Mechanism of Interference Mediated by Human Parainfluenza Virus Type 3 Infection

143

130

Enveloped viruses use multiple mechanisms to inhibit infection of a target cell by more than one virion. These mechanisms may be of particular importance for the evolution of segmented viruses, because superinfection exclusion may limit the frequency of reassortment of viral genes. Here, we show that cellular expression of influenza A virus neuraminidase (NA), but not hemagglutinin (HA) or the M2 proton pump, inhibits entry of HA-pseudotyped retroviruses. Cells infected with H1N1 or H3N2 influenza A virus were similarly refractory to HA-mediated infection and to superinfection with a second influenza A virus. Both HA-mediated entry and viral superinfection were rescued by the neuraminidase inhibitors oseltamivir carboxylate and zanamivir. These inhibitors also prevented the removal of ␣-2,3-and ␣-2,6-linked sialic acid observed in cells expressing NA or infected with influenza A viruses. Our data indicate that NA alone among viral proteins limits influenza A virus superinfection.

Section: Discussionmentioning

confidence: 67%

“…Notably, cell-expressed HA-neuraminidases of several paramyxoviruses mediate such superinfection exclusion by removing the SA receptor from the cell surface (15,27). Other mechanisms of superinfection exclusion have also been described.…”

mentioning

confidence: 99%

Influenza A Virus Neuraminidase Limits Viral Superinfection

Huang

Sui

et al. 2008

143

130

“…Since the low values for C28a were close to the subtracted blank, we cannot be certain whether they denote finite or zero activity. Previous assays of intact HN-expressing cells showed that recombinant wt HN-GFP molecules retained their neuraminidase activity and that C28a HN-GFP-expressing cells had no detectable neuraminidase activity (7). The neuraminidase activity of wt HN-expressing cells, like that of wt HPF3 viral preparations (4), was inhibited by 4-GU-DANA.…”

Section: Isolation Of a New Hpf3 Variant Derived From The Neuraminidamentioning

confidence: 99%

“…HeLa cell lines and CV-1 (African green monkey kidney) cells were maintained with Eagle minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. The generation of monoclonal cell lines stably expressing HN-green fluorescent protein (GFP) of wt and C28a HPF3 was as described elsewhere (7). Briefly, the full-length cDNAs encoding either wt HN or the C28a variant of HN were obtained by PCR amplification of the full-length HN cDNA (19) and subcloned into the corresponding sites of pGFP-C3 (Clontech, Palo Alto, Calif.) to obtain plasmids pwtHN-EGFP and pC28aHN-EGFP, with the amplified HNs fused to the 5Ј end of the EGFP gene.…”

Section: Methodsmentioning

confidence: 99%

Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant

Porotto

Greengard

Poltoratskaia

et al. 2001

Self Cite

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4°C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37°C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.

“…NA assays were performed in transiently transfected 293T cell monolayers, as described previously (9,22).…”

Section: Methodsmentioning

confidence: 99%

Mutations in Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Causing Increased Receptor Binding Activity and Resistance to the Transition State Sialic Acid Analog 4-GU-DANA (Zanamivir)

Murrell

Porotto²,

Weber

et al. 2003

Self Cite

103

Entry and fusion of human parainfluenza virus type 3 (HPF3) require the interaction of the viral hemagglutinin-neuraminidase (HN) glycoprotein with its sialic acid receptor. 4-GU-DANA, a potent inhibitor of influenza virus neuraminidase, inhibits not only HPF3 neuraminidase but also the receptor binding activity of HPF3 HN and thus its ability to promote attachment and fusion. We previously generated a 4-GU-DANAresistant HPF3 virus variant (ZM1) with a markedly fusogenic plaque morphology that harbored two HN gene mutations resulting in amino acid alterations. The present study using cells that express the individual mutations of ZM1 HN shows that one of these mutations is responsible for the increases in receptor binding and neuraminidase activities as well as the diminished sensitivity of both activities to the inhibitory effect of 4-GU-DANA. To examine the hypothesis that increased receptor binding avidity underlies 4-GU-DANA resistance, parallel studies were carried out on the high-affinity HN variant virus C22 and cells expressing the C22 variant HN. This variant also exhibited reduced sensitivity to 4-GU-DANA in terms of receptor binding and infectivity but without concomitant changes in the neuraminidase activity of HN. Another high-affinity HN variant, C0, was not resistant in terms of infectivity; however, a small increase in the receptor binding activity of C0 HN and a partial resistance of this activity to 4-GU-DANA were revealed by sensitive methods that we developed. In each virus variant, one mutation in HN accounted for both increased receptor binding avidity and 4-GU-DANA resistance; the higher affinity for the receptor overcomes the inhibitory effect of 4-GU-DANA. Thus, in contrast to influenza viruses for which 4-GU-DANA escape variants include hemagglutinin mutants with decreased receptor binding avidity that promotes virion release, for HPF3, HN mutants with increased receptor binding avidity are those that can escape the growth inhibitory effect of 4-GU-DANA.