Morphogenesis of human adenovirus type 2 studied with fiber- and fiber and penton base-defective temperature-sensitive mutants.

The nature, polypeptide composition, and antigenic composition of the particles formed by six human adenovirus type 2 temperature-sensitive (ts) mutants were studied. ts115, ts116, and ts125 were phenotypically fiber-defective mutants, and ts103, ts104, and ts136 failed to synthesize detectable amounts of fiber plus penton base at 39.5 degrees C. The mutants belonged to five complementation groups, one group including ts116 and ts125. Except for ts103 and ts136, the other mutants were capable of producing particles at 39.5 degrees C. ts116 and ts125 accumulated light assembly intermediate particles (or top components) at nonpermissive temperatures, with few virus particles. The sodium dodecyl sulfate polypeptide pattern of ts116- or ts125-infected cells, intermediate particles, and virus particles showed that polypeptide IV (fiber) was smaller by a molecular weight of 2,000 than that in the wild-type virion and was glycosylated. In fiber plus penton base-defective ts104-infected cells, equivalent quantities of top components and viruses with a buoyant density (rho) of 1.345 g/ml (rho = 1.345 particles) were produced at 39.5 degrees C. These rho = 1.345 particles corresponded to young virions, as evidenced by the presence of uncleaved precursors to proteins VI, VIII, and VII. These young virions matured upon a shift down. Virus capsid vertex antigenic components underwent a phase of eclipse during their incorporation into mature virus particles. No antigenic penton base or IIa was detected in intermediate particles of all the ts mutants tested. Only hexon and traces of fiber antigens were found in ts104 young virions. Penton base and IIIa appeared as fully antigenically expressed capsid subunits in mature wild-type virions or ts104 virions after a shift down. The ts104 lesion is postulated to affect a regulatory function related in some way to penton base and fiber overproduction and the maturation processing of precursors PVI, PVII, and PVII.     

Phenotypic characterization of temperature-sensitive mutants of vaccinia virus with mutations in a 135,000-Mr subunit of the virion-associated DNA-dependent RNA polymerase

The phenotypic defects of three temperature-sensitive (ts) mutants of vaccinia virus, the ts mutations of which were mapped to the gene for one of the high-molecular-weight subunits of the virion-associated DNA-dependent RNA polymerase, were characterized. Because the virion RNA polymerase is required for the initiation of the viral replication cycle, it has been predicted that this type of mutant is defective in viral DNA replication and the synthesis of early viral proteins at the nonpermissive temperature. However, all three mutants synthesized both DNA and early proteins, and two of the three synthesized late proteins as well. RNA synthesis in vitro by permeabilized mutant virions was not more ts than that by the wild type. Furthermore, only one of three RNA polymerase activities that was partially purified from virions assembled at the permissive temperature displayed altered biochemical properties in vitro that could be correlated with its ts mutation: the ts13 activity had reduced specific activity, increased temperature sensitivity, and increased thermolability under a variety of preincubation conditions. Although the partially purified polymerase activity of a second mutant, ts72, was also more thermolabile than the wild-type activity, the thermolability was shown to be the result of a second mutation within the RNA polymerase gene. These results suggest that the defects in these mutants affect the assembly of newly synthesized polymerase subunits into active enzyme or the incorporation of RNA polymerase into maturing virions; once synthesized at the permissive temperature, the mutant polymerases are able to function in the initiation of subsequent rounds of infection at the nonpermissive temperature.     

Temperature-sensitive mutations in various genes of Escherichia coli K12 can be suppressed by the ssrA gene for 10Sa RNA (tmRNA)

An Escherichia coli strain with a deletion in the ssrA gene that encodes 10Sa RNA (tmRNA) was used to screen for temperature-sensitive (ts) mutants whose ts phenotypes were suppressible by introduction of the wild-type ssrA gene. Mutants in four different genes were isolated. Ts mutants of this type were also obtained in a screen for mutations in thyA, the structural gene for thymidylate synthase. The ThyA activity in crude extracts prepared from the ts mutants was temperature-sensitive. The presence of the ssrA gene caused an increase in the total amount of the temperature-sensitive enzyme expressed, rather than suppressing the ts activity of the enzyme itself. SsrA-DD, a mutant form of 10Sa RNA, suppressed the ts phenotype of a thyA mutant, suggesting that degradation of a tagged peptide was not required for suppression of the ts phenotype. Considering the fact that ssrA-suppressible mutants could be isolated as temperature-sensitive mutants with mutations in different genes, it seems evident that trans-translation can occur on mRNA that is not lacking its stop codon.     

Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.     

Characterization of an Endonuclease Associated with Simian Virus 40 Virions

An endonucleolytic activity associated with purified simian virus 40 (SV40) virions has been found. The enzyme is present in virions prepared from a number of different host lines. The enzyme is present in all early and late temperature-sensitive mutants examined. Some aspects of the endonucleolytic activity have been examined with SV40 deoxyribonucleic acid as substrate.     

Mutation in the matrix protein of Newcastle disease virus can result in decreased fusion glycoprotein incorporation into particles and decreased infectivity.

Virus particles produced in eggs by the group D ts mutants of Newcastle disease virus at permissive temperature display low infectious and hemolytic activities (M.E. Peeples and M. A. Bratt , J. Virol. 42:440-446, 1982). These lower activities correlate with a decreased incorporation of F1+2 (fusion glycoprotein) into virus particles, compared with that for wild type. The incorporation of F1+2 into virus particles of the group D mutants is also lower than that for wild type when grown in chicken embryo cells in culture at either permissive or nonpermissive temperature. The infectivity of virions from these mutants correlates with the amounts of F1+2 in the virus particles, below a certain concentration, indicating that the quantity of F1+2 in virus particles is a determining factor in the infectivity of those particles. In addition, one of these mutants, D1, produces an M (matrix protein) which migrates at a faster rate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of four revertants of D1 have coreverted to wild-type M electrophoretic mobility, associating M with the ts lesion and the other observed phenotypes. In each of these revertants, as well as in three revertants each from D2 and D3, there has been coreversion from the low specific infectious and hemolytic activities to greater, and often wild-type, activities. There is also a coreversion for F1+2 incorporation into virions. All of the revertants incorporate F1+2 into virions more efficiently than their mutant parents. The coreversions associate those phenotypes with the ts lesion and, in the case of D1, with the M lesion as well.     

Complementation between temperature-sensitive and deletion mutants of reovirus.

A very low level of complementation has been found in conventional crosses between various classes of temperature-sensitive (ts) mutants of reovirus. A more definitive test for complementation was devised through a plaque assay on cell monolayers mixedly infected with defective reovirions lacking the L1 segment and prototype ts mutants from one or other of the known classes of reovirus mutants. An increase in the number of plaques on the mixedly infected plates over that on control plates infected with defective virions or ts mutants alone indicated that the ts mutant had been complemented by the defective virus. Class A, B, D, F, and G mutants were complemented at 39 C by the defective viruses, whereas class C and E mutants were not. In tests to determine whether complementation was reciprocal it was found that the defective virions were complemented by a class G mutant but not by the class C mutant. This and previous work (D.A. Spandidos and A. F. Graham, 1975) has therefore shown that of the seven known classes of ts mutants the class C mutant is the only one that neither complements nor is complemented by the defective virions. For this reason the class C ts mutation has been assigned to the L1 segment of the viral genome.     

Protease of adenovirus type 2. Subcellular localization.

The subcellular localization of the adenovirus type 2 core polypeptide specific protease activity was investigated using an in vitro assay system. The protease activity was recovered exclusively from infected cell nuclei and was insoluble, sedimenting with the membrane fraction. Endogenous activity could be demonstrated in young virions which contain precursor PVII molecules. This protease activity only became sensitive to L-1-tosylamide-2-phenylethylchloromethyl ketone- or phenylmethylsulfonyl fluoride-mediated inhibition after disruption of the virus particles by sonication, suggesting that the enzyme was internally located. The putative precursors to virus particles, referred to as top components, which do not contain a full complement of viral DNA, did not contain protease activity. The protease released from sonicated virions converted exogenous PVII substrate molecules to polypeptide VII. The noninfectious H2ts1 virus particles synthesized at the nonpermissive temperature phenotypically resemble young virions, but unlike their wild type counterparts, were devoid of protease activity. The results show that the protease enters the precursor particles concurrently with the viral chromosome and that its presence is a prerequisite for the processing and subsequent maturation of infectious adenovirions.     

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.     

Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein.

We have performed a detailed analysis of the biochemical properties of the human immunodeficiency virus (HIV) type 1 vpu gene product to elucidate its function during virus replication. Our data suggest that vpu is posttranslationally modified by phosphorylation, since a 16-kilodalton phosphoprotein can be specifically immunoprecipitated with both a serum from an HIV-positive individual (HIV-positive serum) and a vpu-specific antiserum. In contrast, our results suggest that vpu is not glycosylated, even though the protein contains a potential glycosylation site. In vitro translation studies demonstrated that vpu is cotranslationally integrated into microsomal membranes, suggesting that vpu is an integral membrane protein. While vpu was found in significant quantities in virus-producing cells, the protein could not be detected in cell-free culture fluids and is therefore most likely not viron associated. Processing of viral precursor proteins was unaffected by the absence of vpu, and no differences were detected in the protein compositions of wild-type and mutant virions. However, virus release from cultures producing vpu-defective virus was found to be delayed, resulting in the intracellular accumulation of viral proteins. Our data suggest that vpu has a function in the release of virus particles from infected cells.     

Selective isolation of mutants of vesicular stomatitis virus defective in production of the viral glycoprotein.

We describe a procedure that enriches for temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV), Indiana serotype, which are conditionally defective in the biosynthesis of the viral glycoprotein. The selection procedure depends on the rescue of pseudotypes of known ts VSV mutants in complementation group V (corresponding to the viral G protein) by growth at 39.5 degrees C in cells preinfected with the avian retrovirus Rous-associated virus 1 (RAV-1). Seventeen nonleaky ts mutants were isolated from mutagenized stocks of VSV. Eight induced no synthesis of VSV proteins at the nonpermissive temperature and hence were not studied further. Four mutants belonged to complementation group V and resembled other ts (V) mutations in their thermolability, production at 39.5 degrees C of noninfectious particles specifically deficient in VSV G protein, synthesis at 39.5 degrees C of normal levels of viral RNA and protein, and ability to be rescued at 39.5 degrees C by preinfection of cells by avian retroviruses. Five new ts mutants were, unexpectedly, in complementation group IV, the putative structural gene for the viral nucleocapsid (N) protein. At 39.5 degrees C these mutants also induced formation of noninfectious particles relatively deficient in G protein, and production of infectious virus at 39.5 degrees C was also enhanced by preinfection with RAV-1, although not to the same extent as in the case of the group V mutants. We believe that the primary effect of the ts mutation is a reduced synthesis of the nucleocapsid and thus an inhibition of synthesis of all viral proteins; apparently, the accumulation of G protein at the surface is not sufficient to envelope all the viral nucleocapsids, or the mutation in the nucleocapsid prevents proper assembly of G into virions. The selection procedure, based on pseudotype formation with glycoproteins encoded by an unrelated virus, has potential use for the isolation of new glycoprotein mutants of diverse groups of enveloped viruses.     

Point Mutations in the Paramyxovirus F Protein That Enhance Fusion Activity Shift the Mechanism of Complement-Mediated Virus Neutralization

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.     

The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication.

In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.     

Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.