Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3

The virion proteins and genomic RNA of human parainfluenza virus 3 have been characterized. The virion contains seven major and two minor proteins. Three proteins of 195 X 10(3) molecular weight (195K), 87K, and 67K are associated with the nucleocapsid of the virion and have been designated L, P, and NP, respectively. Three proteins can be labeled with [14C]glucosamine and have molecular weights of 69K, 60K, and 46K. We have designated these proteins as HN, F0, and F1, respectively. HN protein has interchain disulfide bonds, but does not participate in disulfide bonding to form homomultimeric forms. F1 appears to be derived from a complex, F1,2, that has an electrophoretic mobility similar to that of F0 under nonreducing conditions. A protein of 35K is associated with the envelope components of the virion and aggregates under low-salt conditions; this protein has been designated M. The genome of human parainfluenza virus 3 is a linear RNA molecule with a molecular weight of approximately 4.6 X 10(6).     

Intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides.

The intracellular synthesis of human parainfluenza type 3 virus-specified polypeptides was examined by polyacrylamide gel electrophoresis of [35S]methionine-labeled cell extracts under reducing conditions. All of the virion structural proteins were detected in cell extracts, including: L, 180,000 molecular weight (180K); P, 83K; HN, 69K; NP, 66K; F0, 60K; F1, 51K; and M, 38K. P and NP were phosphorylated. HN and F were glycosylated. The kinetics of intracellular viral protein synthesis did not detect any early or late proteins. Pulse-chase experiments failed to detect any precursor-product relationships. No nonstructural proteins were detected.     

Proteins associated with human parainfluenza virus type 3.

The polypeptides associated with human parainfluenza virus type 3 were identified. Five proteins were present in detergent- and salt-resistant viral cores. Of these, three proteins designated NP0, NP1, and NP2 of 68,000, 58,000, and 52,000 daltons, respectively, were stably associated with 50S RNA in CsCl gradient-purified nucleocapsids. The amounts of NP1 and NP2 were variable, and these proteins were shown to be structurally related to the major nucleocapsid protein (NP0) by partial Staphylococcus aureus V8 protease mapping. The other core proteins included a 240K protein designated L (candidate for the viral polymerase) and an 84K protein designated as the phosphoprotein (P) on the basis of a predominant incorporation of Pi. The viral envelope had four prominent proteins (72, 53, 40, and 12K) under reducing conditions of electrophoresis. The 72 and 53K proteins were specifically labeled with [3H]glucosamine and [3H]mannose. When sulfhydryl reagents were removed, a new 62K protein was visualized in place of the 72, 53, and 12K proteins. The 53 and 12K proteins were interpreted to be the two subunits (F1 and F2) of the fusion protein, and the 72K protein was designated as the HN (hemagglutinin-neuraminidase) glycoprotein. The unglycosylated 40K protein represented the viral matrix protein (M). Immunoprecipitation of infected cell lysates with rabbit hyperimmune antiserum against purified virus confirmed the viral origin of these polypeptides.     

Nucleotide sequence of the bovine parainfluenza 3 virus genome: its 3'' end and the genes of NP, P, C and M proteins.

We present the nucleotide sequence of bovine parainfluenza 3 virus (BPIV3) genome from its 3' end to the opening region of the F gene, through the NP, P plus C, and M genes. Comparison of the sequence with those reported for other paramyxoviruses indicated that BPIV3 was most similar to human parainfluenza 3 virus (HPIV3), and also very similar to Sendai virus in the structural make-up of its genome and the amino acid sequences of its gene products, suggesting that these three viruses constitute a paramyxovirus subgroup from which Newcastle disease and measles viruses are separable. In BPIV3 and Sendai virus, the NP and M proteins, the main structural elements, were more highly conserved than the functionally important P and C proteins. This tendency was also observed even in BPIV3 and HPIV3. Virus-specific amino acid sequences of the NP and M proteins were found at the carboxyl and amino terminal regions, respectively. BPIV3 M mRNA was found to have aberrations in its poly A attachment site.     

Protein-protein interactions within paramyxoviruses identified by native disulfide bonding or reversible chemical cross-linking.

Analysis of native disulfide-bonded protein oligomers in paramyxoviruses showed that some viral proteins are consistently present as covalent complexes. In isolated Sendai virus the hemagglutinating protein HN is present in homodimeric and homotetrameric forms, and the minor nucleocapsid protein P exists partly as a monomer and partly as a disulfide-linked homotrimer. Similar disulfide-linked complexes were observed in Newcastle disease virus (strain HP-16), in which HN exists as a homodimer and some of the major nucleocapsid protein NP exists as a homotrimer. Noncovalent intermolecular interactions between proteins were studied with the reversible chemical cross-linkers dimethyl-3,3'-dithiobispropionimidate and methyl 3-[(p-azidophenyl)dithio]propionimidate, which contain disulfide bridges and a 1.1-nm separation between their functional groups. The same results were achieved with both reagents. The conditions of preparation, isolation, and storage of the viruses affected the protein-protein interactions observed upon cross-linking. Homooligomers of the glycoprotein F, the matrix protein M, and the major nucleocapsid protein NP were produced in both Sendai and Newcastle disease viruses after mild cross-linking of all viral preparations examined, but NP-M heterodimer formation in both viruses was most prevalent in early harvest preparations that were cross-linked soon after isolation. The ability of NP and M to form a heterodimer upon cross-linking indicates that the matrix protein layer lies in close proximity (within 1.1 nm) to the nucleocapsid in the newly formed virion. Some noncovalent intermolecular protein interactions in Sendai and Newcastle disease viruses, i.e., those leading to the formation of F, NP, and M homooliogmers upon cross-linking, are more stable to virus storage than others, i.e., those leading to the formation of an NP-M heterodimer upon cross-linking. The storage-induced loss of the ability of NP and M to form a heterodimer is not accompanied by any apparent loss of infectivity. This indicates that some spacial relationships which form during virus assembly can alter after particle formation and are not essential for the ensuing stages of the infectious process.     

Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.     

The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction

Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.     

汉坦病毒疫苗株Z10与Z37重组核蛋白NP的表达纯化及其特异结合基因组末端反向重复序列的功能研究

汉坦病毒是引起肾综合征出血热(HFRS)和汉坦病毒型肺炎综合征(HPS)的主要病原体.其基因组由三节段的单股负链RNA组成,即S、M与L基因片段.汉坦病毒基因组的一个重要特点是每个基因片段的两个末端都有一段长18个核苷酸的高度保守的反向重复序列,互补可形成双链发夹结构,并且这一特点为不同型病毒所共有.为了研究该基因组末端保守的反向重复序列的功能,首先构建了汉坦病毒中国疫苗株Z10(汉滩型)及Z37(汉城型)的核蛋白原核表达载体,并在大肠杆菌中高效表达.经NI-NAT亲和柱和HiPrep16/10 DEAE离子交换柱液相色谱(FPLC)二步提纯,获得高纯度的重组核蛋白,并分别以胰蛋白酶消化后,用Western blotting进行区分和鉴定.以T4 DNA激酶同位素标记一对人工合成互补的18个核苷酸反向重复序列,制备双链探针.然后将该探针与纯化的Z10、Z37株的核蛋白NP进行非变性凝胶电泳迁移改变实验(EMSA)后发现,重组的Z10、Z37株的核蛋白NP,在体外均可特异地结合其基因组末端反向重复序列形成的双链探针.该结果表明,汉坦病毒基因组末端的反向重复序列是核蛋白重要结合位点,这对理解汉坦病毒核蛋白功能以及病毒复制过程中病毒粒子的包装机制有重要的意义.     

Identification of a uniquely immunodominant, cross-reacting site in the human immunodeficiency virus endonuclease protein.

One of the features of the life cycle of retroviruses is insertion of the proviral DNA into host chromosomes. A protein encoded by the 3' end of the pol gene of the virus genome has been shown to possess endonuclease activity (D. P. Grandgenett, A. C. Vora, and R. D. Schiff, Virology 89:119-132, 1978), which is necessary for DNA integration. Sera from the majority of human immunodeficiency virus (HIV)-infected individuals react with endonuclease protein p31 in serological tests (J. S. Allan, J. E. Coligan, T.-H. Lee, F. Barin, P. J. Kanki, S. M'Boup, M. F. McLane, J. E. Groopman, and M. Essex, Blood 69:331-333, 1987; E. F. Lillehoj, F. H. R. Salazar, R. J. Mervis, M. G. Raum, H. W. Chan, N. Ahmad, and S. Venkatesan, J. Virol. 62:3053-3058, 1988; K. S. Steimer, K. W. Higgins, M. A. Powers, J. C. Stephans, A. Gyenes, G. George-Nascimento, P. A. Liciw, P. J. Barr, R. A. Hallewell, and R. Sanchez-Pescador, J. Virol. 58:9-16, 1986). It is not known, however, which part of the protein represents the target(s) for antibody response. To study this, we synthesized peptides and used them in an enzyme-linked immunosorbent assay system to map the reactivity of human immunodeficiency virus type 1 (HIV-1) antibody-positive sera to the different regions of the HIV endonuclease. A uniquely antigenic, HIV-1- and HIV-2-cross-reacting site was identified in the central part of this protein from Phe-663 to Trp-670.     

Nucleotide sequence of the bovine parainfluenza 3 virus genome: the genes of the F and HN glycoproteins.

By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins.     

Nucleocapsid incorporation into parainfluenza virus is regulated by specific interaction with matrix protein

The paramyxovirus nucleoproteins (NPs) encapsidate the genomic RNA into nucleocapsids, which are then incorporated into virus particles. We determined the protein-protein interaction between NP molecules and the molecular mechanism required for incorporating nucleocapsids into virions in two closely related viruses, human parainfluenza virus type 1 (hPIV1) and Sendai virus (SV). Expression of NP from cDNA resulted in in vivo nucleocapsid formation. Electron micrographs showed no significant difference in the morphological appearance of viral nucleocapsids obtained from lysates of transfected cells expressing SV or hPIVI NP cDNA. Coexpression of NP cDNAs from both viruses resulted in the formation of nucleocapsid composed of a mixture of NP molecules; thus, the NPs of both viruses contained regions that allowed the formation of mixed nucleocapsid. Mixed nucleocapsids were also detected in cells infected with SV and transfected with hPIV1 NP cDNA. However, when NP of SV was donated by infected virus and hPIV1 NP was from transfected cDNA, nucleocapsids composed of NPs solely from SV or solely from hPIVI were also detected. Although almost equal amounts of NP of the two viruses were found in the cytoplasm of cells infected with SV and transfected with hPIV1 NP cDNA, 90% of the NPs in the nucleocapsids of the progeny SV virions were from SV. Thus, nucleocapsids containing heterologous hPIV1 NPs were excluded during the assembly of progeny SV virions. Coexpression of hPIV1 NP and hPIV1 matrix protein (M) in SV-infected cells increased the uptake of nucleocapsids containing hPIV1 NP; thus, M appears to be responsible for the specific incorporation of the nucleocapsid into virions. Using SV-hPIV1 chimera NP cDNAs, we found that the C-terminal domain of the NP protein (amino acids 420 to 466) is responsible for the interaction with M.     

Sequence of 3,687 nucleotides from the 3' end of Sendai virus genome RNA and the predicted amino acid sequences of viral NP, P and C proteins.

The sequence of 3,687 nucleotides from the 3' end of the Sendai virus genome (Z strain) was determined by a molecular cloning technique followed by rapid sequence analysis. Two large open reading frames, one consisting of 1,572 nucleotides and the other of 1,704 nucleotides, were observed in the region, that is OP-1 and OP-2 from the 3' end of the genome. The amino acid sequences of the gene products were predicted from the observed sequence. Determination of amino acid compositions of viral proteins, P, HN, Fo, NP and M, led us to conclude that NP and P are the gene products of OP-1 and OP-2, respectively. An additional open reading frame consisting of 612 nucleotides (OP-3) was discovered in the 3' most proximal region of OP-2. The predicted product of OP-3 was considered to be viral non-structural protein C. The leader sequence of 51 nucleotides at the 3' terminal of the genome and consensus sequences at 3' and 5' ends of each gene for proteins NP and P were identified.     

Requirements for budding of paramyxovirus simian virus 5 virus-like particles

Enveloped viruses are released from infected cells after coalescence of viral components at cellular membranes and budding of membranes to release particles. For some negative-strand RNA viruses (e.g., vesicular stomatitis virus and Ebola virus), the viral matrix (M) protein contains all of the information needed for budding, since virus-like particles (VLPs) are efficiently released from cells when the M protein is expressed from cDNA. To investigate the requirements for budding of the paramyxovirus simian virus 5 (SV5), its M protein was expressed in mammalian cells, and it was found that SV5 M protein alone could not induce vesicle budding and was not secreted from cells. Coexpression of M protein with the viral hemagglutinin-neuraminidase (HN) or fusion (F) glycoproteins also failed to result in significant VLP release. It was found that M protein in the form of VLPs was only secreted from cells, with an efficiency comparable to authentic virus budding, when M protein was coexpressed with one of the two glycoproteins, HN or F, together with the nucleocapsid (NP) protein. The VLPs appeared similar morphologically to authentic virions by electron microscopy. CsCl density gradient centrifugation indicated that almost all of the NP protein in the cells had assembled into nucleocapsid-like structures. Deletion of the F and HN cytoplasmic tails indicated an important role of these cytoplasmic tails in VLP budding. Furthermore, truncation of the HN cytoplasmic tail was found to be inhibitory toward budding, since it prevented coexpressed wild-type (wt) F protein from directing VLP budding. Conversely, truncation of the F protein cytoplasmic tail was not inhibitory and did not affect the ability of coexpressed wt HN protein to direct the budding of particles. Taken together, these data suggest that multiple viral components, including assembled nucleocapsids, have important roles in the paramyxovirus budding process.