicistroviruses (when based on sequence similarity). Blastp analysis of SINV-1 VP1, VP2, and VP3 revealed significant identity with corresponding structural capsid proteins of positive-strand RNA viruses, particularly acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Amino acid residues about the scissile bonds for VP1 and VP3 were consistent with dicistroviruses and insect-infecting picorna-like viruses. N-terminal sequencing of VP2 also established that translation initiation of the SINV-1 structural polyprotein was mediated by an internal ribosomal entry site and is AUG-independent.     

Detection and genotyping of human papillomavirus DNA by SPF10 and MY09/11 primers in cervical cells taken from women attending a colposcopy clinic

Diagnosis of erythrovirus B19 (B19) relies on serology and the detection of viral DNA. Recently, a distinct erythrovirus isolate termed V9, markedly different from erythrovirus B19 (> 11% nucleotide disparity), was isolated. Standard B19 PCR assays were inconclusive and serologic tests failed to categorize V9 as an acute B19-like infection. Sequencing, combined with PCR studies, have since demonstrated the need for specific and differentiated techniques when examining samples for possible B19 or V9 viremia. The antigenic properties of the V9 capsid proteins have not been characterized previously. To address this question, V9 VP1 and VP2 open reading frames were cloned and expressed in insect cells using a baculovirus vector. Large quantities of purified recombinant V9 capsid protein were produced and electron micrographs revealed self-assembly of V9 VP1/VP2 and VP2 capsids into empty icosahedral erythrovirus-like particles with a diameter of approximately 23 nm. Screening of a panel of 270 clinical samples for the presence of V9 IgM and IgG antibodies in ELISA showed 100% serologic cross-reactivity between B19 and V9 when comparing V9 VP2 capsids to a commercial B19 VP2 assay. This suggests that both a V9 and a B19 antibody response may be diagnosed equally well by ELISA using either V9 or B19 recombinant capsids as antigen source. Retrospectively, translation of the V9 sequence indicates that despite a significant genetic variation on the DNA level, the majority of the discrepant DNA sequence represents silent mutations leading to an amino acid sequence very similar to the known B19 strains (96-97% homology).     

Rotavirus VP6 Expressed by PVX Vectors in Nicotiana benthamiana Coats PVX Rods and Also Assembles into Viruslike Particles

The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external “overcoat” of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.     

Characterization of infectious particles of grass carp reovirus by treatment with proteases

Proteolytic cleavages play an important role in reovirus infection during entry into cells. The effects of protease digestion on the morphology, infectivity and polypeptide composition of grass carp reovirus (GCRV) were investigated. Following treatment with chymotrypsin, the different subviral particles of GCRV were isolated using density gradient centrifugation and examined by electron microscope (EM). Analysis of protein components revealed that the viral outer capsid was composed of VP5 and VP7. Of particular note, VP5 was found to primarily exist within virions as cleaved fragments, which was consistent with observations for its analogue μ1/μ1C, generated by autolysis of μ1 at the μ1N/μ1C junction for mammalian orthoreoviruses (MRVs). Meanwhile, both trypsin- and chymotrypsin-treated GCRV particles appeared to have an enhanced infectivity. Moreover, the corresponding assays between infectivity and protein component indicated that the enhancement of infectivity was correlated to the complete digestion of the outer capsid protein VP7 and partial cleavage of VP5. Overall, the results presented in this paper provided strong evidence that the proteins VP5 and VP7 of GCRV play an indispensable role in viral infection.     

Capsid Protein-Encoding Genes of Hamster Polyomavirus and Properties of the Viral Capsid

On the basis of its genome organization the hamster polyomavirus (HaPV) is closely related to the murine polyomavirus Py. But HaPV infection, in contrast to Py infection, gives rise to two different tumor types; depending on the hamster strain used for infection, HaPV induces either epitheliomas or lymphomas. Although the HaPV virions were shown to be similar to those of Py and SV40, more precise information about the structure and protein composition of the HaPV capsid was still missing. Here we describe the primary structure of the capsid protein-encoding HaPV genes and the structure and protein composition of the HaPV capsid. Virions isolated from epitheliomas in HaPV-infected hamsters were shown by electron microscopy to be spherical particles with the typical icosahedral structure of polyomaviruses. However, in contrast to the capsids of SV40 and Py, a T = 7 laevo symmetry of HaPV capsids was observed. Separation of HaPV virions in SDS polyacrylamide gels and Western blotting with VP1-specific antisera identified VP1 as the major capsid protein species corresponding in its molecular weight to the predicted value of 41.8 kDa. Because of the presence of two potential translational initiation sites in the VP1 gene, the N-terminal amino acid sequence of virion VP1 was determined and found to start at the second initiation site. The amino acid homologies of HaPV capsid proteins shared with Py varied between 65.5% (VP1), 45.4% (VP3) and 44.6% (VP2), whereas the homologies to the relevant proteins of other polyomaviruses were found to range between 49.6–57.9% for VP1 and 28.9–41% for VP2/VP3.     

Different architectures in the assembly of infectious bursal disease virus capsid proteins expressed in insect cells

Infectious bursal disease virus (IBDV) capsid is formed by the processing of a large polyprotein and subsequent assembly of VPX/VP2 and VP3. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructs were made using two baculovirus transfer vectors, pFastBac and pAcYM1. Surprisingly, the expression of the capsid proteins gave rise to different types of particles in each system, as observed by electron microscopy and immunofluorescence. FastBac expression led to the production of only rigid tubular structures, similar to those described as type I in viral infection. Western blot analysis revealed that these rigid tubules are formed exclusively by VPX. These tubules revealed a hexagonal arrangement of units that are trimer clustered, similar to those observed in IBDV virions. In contrast, pAcYM1 expression led to the assembly of virus-like particles (VLPs), flexible tubules, and intermediate assembly products formed by icosahedral caps elongated in tubes, suggesting an aberrant morphogenesis. Processing of VPX to VP2 seems to be a crucial requirement for the proper morphogenesis and assembly of IBDV particles. After immunoelectron microscopy, VPX/VP2 was detected on the surface of tubules and VLPs. We also demonstrated that VP3 is found only on the inner surfaces of VLPs and caps of the tubular structures. In summary, assembly of VLPs requires the internal scaffolding of VP3, which seems to induce the closing of the tubular architecture into VLPs and, thereafter, the subsequent processing of VPX to VP2.     

Effect of foot-and-mouth disease virus capsid precursor protein and 3C protease expression on bovine herpesvirus 1 replication

Several reports have previously shown that expression of the foot-and-mouth disease virus (FMDV) capsid precursor protein encoding region P1-2A together with the 3C protease (P1-2A/3C) results in correct processing of the capsid precursor into VP0, VP1 and VP3 and formation of FMDV capsid structures that are able to induce a protective immune response against FMDV challenge after immunization using naked DNA constructs or recombinant viruses. To elucidate whether bovine herpesvirus 1 (BHV-1) might also be suitable as a viral vector for empty capsid generation, we aimed to integrate a P1-2A/3C expression cassette into the BHV-1 genome, which, however, failed repeatedly. In contrast, BHV-1 recombinants that expressed an inactive 3C protease or the P1-2A polyprotein alone could be easily generated, although the recombinant that expressed P1-2A exhibited a defect in direct cell–cell spread and release of infectious particles. These results suggested that expression of the original, active FMDV 3C protease is not compatible with BHV-1 replication. This conclusion is supported by the isolation of recombinant BHV-1/3C*, which contained mutations within the 3C ORF (3C* ORF)—probably introduced spontaneously during generation of BHV-1/3C*—instead of the authentic 3C ORF contained in the transfer plasmids. Within the 3C* ORF, the codons for glycine 38 and phenylalanine 48 were both substituted by codons for serine. The resulting 3C* protease exhibits a highly reduced activity for proteolytic processing of the P1-2A polyprotein and thus might be a good candidate for the generation of live attenuated FMDV variants.     

Capsid assembly and DNA packaging in herpes simplex virus

The genome of HSV-1 contains 80–85 open reading frames. Genetic and biochemical evidence suggests that at least 39 of these genes encode proteins that are components of the HSV-1 virion. The architecture of the HSV-1 virion consists of a trilaminar lipid envelope, an amorphous layer known as the tegument, a capsid shell, and a DNA-containing core. The capsid is an icosahedral shell whose major morphological features are 162 capsomers. It is composed of a major capsid protein called VP5 and three less abundant proteins, VP19C, VP23 and VP26. VP5 is the structural subunit of all 162 capsomers while VP19C and VP23 are located in the space between the capsomers. In addition to the structural proteins, capsid assembly involves participation of the HSV-1-encoded protease and the scaffolding protein, preVP22a. DNA packaging involves participation of DNA, empty capsids, and at least seven additional HSV-1-encoded proteins. Considerable advances have been made in understanding the structure of the capsid shell, largely as the result of applying cryoelectron microscopy techniques. Use of recombinant baculoviruses has allowed for a detailed analysis of the proteins required for capsid assembly. More recently, an in vitro system has been developed which has aided in defining the assembly pathway by identifying intermediates in the assembly of intact capsids. The in vitro system has identified a fragile roundish procapsid which matures into the polyhedral capsid in a transition similar to that undergone by bacteriophage proheads. This review is a summary of our present knowledge with respect to the structure and assembly of the HSV-1 capsid and what is known about the seven genes involved in DNA packaging. © 1997 John Wiley & Sons Ltd.     

Hamster polyomavirus-derived virus-like particles are able to transfer in vitro encapsidated plasmid DNA to mammalian cells

The authentic major capsid protein 1 (VP1) of hamster polyomavirus (HaPyV) consists of 384 amino acid (aa) residues (42 kDa). Expression from an additional in-frame initiation codon located upstream from the authentic VP1 open reading frame (at position −4) might result in the synthesis of a 388 aa-long, amino-terminally extended VP1 (aa −4 to aa 384; VP1^ext). In a plasmid-mediated Drosophila Schneider (S2) cell expression system, both VP1 derivatives as well as a VP1^ext variant with an amino acid exchange of the authentic Met1Gly (VP1^ext-M1) were expressed to a similar high level. Although all three proteins were detected in nuclear as well as cytoplasmic fractions, formation of virus-like particles (VLPs) was observed exclusively in the nucleus as confirmed by negative staining electron microscopy. The use of a tryptophan promoter-driven Escherichia coli expression system resulted in the efficient synthesis of VP1 and VP1^ext and formation of VLPs. In addition, establishment of an in vitro disassembly/reassembly system allowed the encapsidation of plasmid DNA into VLPs. Encapsidated DNA was found to be protected against the action of DNase I. Mammalian COS-7 and CHO cells were transfected with HaPyV-VP1-VLPs carrying a plasmid encoding enhanced green fluorescent protein (eGFP). In both cell lines eGFP expression was detected indicating successful transfer of the plasmid into the cells, though at a still low level. Cesium chloride gradient centrifugation allowed the separation of VLPs with encapsidated DNA from “empty” VLPs, which might be useful for further optimization of transfection. Therefore, heterologously expressed HaPyV-VP1 may represent a promising alternative carrier for foreign DNA in gene transfer applications.     

Altered virion proteins of a temperature-sensitive mutant of polyoma virus, ts59.

The ts59 mutant of polyoma virus is blocked in a late step of infection at the restrictive temperature. Cellular and viral DNA synthesis proceed normally in ts59-infected cells at the restrictive temperature, but infectious progency virus particles are not assembled. The ts59 mutant complements early tsA mutants in mixed infection, and the temperature-sensitive mutation maps in the late region of the polyoma genome. The infectivity of ts59 virions is much heat labile than wild-type polyoma. All three nonhistone capsid proteins of ts59, VP1 (45,000 daltons) and the overlapping proteins VP2 (30,000 daltons) and VP3 (20,000 daltons), show altered mobilities when analyzed by SDS-polyacrylamide gel electrophoresis. The tryptic peptide patterns of all three ts59 virion proteins also differ from the tryptic peptide patterns of wild-type proteins. Analysis of the ts59 proteins synthesized in vitro and in infected cells suggests that the alterations in the ts59 virion proteins are caused by differences in primary structure rather than by post-translational modifications. The capsid proteins of convertant virions produced by marker rescue of the ts59 temperature-sensitive mutation, using various restriction endonuclease fragments of wild-type DNA, have been analyzed. Results of these studies suggest that (i) 26 map units is the furthest point, in a clockwise direction on the genetic map, that the information for the C-terminus of VP1 can be from the Eco·R1 cleavage site; (ii) the N-terminal end of VP2 extends beyond the N-terminal end of VP3; (iii) the temperature-sensitive phenotype of ts59 is correlated with a peptide alteration common to VP2 and VP3. The ts59 mutant contains two further peptide alterations not related to the temperature-sensitive phenotype: a C-terminal alteration in VP1 and an alteration unique to VP2. Cells infected by ts59 contain approximately fourfold lower amounts of viral capsid proteins and virus-specific messenger RNA at the restrictive temperature compared to the permissive temperature.     

Analysis of two VP60 capsid protein genes of rabbit hemorrhagic disease virus from viruses obtained from the same farm

Two distinct clones of the VP60 capsid protein gene of rabbit hemorrhagic disease virus were amplified from mixed liver tissue of rabbits collected from the same farm in the Xinjiang Uygur Autonomous Region of China in 2002. The results of DNA sequence analysis showed that the length of the VP60 gene in the first clone was 1,740 bp, similar to other VP60 genes. The length of the VP60 gene in the second clone was only 1,536 bp. The two clones were predicted to encode 579 and 511 amino acids, respectively.     

Scaffold-free vascular tissue engineering using bioprinting

Current limitations of exogenous scaffolds or extracellular matrix based materials have underlined the need for alternative tissue-engineering solutions. Scaffolds may elicit adverse host responses and interfere with direct cell–cell interaction, as well as assembly and alignment of cell-produced ECM. Thus, fabrication techniques for production of scaffold-free engineered tissue constructs have recently emerged. Here we report on a fully biological self-assembly approach, which we implement through a rapid prototyping bioprinting method for scaffold-free small diameter vascular reconstruction. Various vascular cell types, including smooth muscle cells and fibroblasts, were aggregated into discrete units, either multicellular spheroids or cylinders of controllable diameter (300–500 μm). These were printed layer-by-layer concomitantly with agarose rods, used here as a molding template. The post-printing fusion of the discrete units resulted in single- and double-layered small diameter vascular tubes (OD ranging from 0.9 to 2.5 mm). A unique aspect of the method is the ability to engineer vessels of distinct shapes and hierarchical trees that combine tubes of distinct diameters. The technique is quick and easily scalable.