Kinetics of Thermal Inactivation of Lactate Dehydrogenase from Rabbit Muscle

The kinetics of thermal inactivation of rabbit muscle lactate dehydrogenase at different temperatures has been studied using the kinetic method for the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou [Adv. Enzymol. Relat. Areas Mol. Biol. (1988), 61, 381–436]. The results show that thermal inactivation of the enzyme is an irreversible reaction. Microscopic rate constants were determined for thermal inactivation of the free enzyme and the enzyme–substrate complex. The inactivation rate constant of the free enzyme is much larger than the rate constant of the enzyme–substrate complex. The results suggest that the presence of the substrate has a certain protective effect against thermal inactivation of the enzyme.     

Relationship between hemagglutinin stability and influenza virus persistence after exposure to low pH or supraphysiological heating

The hemagglutinin (HA) surface glycoprotein is triggered by endosomal low pH to cause membrane fusion during influenza A virus (IAV) entry yet must remain sufficiently stable to avoid premature activation during virion transit between cells and hosts. HA activation pH and/or virion inactivation pH values less than pH 5.6 are thought to be required for IAV airborne transmissibility and human pandemic potential. To enable higher-throughput screening of emerging IAV strains for “humanized” stability, we developed a luciferase reporter assay that measures the threshold pH at which IAVs are inactivated. The reporter assay yielded results similar to TCID50 assay yet required one-fourth the time and one-tenth the virus. For four A/TN/09 (H1N1) HA mutants and 73 IAVs of varying subtype, virion inactivation pH was compared to HA activation pH and the rate of inactivation during 55°C heating. HA stability values correlated highly with virion acid and thermal stability values for isogenic viruses containing HA point mutations. HA stability also correlated with virion acid stability for human isolates but did not correlate with thermal stability at 55°C, raising doubt in the use of supraphysiological heating assays. Some animal isolates had virion inactivation pH values lower than HA activation pH, suggesting factors beyond HA stability can modulate virion stability. The coupling of HA activation pH and virion inactivation pH, and at a value below 5.6, was associated with human adaptation. This suggests that both virologic properties should be considered in risk assessment algorithms for pandemic potential.     

Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities

The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the "monomer" of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Downstream processing of MDCK cell-derived equine influenza virus

A microcarrier-based process was used to produce equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93) in Madin Darby Canine kidney (MDCK) cells. The virus was purified in a sequence of downstream processing steps comprising of depth filtration, inactivation, ultrafiltration (UF) and gel filtration. In the ultrafiltration step, the hemagglutinin (HA) was recovered to 100%. A high increase of neuraminidase (NA) activity indicated the removal of some inhibitory compounds during this step. At the same time, the level of contaminating proteins and DNA was reduced by more than 88%. In the subsequent size exclusion chromatography (Sepharose CL 2B), the recovery of HA and NA in the "virus peak" was 37.8 and 59.8%, respectively compared to the concentrated feed material. Inconsistencies in the overall mass balance for HA and NA (70.0 and 69.2%) during gel filtration indicated non-specific interactions of the inactivated virus to the gel matrix which is supported by a HA recovery of about 50% in shake flask experiments performed as a control. Overall 35.8% of HA and 291.6% of NA were recovered. More than 95.7% of the host cell proteins and 98.7% of the host cell DNA were removed during downstream processing.     

Full Inactivation of Human Influenza Virus by High Hydrostatic Pressure Preserves Virus Structure and Membrane Fusion While Conferring Protection to Mice against Infection

Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses.     

Inactivation and stability of viral diagnostic reagents treated by gamma radiation

The objective of this study was to apply the pertinent findings from gamma inactivation of virus infectivity to the production of high quality diagnostic reagents. A Gammacell 220 (Atomic Energy of Canada, Ltd., Ottawa, Canada) was used to subject 38 viruses grown in either susceptible tissue cultures or embryonated chicken eggs to various doses of gamma radiation from a cobalt-60 source. The radiation required to reduce viral infectivity was 0.42 to 3.7 megarads (Mrad). The effect of gamma treatment on the antigenic reactivity of reagents for the complement fixation (CF), hemagglutination (HA) and neuraminadase assays was determined. Influenza antigens inactivated with 1.7 Mrad displayed comparable potency, sensitivity, specificity and stability to those inactivated by standard procedures with beta-propiolactone (BPL). Significant inactivation of influenza N1 and B neuraminidase occurred with greater than 2.4 Mrad radiation at temperatures above 4 degrees C. All 38 viruses were inactivated, and CF or HA antigens were prepared successfully. Antigenic potency remained stable with all antigens for 3 years and with 83% after 5 years storage. Influenza HA antigens evaluated after 9 years of storage demonstrated 86% stability. Gamma radiation is safer than chemical inactivation procedures and is reliable and effective replacement for BPL in preparing diagnostic reagents.     

Enhancement of the Influenza A Hemagglutinin (HA)-Mediated Cell-Cell Fusion and Virus Entry by the Viral Neuraminidase (NA)

Background

The major role of the neuraminidase (NA) protein of influenza A virus is related to its sialidase activity, which disrupts the interaction between the envelope hemagglutin (HA) protein and the sialic acid receptors expressed at the surface of infected cells. This enzymatic activity is known to promote the release and spread of progeny viral particles following their production by infected cells, but a potential role of NA in earlier steps of the viral life cycle has never been clearly demonstrated. In this study we have examined the impact of NA expression on influenza HA-mediated viral membrane fusion and virion infectivity.

Methodology/Principal Findings

The role of NA in the early stages of influenza virus replication was examined using a cell-cell fusion assay that mimics HA-mediated membrane fusion, and a virion infectivity assay using HIV-based pseudoparticles expressing influenza HA and/or NA proteins. In the cell-cell fusion assay, which bypasses the endocytocytosis step that is characteristic of influenza virus entry, we found that in proper HA maturation conditions, NA clearly enhanced fusion in a dose-dependent manner. Similarly, expression of NA at the surface of pseudoparticles significantly enhanced virion infectivity. Further experiments using exogeneous soluble NA revealed that the most likely mechanism for enhancement of fusion and infectivity by NA was related to desialylation of virion-expressed HA.

Conclusion/Significance

The NA protein of influenza A virus is not only required for virion release and spread but also plays a critical role in virion infectivity and HA-mediated membrane fusion.     

Envelope Proteins Pertain with Evolution and Adaptive Mechanism of the Novel Influenza A/H1N1 in Humans

The novel swine-origin influenza A/H1N1 virus (S-OIV) first detected in April 2009 has been identified to transmit from human to human directly and is the cause of currently emerged pandemic. In this study, nucleotide and deduced amino acid sequences of hemagglutinin (HA) and neuraminidase (NA) of the S-OIV and other influenza A viruses were analyzed through bioinformatic tools for phylogenetic analysis, genetic recombination and point mutation to investigate the emergence and adaptation of the S-OIV in human. The phylogenetic analysis showed that the HA comes from triple reassortant influenza A/H1N2 and the NA from Eurasian swine influenza A/H1N1 indicating HA and NA to descend from different lineages during the genesis of the S-OIV. Recombination analysis nullified the possibility of occurrence of recombination in HA and NA denoting the role of reassortment in the outbreak. Several conservative mutations are observed in the amino acid sequences of the HA and NA and this mutated residues are identical in the S-OIV. The results reported herein suggested the notion that the recent pandemic is the result of reassortment of different genes from different lineages of two envelope proteins, HA and NA which are responsible for antigenic activity of virus. This study further suggests that the adaptive capability of the S-OIV in human is acquired by the unique mutations generated during emergence.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

Functional Interactions of the Influenza Virus Glycoproteins

Hemagglutinin (HA) and neuraminidase (NA) are functionally related envelope glycoproteins of the influenza virus (Flu). HA interacts with terminal sialyl residues of oligosaccharides and ensures the binding of the virus particle to the cell surface. NA is a receptor-destroying enzyme that removes sialyl residues from oligosaccharides contained in cell and virus components and thereby prevents aggregation of virus particles. Analysis of reassortants combining low-functional NA of human Flu with HA of avian Flu showed that sialyl residues are not completely removed in some cases. With high HA affinity for sialyl substrates, such virus particles aggregate, aggregates accumulate on the cell surface, and virus yield decreases. Serial passaging of low-yield aggregating reassortants may lead to selection of high-yield variants, which do not aggregate. The loss of aggregation is due to a decrease in HA affinity for high-molecular-weight sialyl substrates. On evidence of sequencing of the HA gene in original reassortants and their nonaggregating variants, for different HA antigenic subtypes (H2, H3, H4, and H13) the affinity is reduced and aggregation lost through a common mechanism: an increase in the negative charge as a result of an amino acid substitution in the vicinity of the receptor-binding pocket of HA. Taken together, these findings suggest a way of postreassortment adaptation, which improves the functional match of HA and NA. The experimental system employed provides a model of natural processes associated with emergence of Flu variants having a pandemic potential.     

Influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of M1 protein

Influenza virus matrix protein (M1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. However, because of the inherent membrane-binding ability of M1 protein, it has been difficult to demonstrate the specific interaction of M1 protein with hemagglutinin (HA) or neuraminidase (NA), the influenza virus envelope glycoproteins. Using Triton X-100 (TX-100) detergent treatment of membrane fractions and floatation in sucrose gradients, we observed that the membrane-bound M1 protein expressed alone or coexpressed with heterologous Sendai virus F was totally TX-100 soluble but the membrane-bound M1 protein expressed in the presence of HA and NA was predominantly detergent resistant and floated to the top of the density gradient. Furthermore, both the cytoplasmic tail and the transmembrane domain of HA facilitated binding of M1 to detergent-resistant membranes. Analysis of the membrane association of M1 in the early and late phases of the influenza virus infectious cycle revealed that the interaction of M1 with mature glycoproteins which associated with the detergent-resistant lipid rafts was responsible for the detergent resistance of membrane-bound M1. Immunofluorescence analysis by confocal microscopy also demonstrated that, in influenza virus-infected cells, a fraction of M1 protein colocalized with HA and associated with the HA in transit to the plasma membrane via the exocytic pathway. Similar results for colocalization were obtained when M1 and HA were coexpressed and HA transport was blocked by monensin treatment. These studies indicate that both HA and NA interact with influenza virus M1 and that HA associates with M1 via its cytoplasmic tail and transmembrane domain.     

Hemagglutinin and neuraminidase matching patterns of two influenza A virus strains related to the 1918 and 2009 global pandemics

The current pandemic influenza A (H1N1) virus has revealed a complicated reassortment of various influenza A viruses. The biological study of these viruses, especially of the viral envelope proteins hemagglutinin (HA) and neuraminidase (NA), is urgently needed for the control and prevention of H1N1 viruses. We have generated H1N1-2009 and H1N1-1918 pseudotyped particles (pp) with high infectivity. Combinations of HA1918 + NA2009 and HA2009 + NA1918 also formed infectious H1N1pps, among which the HA2009 + NA1918 combination resulted in the most highly infectious pp. Our study demonstrated that some reassortments of H1N1 viruses may hold the potential to produce higher infectivity than do their ancestors.     

Novel function of plasminogen-binding activity of the NA determines the pathogenicity of influenza A virus

Because cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for infectivity of influenza A viruses, this molecule is a major determinant of viral pathogenicity. Although well documented in the pathogenicity of avian influenza viruses, the role of HA cleavage in the pathogenicity of mammalian viruses is not well understood. Therefore, we studied a mouse-adapted human isolate A/WSN/33 (WSN), a neurovirulent influenza virus strain that causes systemic infection when inoculated intranasally into mice. We found a novel mechanism of HA cleavage for WSN virus: the neuraminidase (NA) of WSN virus binds and sequesters plasminogen on the cell surface, leading to enhanced cleavage of the HA. The structural basis of this novel function of the NA molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146. To obtain direct evidence that the plasminogen-binding activity of the NA enhances the pathogenicity of WSN virus, we generated mutant viruses that are deficient in plasminogen-binding activity by reverse genetics. The mutant viruses showed attenuated growth in mice and failed to grow at all in the brains of these animals. Therefore, we concluded that the novel function of plasminogen-binding activity of the NA determines the pathogenicity of WSN virus in mice.     

Apical budding of a recombinant influenza A virus expressing a hemagglutinin protein with a basolateral localization signal

Influenza virions bud preferentially from the apical plasma membrane of infected epithelial cells, by enveloping viral nucleocapsids located in the cytosol with its viral integral membrane proteins, i.e., hemagglutinin (HA), neuraminidase (NA), and M2 proteins, located at the plasma membrane. Because individually expressed HA, NA, and M2 proteins are targeted to the apical surface of the cell, guided by apical sorting signals in their transmembrane or cytoplasmic domains, it has been proposed that the polarized budding of influenza virions depends on the interaction of nucleocapsids and matrix proteins with the cytoplasmic domains of HA, NA, and/or M2 proteins. Since HA is the major protein component of the viral envelope, its polarized surface delivery may be a major force that drives polarized viral budding. We investigated this hypothesis by infecting MDCK cells with a transfectant influenza virus carrying a mutant form of HA (C560Y) with a basolateral sorting signal in its cytoplasmic domain. C560Y HA was expressed nonpolarly on the surface of infected MDCK cells. Interestingly, viral budding remained apical in C560Y virus-infected cells, and so did the location of NP and M1 proteins at late times of infection. These results are consistent with a model in which apical viral budding is a shared function of various viral components rather than a role of the major viral envelope glycoprotein HA.     

Improvement of influenza A/Fujian/411/02 (H3N2) virus growth in embryonated chicken eggs by balancing the hemagglutinin and neuraminidase activities, using reverse genetics

The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.     

Characterization of a temperature-sensitive influenza B virus mutant defective in neuraminidase.

ts5, a temperature-sensitive mutant of influenza B virus, belongs to one of seven recombination groups. When the mutant infected MDCK cells at the nonpermissive temperature (37.5 degrees C), infectious virus was produced at very low levels compared with the yield at the permissive temperature (32 degrees C) and hemagglutinating and enzymatic activities were undetectable. However, viral protein synthesis and transport of hemagglutinin (HA) and neuraminidase (NA) to the cell surface were not affected. The NA was found as a monomer within cells even at 32 degrees C, in contrast to wild-type virus NA, existing mostly as an oligomer, but the mutant had oligomeric NA, like the wild-type virus. Its enzymatic activity was more thermolabile than that of wild-type virus. Despite the low yield, large aggregates of progeny virus particles were found to accumulate on the cell surface at the nonpermissive temperature, and these aggregates were broken by treatment with bacterial neuraminidase, with the concomitant appearance of hemagglutinating activity, suggesting that NA prevents the aggregation of progeny virus by removal of neuraminic acid from HA and cell receptor, allowing its release from the cells. Further treatment with trypsin resulted in the recovery of infectivity. When bacterial NA was added to the culture early in infection, many hemagglutinable infectious virus was produced. We also suggest that the removal of neuraminic acid from HA by NA is essential for the subsequent cleavage of HA by cellular protease. Nucleotide sequence analysis of RNA segment 6 revealed that ts5 encoded five amino acid changes in the NA molecule but not in NB.     

Thymineless Death in Escherichia coli: Inactivation and Recovery

The effects of chloramphenicol (CAP) on the progress of thymineless death (TLD), nalidixic acid (NA) inactivation, ultraviolet (UV) irradiation, and mitomycin C (MC) inactivation were studied in Escherichia coli B, B(s-1), B(s-3), B(s-12), and B/r. This was done before, during, and after inactivation. During the progress of inactivation, it was found that at 10 to 20 mug of CAP per ml, up to 50% of the UV-sensitive bacteria survived TLD and about 10% survived NA. In E. coli B/r, at these concentrations of CAP, about 10 to 15% of the cells survived TLD and about 20 to 25% survived NA. Concentrations of CAP greater than 25 mug/ml actually increased the sensitivity of E. coli B, B(s-1), B(s-3), and B(s-12) to inactivation by either TLD or NA; at 150 mug of CAP per ml, the sensitivity of E. coli B/r to inactivation also increased. When E. coli B cells were incubated in CAP prior to inactivation, the longer the preincubation the longer onset of TLD was delayed; NA inactivation was also affected in that the rate of inactivation after CAP incubation was greatly decreased. Preincubation of E. coli B/r with CAP had much less effect on the progress of inactivation. After thymineless death, incubation in CAP plus thymine led to a rapid and almost complete recovery of E. coli B and B(s-12). Lesser recoveries were observed after inactivation due to UV, NA, or MC inactivation. E. coli B(s-1) and B/r did not recover viability after any mode of inactivation, and E. coli B(s-3) and B(s-12) recovered from UV to about 20% of the initial titer. It was suggested that protein synthesis, in particular proteins involved in deoxyribonucleic synthesis, was a determining factor in these inactivating and recovery events.     

静注丙球低pH孵放灭活病毒效果验证

以水泡性口炎病毒（ＶＳＶ）为指示病毒,考察了低ｐＨ孵放不同时间对低温乙醇法生产的静注丙球（ＩＶＩＧ）中ＶＳＶ的灭活效果,并对不同厂家及不同批号ＩＶＩＧ中病毒灭活情况进行了比较。结果表明,液体ＩＶＩＧ在ＰＨ４．１±０．３,２０－２５℃,孵放２１天可灭活ＶＳＶ达６Ｌｏｇｓ以上（即低于实验检测限）,但不同厂家及不同批号的ＩＶＩＧ在病毒灭活的发生上有所不同     

Protection of Vaccinia from Heat Inactivation by Nucleotide Triphosphates

The presence of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, or uridine triphosphate reduced the rate of inactivation of vaccinia when heated at 50 C. The virus-associated nucleoside triphosphate phosphohydrolases (adenosine triphosphatase, guanosine triphosphatase, cytosine triphosphatase, and uridine triphosphatase) and ribonucleic acid polymerase were also protected from heat inactivation by these compounds. These obervations are best explained by postulating that ribonucleoside triphosphates bind to enzymes in the virus particle, and that these enzyme-substrate complexes are more resistant to thermal denaturation than are the enzymes without their substrates. The kinetics of heat inactivation of the vaccinia ATP phosphohydrolase activity is biphasic, suggesting that there are two proteins in the vaccinia particle that have this enzyme activity but they have different kinetics of heat inactivation. Any of the vaccinia-associated nucleotide phosphohydrolase activities are protected from heat inactivation by the presence of any one of the respective nucleoside triphosphates. This observation suggests that there is a single enzymatic site in vaccinia that is able to react with any ribonucleoside triphosphate.     

Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice

Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.