Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells

Human parainfluenza virus type 3 (HPIV-3) is an airborne pathogen that infects human lung epithelial cells from the apical (luminal) plasma membrane domain. In the present study, we have identified cell surface-expressed nucleolin as a cellular cofactor required for the efficient cellular entry of HPIV-3 into human lung epithelial A549 cells. Nucleolin was enriched on the apical cell surface domain of A549 cells, and HPIV-3 interacted with nucleolin during entry. The importance of nucleolin during HPIV-3 replication was borne out by the observation that HPIV-3 replication was significantly inhibited following (i). pretreatment of cells with antinucleolin antibodies and (ii). preincubation of HPIV-3 with purified nucleolin prior to its addition to the cells. Moreover, HPIV-3 cellular internalization and attachment assays performed in the presence of antinucleolin antibodies and purified nucleolin revealed the requirement of nucleolin during HPIV-3 internalization but not during attachment. Thus, these results suggest that nucleolin expressed on the surfaces of human lung epithelial A549 cells plays an important role during HPIV-3 cellular entry.     

Natural killer cells regulate T-cell proliferation during human parainfluenza virus type 3 infection

Human parainfluenza virus type 3 (HPIV3) is a major respiratory pathogen in humans. Failure to induce immunological memory associated with HPIV3 infection has been attributed to inhibition of lymphocyte proliferation. We demonstrate that the inability of mixed lymphocytes to respond to virally infected antigen-presenting cells is due to an interleukin-2-dependent, nonapoptotic mechanism involving natural killer (NK) cells and their influence is exerted in a contact-dependent manner. These results suggest a novel regulatory mechanism for NK cells during HPIV3 infection, offering an explanation for viral persistence and poor memory responses.     

Expression of the surface glycoproteins of human parainfluenza virus type 3 by bovine parainfluenza virus type 3, a novel attenuated virus vaccine vector

Bovine parainfluenza virus type 3 (bPIV3) is being evaluated as an intranasal vaccine for protection against human PIV3 (hPIV3). In young infants, the bPIV3 vaccine appears to be infectious, attenuated, immunogenic, and genetically stable, which are desirable characteristics for an RNA virus vector. To test the potential of the bPIV3 vaccine strain as a vector, an infectious DNA clone of bPIV3 was assembled and recombinant bPIV3 (r-bPIV3) was rescued. r-bPIV3 displayed a temperature-sensitive phenotype for growth in tissue culture at 39 degrees C and was attenuated in the lungs of Syrian golden hamsters. In order to test whether r-bPIV3 could serve as a vector, the fusion and hemagglutinin-neuraminidase genes of bPIV3 were replaced with those of hPIV3. The resulting bovine/human PIV3 was temperature sensitive for growth in Vero cells at 37 degrees C. The replication of bovine/human PIV3 was also restricted in the lungs of hamsters, albeit not as severely as was observed for r-bPIV3. Despite the attenuation phenotypes observed for r-bPIV3 and bovine/human PIV3, both of these viruses protected hamsters completely upon challenge with hPIV3. In summary, bPIV3 was shown to function as a virus vector that may be especially suitable for vaccination of infants and children against PIV3 and other viruses.     

人3型副流感病毒疫苗的研究进展

人3型副流感病毒是一种主要感染人类肺部上皮细胞的副黏病毒,可引起肺炎和支气管炎,在婴幼儿和免疫力低下的成人中有较高的发病率。经过多年的研究,对人3型副流感病毒疫苗的研究取得了重要的进展,但还没有有效的抗病毒药物和批准的疫苗上市。目前研究主要集中在减毒活疫苗及亚单位疫苗等,对人3型副流感病毒当前疫苗的研究情况做简要的综述。     

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.     

Effects of hemagglutinin-neuraminidase protein mutations on cell-cell fusion mediated by human parainfluenza type 2 virus

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.     

人3型副流感病毒及其疫苗的研究进展

人3型副流感病毒(Human Parainfluenza Virus type 3,HPIV-3)是引起婴幼儿严重细支气管炎及肺炎等下呼吸道疾病的主要病原体,其在发达国家和发展中国家都造成了沉重的疾病负担。迄今,对HPIV-3感染的预防和治疗都还没有有效的疫苗和药物,因此WHO将HPIV-3疫苗列为未来重点研发的疫苗。近年来,随着重组技术和反向遗传学的发展,HPIV-3疫苗的研制取得了重要进展,部分疫苗已进入临床评价阶段。就HPIV-3的生物学特性如病毒结构特征、复制过程、流行病学特征,以及近年来传统冷适应减毒活疫苗、亚单位疫苗、以反向遗传学为基础的新型减毒活疫苗的研制成果及临床试验进展作简要综述。     

Recombinant type 5 adenoviruses expressing bovine parainfluenza virus type 3 glycoproteins protect Sigmodon hispidus cotton rats from bovine parainfluenza virus type 3 infection.

Cotton rats were used to study the replication and pathogenesis of bovine parainfluenza virus type 3 (bPIV3) and to test the efficacy of the F and HN glycoproteins in modulating infection. In vitro cultures of cotton rat lung cells supported the growth of bPIV3 as shown by virus recovery, immunofluorescence, immunoprecipitation, and syncytium induction. Intranasal (i.n.) inoculation of cotton rats with 10(7) PFU resulted in peak recovery of virus after 2 days (8 x 10(4) PFU/g of lung tissue) and significant bronchiolitis with lymphocyte infiltration 5 to 7 days postinfection. Immunohistochemical staining of lungs and trachea demonstrated that virus antigen-positive cells increased in frequency over the course of infection to a maximum on day 5. Serum antibody responses were evaluated by enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HAI), and serum neutralization (SN). Following a single i.n. inoculation, serum antibody levels were 1/40,960, 1/32, and 1/80, as detected by ELISA, HAI, and SN, respectively. When an intramuscular inoculation of 10(7) PFU was administered 10 days prior to the i.n. inoculation, a secondary response which resulted in an ELISA titer of 1/163,000, an HAI titer of 1/640, and an SN titer of 1/512 was induced. IN inoculation of recombinant adenoviruses type 5 containing the bPIV3 F or HN protein or a combination of the two viruses protected cotton rats from bPIV3 challenge. Protection was evaluated serologically by ELISA, HAI, and SN titers, histopathology, immunohistochemistry, and virus recovery.     

人3型副流感病毒减毒活疫苗的研究进展

人副流感病毒(human parainfluenza viruses,HPIVs)是引起婴幼儿支气管炎和肺炎的主要病原体,人3型副流感病毒(human parainfluenza viruses type 3,HPIV-3)是HPIVs中最主要的型别。目前尚无针对HPIVs的上市疫苗和有效的抗病毒药物,研发减毒活疫苗是目前开发HPIVs疫苗的方向。本文主要对HPIV-3减毒活疫苗的研究进展作一综述。     

Characterization of bovine parainfluenza virus type 3.

Bovine parainfluenza virus type 3 (PIV-3) has a buoyant density of 1.197. The RNA of PIV-3, like that of Sendai virus, is a single continuous chain which lacks polyadenylic acid sequences and tends to self-anneal to a marked extent. It has a sedimentation coefficient of 42S and a molecular weight of 4.5 X 10(6), being slightly smaller than Sendai virus RNA (47S, 5.3 X 10(6)). PIV-3 has 5 main structural proteins, of which 2 are glycoproteins. The molecular weights of protein 1, protein 2, protein 3, glycoprotein 1, and glycoprotein 2 were estimated to be 79,000, 68,000, 35,000, 69,000, and 55,000, respectively. Protein 2 was suggested to be nucleocapsid protein.     

Specific inhibition of granzyme B by parainfluenza virus type 3.

T-cell-mediated cytotoxicity is an important means of defense against viral pathogens; however, several viruses possess mechanisms to disrupt cytotoxicity, thereby allowing them to avoid immune clearance. These viruses have been shown to inhibit cytotoxicity by interfering with the capacity of T lymphocytes to specifically recognize infected cells. An alternative mechanism for virally induced cytotoxic dysfunction is identified in this report. We show that parainfluenza virus type 3, a negative-stranded RNA virus, can inhibit cytotoxicity by causing a defect in the cytotoxic effector apparatus. This defect is identified as a selective inhibition of granzyme B mRNA.     

Mechanism of fusion triggering by human parainfluenza virus type III: communication between viral glycoproteins during entry

Parainfluenza viruses enter host cells by fusing the viral and target cell membranes via concerted action of their two envelope glycoproteins: the hemagglutinin-neuraminidase (HN) and the fusion protein (F). Receptor-bound HN triggers F to undergo conformational changes that render it fusion-competent. To address the role of receptor engagement and to elucidate how HN and F interact during the fusion process, we used bimolecular fluorescence complementation to follow the dynamics of human parainfluenza virus type 3 (HPIV3) HN/F pairs in living cells. We show that HN and F associate before receptor engagement. HN drives the formation of HN-F clusters at the site of fusion, and alterations in HN-F interaction determine the fusogenicity of the glycoprotein pair. An interactive site, at the HN dimer interface modulates HN fusion activation property, which is critical for infection of the natural host. This first evidence for the sequence of initial events that lead to viral entry may indicate a new paradigm for understanding Paramyxovirus infection.     

Lentiviral vectors pseudotyped with envelope glycoproteins derived from human parainfluenza virus type 3

We describe the generation of lentiviruses pseudotyped with human parainfluenza type 3 envelope (HPIV3) glycoproteins. Lentivirus particles, expressed in 293T/17 cells, incorporate HPIV3 hemagglutinin-neuraminidase (HN) and fusion (F) proteins into their lipid bilayers and are able to transduce human kidney epithelial cells and polarized MDCK cells. Neuraminidase, AZT, and anti-HPIV3 antisera block transduction, which is consistent with lentiviral-mediated transduction via sialated receptors for HPIV3. Our findings show that HPIV3 pseudotyped lentiviruses can be formed and may have a number of useful properties for human gene transfer.