Detection of all known filovirus species by reverse transcription-polymerase chain reaction using a primer set specific for the viral nucleoprotein gene

The filoviruses, Marburg virus (MARV) and Ebola virus (EBOV), are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates. Sporadic outbreaks of filovirus infection have occurred in Central Africa and parts of Asia. Identification of the natural reservoir animals that are unknown yet and epidemiological investigations are current challenges to forestall outbreaks of filovirus diseases. The filovirus species identified currently include one in the MARV group and five in the EBOV group, with large genetic variations found among the species. Therefore, it has been difficult to develop a single sensitive assay to detect all filovirus species, which would advance laboratory diagnosis greatly in endemic areas. In this study, a highly sensitive universal RT-PCR assay targeting the nucleoprotein (NP) gene of filoviruses was developed. The genomic RNAs of all known MARV and EBOV species were detected by using an NP-specific primer set. In addition, this RT-PCR procedure was verified further for its application to detect viral RNAs in tissue samples of animals infected experimentally and blood specimens of infected patients. This assay will be a useful method for diagnostics and epidemiological studies of filovirus infections.     

Uncatalyzed assembly of spherical particles from SV40 VP1 pentamers and linear dsDNA incorporates both low and high cooperativity elements

The capsid of SV40 virion is comprised of 72 pentamers of the major capsid protein, VP1. We examined the synergism between pentamer-pentamer interaction and pentamer-DNA interaction using a minimal system of purified VP1 and a linear dsDNA 600-mer, comparing electrophoresis with electron microscopy and size exclusion chromatography. At low VP1/DNA ratios, large tubes were observed that apparently did not survive native agarose gel electrophoresis. As the VP1 concentration increased, electrophoretic migration was slower and tubes were replaced by 200 Å diameter particles and excess free pentamer. At high VP1/DNA ratios, a progressively larger fraction of particles was similar to 450 Å diameter virions. VP1 association with DNA is very strong compared to the concentrations in these experiments yet, paradoxically, stable complexes appear only at high ratios of VP1 to DNA. These data suggest a DNA saturation-dependent nucleation event based on non-specific pentamer-DNA interaction that controls assembly and the ultimate capsid geometry.     

Identification of a neutralization epitope in the VP1 capsid protein of SV40

Three SV40 escape mutants were identified by selection in the presence of monoclonal antibodies with neutralizing activity. The VP1 amino acid alterations in these mutants were: (1) K73 → E (in loop BC); (2) D77 → E (in loop BC); (3) K171 → R (in loop EF); and (4) Q175 → H (in loop EF). These residues are clustered in close proximity to each other on the surface of the native capsid protein, strongly suggesting that they form a conformational epitope directly recognized by the neutralizing antibody. To our knowledge, the present study represents the first experimental mapping of a neutralization epitope of a polyomavirus family member. Structural information regarding the neutralization epitope should be useful for clarifying the extent of cross-reactivity exhibited by the humoral immune response towards related primate polyomaviruses (e.g., SV40, BKV, and JCV).     

Residues F593 and E596 of HSV-1 tegument protein pUL36 (VP1/2) mediate binding of tegument protein pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument proteins pUL36 (VP1/2) and pUL37 are essential for secondary envelopment during the egress of viral particles. Our laboratory has previously shown that HSV-1 pUL36(512-767) fragment interacts with full-length pUL37. A number of single and double amino acid changes of conserved residues in the pUL36(512-767) fragment were generated using alanine-scanning site-directed mutagenesis. The interaction of pUL36(512-767) and pUL37 was then assessed using a combination of yeast two-hybrid and coimmunoprecipitation assays. Single changes to alanine of pUL36 residues F593 and E596 impaired binding of pUL37 with the greatest effect observed for the substitution E596A. Double mutations involving either of these residues in combination with the substitution E580A essentially blocked binding of pUL37. This information will provide the basis for generation of viral mutants to further define the importance of the pUL36/pUL37 interaction in assembly of HSV-1.     

Characterization of Marburg virus glycoprotein in viral entry

One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289-501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP.     

Role of arginine-56 within the structural protein VP3 of foot-and-mouth disease virus (FMDV) O1 Campos in virus virulence

FMDV O1 subtype undergoes antigenic variation under diverse growth conditions. Of particular interest is the amino acid variation observed at position 56 within the structural protein VP3. Selective pressures influence whether histidine (H) or arginine (R) is present at this position, ultimately influencing in vitro plaque morphology and in vivo pathogenesis in cattle. Using reverse genetics techniques, we have constructed FMDV type O1 Campos variants differing only at VP3 position 56, possessing either an H or R (O1Ca-VP3-56H and O1Ca-VP3-56R, respectively), and characterized their in vitro phenotype and virulence in the natural host. Both viruses showed similar growth kinetics in vitro. Conversely, they had distinct temperature-sensitivity (ts) and displayed significantly different pathogenic profiles in cattle and swine. O1Ca-VP3-56H was thermo stable and induced typical clinical signs of FMD, whereas O1Ca-VP3-56R presented a ts phenotype and was nonpathogenic unless VP3 position 56 reverted in vivo to either H or cysteine (C).     

Low-abundance envelope protein VP12 of white spot syndrome virus interacts with envelope protein VP150 and capsid protein VP51

VP12 and VP150 are two minor envelope proteins of white spot syndrome virus (WSSV). In our previous studies, VP12 was found to co-migrate with 53-kDa form of VP150 on two-dimensional Blue Native/SDS–PAGE, suggesting that there is an interaction between them. In this study, we confirmed the interaction by co-immunoprecipitation assay and demonstrated that the binding region with VP12 is located between residues 207 and 803 of VP150. Further studies found that VP12 can be attached to WSSV capsids by interacting with capsid protein VP51. These findings suggest that VP12 may function as a linker protein participating in the linkage between VP12/VP150 complex and viral nucleocapsid.     

Virion incorporation of the herpes simplex virus type 1 tegument protein VP22 is facilitated by trans-Golgi network localization and is independent of interaction with glycoprotein E

HSV-1 virions contain a proteinaceous layer termed the tegument that lies between the nucleocapsid and viral envelope. The molecular mechanisms that facilitate incorporation of tegument proteins are poorly characterized. The tegument protein VP22 interacts with VP16 and the cytoplasmic tail of glycoprotein E (gE). Virion incorporation of VP22 occurs independently of interaction with VP16; however, the contribution of gE binding remains undefined. Site-directed mutagenesis was used to identify VP22 mutants which abrogate interaction with gE but retain VP16 binding. Virion incorporation assays demonstrated that failure to bind gE did not abrogate VP22 packaging. A region of VP22 which binds to both VP16 and gE failed to be packaged efficiently, with wild-type levels of incorporation only attained when residues 43-86 of VP22 were present. Mutational analysis of an acidic cluster of amino acids within this region indicates that this motif facilitates trans-Golgi network (TGN) localization and optimal virion incorporation of VP22.     

Identification of a mutation in the SV40 capsid protein VP1 that influences plaque morphology, vacuolization, and receptor usage

A plaque variant of SV40 that was first isolated in the 1960s, designated SV40-LP(KT), was molecularly cloned and subjected to sequence analysis. The genome of SV40-LP(KT) was found to be nearly identical to the previously described isolate known as 777. However, SV40-LP(KT) contained a mutation in the VP1 coding region resulting in a change of histidine 136 to tyrosine. This VP1 mutation was identified as a genetic determinant influencing a number of phenotypes associated with SV40-LP(KT) such as plaque morphology, intracellular vacuole formation, and ganglioside receptor usage.     

Herpes simplex virus type 1 tegument proteins VP1/2 and UL37 are associated with intranuclear capsids

The assembly of the tegument of herpes simplex virus type 1 (HSV-1) is a complex process that involves a number of events at various sites within virus-infected cells. Our studies focused on determining whether tegument proteins, VP1/2 and UL37, are added to capsids located within the nucleus. Capsids were isolated from the nuclear fraction of HSV-1-infected cells and purified by rate-zonal centrifugation to separate B capsids (containing the scaffold proteins and no viral DNA) and C capsids (containing DNA and no scaffold proteins). Western blot analyses of these capsids indicated that VP1/2 associated primarily with C capsids and UL37 associated with B and C capsids. The results demonstrate that at least two of the tegument proteins of HSV-1 are associated with capsids isolated from the nuclear fraction, and these capsid-tegument protein interactions may represent initial events of the tegumentation process.     

The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4

Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting β-hairpin observed in other polyomaviruses. We postulate that the terminal β-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.     

Changes in VP3 and VP5 genes during the attenuation of the very virulent infectious bursal disease virus strain Gx isolated in China

A very virulent infectious bursal disease virus (vvIBDV) Gx strain with high pathogenicity was attenuated through replication in specific-pathogen free (SPF) chicken embryos and in chicken embryo fibroblast (CEF) cell cultures. The changes in nucleotide sequences and the deduced amino acid sequences of VP3 and VP5 genes during attenuation were obtained. Sequence analysis of selected passages from numbers 0 to 20 in CEF’s (designated here Gx to CEF-20) showed that there were no amino acid changes detected in the VP3 and VP5 genes before CEF-9. There were some changes in the nucleotide sequence and amino acid substitutions in the VP3 and VP5 genes at CEF-9. CEF-9 was an intermediate with some amino acid changes which were possibly related to virulence. The amino acid sequences of VP2 and VP5 genes remained unchanged from CEF-10 to CEF-20. The results of pathogenicity test showed that the mortalities of vvIBDV-Gx, CEF-5, CEF-8, and CEF-9 were 64, 60, 60, and 28%, respectively, while there were no mortalities observed for CEF-10, CEF-15 and CEF-20. There was also no bursal atrophy when chickens were inoculated with CEF-10, CEF-15, and CEF-20. Virus neutralization tests with the Gx strain and sera from inoculated chickens showed that the antigenicity was similar from Gx to CEF-20. The implications of these findings for the study of IBDV virulence and a more effective control of vvIBDV are discussed.     

In vitro binding of simian immunodeficiency virus matrix protein to the cytoplasmic domain of the envelope glycoprotein

Incorporation of the envelope (Env) glycoprotein into budding virions is a key step in the replication cycle of lentiviruses. Previously, we provided genetic and biochemical evidence indicating that Env packaging into simian immunodeficiency virus (SIV) particles is mediated by the association of the Env cytoplasmic domain (CD) with the matrix (MA) domain of Gag. In this study, we developed an in vitro binding assay that, based on recombinant proteins expressed in bacteria, allowed us to demonstrate the physical interaction between the SIV Env CD and the MA in the absence of other viral or cellular proteins. We show that this association is blocked by mutations in each of the interacting domains that have been reported to interfere in vivo with the incorporation of Env into SIV virions. Moreover, we determined that the binding of SIV MA to the Env CD is saturable with a dissociation constant of 7x10(-7) M. Interestingly, the SIV MA is capable of specifically interacting in vitro with the human immunodeficiency virus type 1 Env CD, but not with that of the distantly related feline immunodeficiency virus. Our results strongly support the notion that the association between the SIV MA and Env CD plays a central role in the process of SIV Env incorporation into Gag-made particles.     

Interaction of white spot syndrome virus VP26 protein with actin

VP26 protein, the product of the WSV311 gene of white spot syndrome virus (WSSV), is one of major structural proteins of virus. In this study, when purified virions were treated with Triton X-100 detergent, VP26 protein was present in both the envelope and the nucleocapsid fraction. We have rationalized this finding by suggesting that VP26 protein might be located in the space between the envelope and the nucleocapsid. By using a fluorescent probe method, we have investigated the interaction between VP26 protein and some proteins of host cells. Three major VP26-binding proteins were purified from crayfish hemocytes by affinity-chromatography, in which the protein with an apparent molecular mass of 42 kDa was identified as actin by mass spectrometry (MS). Moreover, the association of VP26 protein with actin microfilaments was confirmed by coimmunoprecipitation.     

Sequence and structure relatedness of matrix protein of human respiratory syncytial virus with matrix proteins of other negative-sense RNA viruses

Matrix proteins of viruses within the order Mononegavirales have similar functions and play important roles in virus assembly. Protein sequence alignment, phylogenetic tree derivation, hydropathy profiles and secondary structure prediction were performed on selected matrix protein sequences, using human respiratory syncytial virus matrix protein as the reference. No general conservation of primary, secondary or tertiary structure was found, except for a broad similarity in the hydropathy pattern correlating with the fact that all the proteins studied are membrane-associated. Interestingly, the matrix proteins of Ebola virus and human respiratory syncytial virus shared secondary structure homology.     

Ebola and Marburg viruses: II. Thier development within Vero cells and the extra-cellular formation of branched and torus forms.

The development of Marburg virus and the Sudanese and Zaire strains of Ebola virus in Vero cells as visualized by electron microscopy is described. Despite differences in timing, all three strains appear to pass through identical stages of development. Initially there is a large increase in nucleolus material, and viral precursor material arranges itself in spirals and then into tubes. The cells fill with core material, which passes to the plasmalemma, which often proliferates. Each virion passes through the plasmalemma, acquiring a coat of host material. The formation of torus forms is discussed; the branched appearance that is often seen is believed to be an aberrant form. The reasons for this view are put forward.     

Mapping the site of guanylylation on VP1, the protein primer for infectious pancreatic necrosis virus RNA synthesis

VP1, the putative virion-associated RNA-dependent RNA polymerase (RdRp) of infectious pancreatic necrosis virus (IPNV) can be guanylylated in vitro whereupon it becomes a primer for in vitro RNA synthesis [Virology 208 (1995) 19]. The role of a template or other virion polypeptides in the reaction is unknown. To shed light on this question, his-tagged recombinant VP(1) (rVP1) was expressed both in Escherichia coli and insect cells and used in the guanylylation reaction. Unlike other viral VPg polypeptides, the purified rVP1 alone could guanylylate itself in vitro in a template-independent manner. Chemical and enzymatic cleavage in combination with site-directed mutagenesis mapped the site of guanylylation to serine 163. The purified rVP1 functioned as a primer as well as an RdRp in vitro, producing labeled dsRNA in the presence of [alpha(32)P] NTP and synthetically produced viral ss + RNA as a template. Only a single cycle of replication was observed and labeled VPg could be recovered from the dsRNA by RNase V(1) digestion. Denaturation of the dsRNA yielded genome-length labeled ssRNA, indicating that RNA synthesis was not initiated by 3'-end snap-back self-priming. Mutating serine 163 to alanine of rVP1 abolished both its self-guanylylating and polymerizing activity.