NC Mutants Research Articles

HIV-1 nucleocapsid protein binds double-stranded DNA in multiple modes to regulate compaction and capsid uncoating.

The HIV-1 nucleocapsid protein (NC) is a multi-functional protein necessary for viral replication. Recent studies have demonstrated reverse transcription occurs inside the fully intact viral capsid and that the timing of reverse transcription and uncoating are correlated. How a nearly 10 kbp viral DNA genome is stably contained within a narrow capsid with diameter similar to the persistence length of double-stranded (ds) DNA, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates the DNA substrate in a non-specific binding mode that triggers uniform DNA self-attraction, condensing the DNA into a tight globule at a constant force up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to observe the activity of basic residue mutations of NC. All basic residue mutations lead to a defective interaction with the DNA substrate. Notably, the constant force “plateau” observed with wild-type NC is missing or largely diminished. However, the NC mutants that are still able to compact dsDNA, at least to some extent, create a DNA condensate that persists even at high forces in our stretching experiments. We investigate the nature of this high kinetic barrier for DNA decompaction, which may come from either direct NC-induced DNA crosslinking or, alternatively, from the steric entanglement of DNA in its compacted state. In either case, the slow kinetics of DNA decompaction from its NC-condensed state is likely responsible for the few hours delay between the completion of HIV-1 DNA synthesis inside the mature capsid, and subsequent capsid breaking, i.e. uncoating.

Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching.

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.

Uncovering Principles That Control Septin-Septin Interactions

Septins comprise a conserved family of GTPases important in cytokinesis. These proteins polymerize into filaments from rod-shaped heteromeric septin complexes. Septins interact with one another at two interfaces (NC and G) that alternate within the complex. Here, we show that small mutations at the N terminus greatly enhance the formation of SEPT2 homopolymers. Taking advantage of this mutation to examine polymer formation using SEPT2 alone, we show that both NC and G interfaces are required for filament formation. However, co-expression of wild type SEPT2 with SEPT2 containing mutations at either NC or G interfaces revealed that only the NC mutant suppressed filament formation. NC mutants are able to interact with one another at putative G interfaces, whereas G mutants fail to interact at NC interfaces. In addition, all promiscuous septin pairwise interactions occur at the G interface. These findings suggest that G interface interactions must occur before NC interactions during polymer formation.

Nucleic Acid Chaperone Activity of the Yeast Ty3 Retrotransposon Nucleocapsid Protein

Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and nucleic acid aggregation, duplex destabilization, and rapid protein binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. We used single molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. Wild type Ty3 NC strongly aggregates both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), and melted DNA exhibits rapid reannealing in its presence. We also studied several Ty3 NC mutants to identify the roles of functional regions of the protein. We found that the N-terminal basic residues contribute to duplex stabilization, while the zinc finger at the C-terminus counteracts this effect. The mutants examined lack the rapid kinetics of wild type Ty3 NC, indicating that both the basic residues and the zinc finger are required for optimum chaperone activity, which is consistent with previous biochemical experiments. Ty3 NC therefore has a chaperone mechanism similar to that of HIV-1 NC. Although Ty3 NC does not exhibit the strong duplex destabilization of HIV-1 NC, this is consistent with the weaker secondary structure of the Ty3 long-terminal repeat region, which suggests that strong duplex destabilization is not needed for NC to facilitate minus-strand transfer during reverse transcription. This research was supported in part by funding from INSERM and ANRS (France).

A homeotic mutation influences the wing vibration patterns during mating in males of the silkworm moth Bombyx mori

An abnormality in the wing vibration pattern in males of the E Nc homeotic mutant of Bombyx mori was investigated. The wild-type (+/+) males show a switching of the rhythmic wing vibrations from a sequential pattern to an intermittent pattern during mating, whereas the E Nc mutants show a sequential pattern both before and during mating. Wing motions in +/+ males became small during mating, but those in +/ E Nc males did not. Ablation of the head ganglia of +/+ and +/ E Nc males during mating caused no change in the motor patterns of wing vibrations. Ablation or cooling of the posterior abdomen in the +/+ males during mating caused sequential wing vibrations, suggesting that the change in wing vibrations is induced by signals from the posterior abdomen. The pterothoracic ganglion in the +/ E Nc males is separated into two ganglia, in contrast to the complete ganglionic fusion in the +/+ males. The neurons in the pterothoracic ganglion stained from abdominal nerve cords are homologus in +/+ and +/ E Nc males, but many of these in +/ E Nc males are elongated along the anteroposterior axis. These results suggest that the wing vibration pattern is restricted by genetic factors through reconstruction of the thoracic nervous system during metamorphosis.

DNA Interaction Properties of Nucleic Acid Chaperone Proteins from Retrotransposons

Nucleic acid chaperone activity is an essential component of reverse transcription in retroviruses and retrotransposons. Using DNA stretching with optical tweezers, we have developed a method for detailed characterization of nucleic acid chaperone proteins, which facilitate the rearrangement of nucleic acid secondary structure. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) have been extensively studied, and duplex destabilization, nucleic acid aggregation, and rapid protein binding kinetics have been identified as major components of its activity. The chaperone properties of other nucleic acid chaperone proteins, such as those from the retrotransposons LINE-1 and Ty3, ORF1p and Ty3 NC, are not well understood. We used single molecule DNA stretching to characterize the activity of wild type and mutant ORF1p and Ty3 NC. ORF1p binds both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) with high affinity, and strongly aggregates both forms. It is therefore an excellent chaperone, and altering certain residues has dramatic effects on chaperone activity. Wild type Ty3 also strongly aggregates both dsDNA and ssDNA, and melted DNA exhibits more rapid reannealing in the presence of Ty3 NC, relative to that observed in the presence of ORF1p. We examine several Ty3 NC mutants to identify the roles of functional regions of the protein in its chaperone activity. This research was supported in part by funding from INSERM and ANRS (France).

Mutations in Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Zinc Fingers Cause Premature Reverse Transcription

Human immunodeficiency virus type 1 (HIV-1) requires that its genome be reverse transcribed into double-stranded DNA for productive infection of cells. This process requires not only reverse transcriptase but also the nucleocapsid protein (NC), which functions as a nucleic acid chaperone. Reverse transcription generally begins once the core of the virion enters the cytoplasm of a newly infected cell. However, some groups have reported the presence of low levels of viral DNA (vDNA) within particles prior to infection, the significance and function of which is controversial. We report here that several HIV-1 NC mutants, which we previously identified as being replication defective, contain abnormally high levels of intravirion DNA. These findings were further reinforced by the inability of these NC mutants to perform endogenous reverse transcription (ERT), in contrast to the readily measurable ERT activity in wild-type HIV-1. When either of the NC mutations is combined with a mutation that inactivates the viral protease, we observed a significant reduction in the amount of intravirion DNA. Interestingly, we also observed high levels of intravirion DNA in the context of wild-type NC when we delayed budding by means of a PTAP((-)) (Pro-Thr-Ala-Pro) mutation. Premature reverse transcription is most probably occurring before these mutant virions bud from producer cells, but we fail to see any evidence that the NC mutations alter the timing of Pr55(Gag) processing. Critically, our results also suggest that the presence of intravirion vDNA could serve as a diagnostic for identifying replication-defective HIV-1.

Characterization of human immunodeficiency virus type 1 (HIV-1) containing mutations in the nucleocapsid protein at a putative HIV-1 protease cleavage site

The HIV-1 nucleocapsid protein (NC) has been hypothesized to be cleaved by the viral protease (PR) during early infection. Characterization of viruses, with amino-acid substitutions that modulate PR cleavage of NC in vitro, was performed in cell culture. Two of the NC mutants, NC N17F and NC N17G, had decreased infectivity and exhibited severe H9 replication defects. Examination of viral DNA after infections revealed defects in reverse transcription and integration, although integration defects were cell-type dependent. However, while the defects in reverse transcription and integration correlate with lowered infectivity in a single-round of infection, they did not approach the magnitude of the replication defect measured in H9 cells over multiple rounds. Importantly, we fail to see evidence that H9 cells are re-infected with the NC N17G and NC N17F viruses 24 h after the initial infection, which suggests that the principal defect caused by these NC mutations occurs during late events of viral replication.

Specific Interactions Between HIV-1 Nucleocapsid Protein and the TAR Element

During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer.

Structural Determinants of HIV-1 Nucleocapsid Protein for cTAR DNA Binding and Destabilization, and Correlation with Inhibition of Self-primed DNA Synthesis

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

Role of Murine Leukemia Virus Nucleocapsid Protein in Virus Assembly

The retroviral nucleocapsid protein (NC) originates by cleavage of the Gag polyprotein. It is highly basic and contains one or two zinc fingers. Mutations in either the basic residues or the zinc fingers can affect several events of the virus life cycle. They frequently prevent the specific packaging of the viral RNA, affect reverse transcription, and impair virion assembly. In this work, we explore the role of NC in murine leukemia virus (MLV) particle assembly and release. A panel of NC mutants, including mutants of the zinc finger and of a basic region, as well as truncations of the NC domain of Gag, were studied. Several of these mutations dramatically reduce the release of virus particles. A mutant completely lacking the NC domain is apparently incapable of assembling into particles, although its Gag protein is still targeted to the plasma membrane. By electron microscopy on thin sections of virus-producing cells, we observed that some NC mutants exhibit various stages of budding defects at the plasma membrane and have aberrant particle morphology; electron micrographs of cells expressing some of these mutants are strikingly similar to those of cells expressing "late-domain" mutants. However, the defects of NC mutants with respect to virus release and infectivity could be complemented by an MLV lacking the p12 domain. Therefore, the functions of NC in virus budding and infectivity are completely distinct from viral late-domain function.

Specific zinc-finger architecture required for HIV-1 nucleocapsid protein's nucleic acid chaperone function.

The nucleocapsid protein (NC) of HIV type 1 (HIV-1) is a nucleic acid chaperone that facilitates the rearrangement of nucleic acid secondary structure during reverse transcription. HIV-1 NC contains two CCHC-type zinc binding domains. Here, we use optical tweezers to stretch single lambda-DNA molecules through the helix-to-coil transition in the presence of wild-type and several mutant forms of HIV-1 NC with altered zinc-finger domains. Although all forms of NC lowered the cooperativity of the DNA helix-coil transition, subtle changes in the zinc-finger structures reduced NC's effect on the transition. The change in cooperativity of the DNA helix-coil transition correlates strongly with in vitro nucleic acid chaperone activity measurements and in vivo HIV-1 replication studies using the same NC mutants. Moreover, Moloney murine leukemia virus NC, which contains a single zinc finger, had little effect on transition cooperativity. These results suggest that a specific two-zinc-finger architecture is required to destabilize nucleic acids for optimal chaperone activity during reverse transcription in complex retroviruses such as HIV-1.

Host cell-dependent replication of HIV-1 gag MA, CA, and NC mutants are independent of the functions of Vif and Vpu.

We have previously shown that some gag gene mutants of human immunodeficiency virus type 1 (HIV-1) display a replication-defect in a cell-dependent manner. We and others have also demonstrated that the requirement of vif and vpu genes for HIV-1 replication is cell-dependent. To determine whether the cell-dependent growth of the HIV-1 gag mutants is related to the functions of Vif and Vpu, double mutants of gag-vif and gag-vpu were constructed, and monitored for their replication in various cell lines. The results obtained showed that the mutations in gag do not affect the cell-dependent functions of Vif and Vpu.

Binding of Ca2+ and Zn2+ to Human Nuclear S100A2 and Mutant Proteins

The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.

Developmental expression of the Bombyx Antennapedia homologue and homeotic changes in the Nc mutant.

Abundant availability of homeotic mutants in Bombyx mori provides us with clues to study the body plan of the silkworm. Previous studies indicated that the ECa/ECa and EN/EN embryos, which reveal drastic morphological changes (Itikawa 1943, 1952) lack the homologue of abd-A and the homologues of abd-A and Ubx, respectively (Ueno et al. 1992). It will be interesting to characterize a mutant named Nc, the locus of which was mapped about 1.4 cm apart from the E loci (Itikawa 1944, 1952). In the present study we cloned and identified the Bombyx Antennapedia cDNA from a library of larval middle silk gland. The developmental expression patterns of the Bombyx Antennapedia were analysed in embryos and larval middle silk glands. We found the Bombyx Antennapedia mRNA of the Nc mutant lacks the homeobox region and its downstream region. This defect comes from a corresponding deletion on the Nc chromosome. Development of the Nc/Nc embryos terminates at stage 26, revealing a transformation of the prothoracic legs into antennae, milder changes in the mesothoracic legs, compression between the prothoracic and mesothoracic segments as well as in the gnathocephalon region, and a severe growth suppression of the silk glands. These results give a molecular basis and detailed morphological abnormalities of the Nc mutant known since 1944.

Effect of rearrangements and duplications of the Cys-His motifs of Rous sarcoma virus nucleocapsid protein

It has been widely documented that the nucleocapsid protein p12 (NC) of Rous sarcoma virus (RSV) has a role in the encapsidation and maturation of the virus genomic RNA during particle formation, and particularly important appear to be the Cys-His motifs of this protein. Since some retroviruses only have one such motif, we have investigated the significance of the two distinct Cys-His motifs of RSV NC. The analysis of the phenotype of virus NC mutants with precise rearrangements or duplications of the motifs highlights the following features. (i) The two motifs are not functionally equivalent. (ii) The order and number of Cys-His motifs are less important for RSV NC than the presence of two distinct motifs for both the encapsidation of virus genomic RNA and maintenance of the integrity of the RNA after particle formation. (iii) The proximal motif has a distinct function in the virus replication cycle other than RNA encapsidation and dimerization. (iv) The presence of three Cys-His motifs reduces virus infectivity and leads to high-frequency deletion events (of one of the motifs) after infection: the resulting RNA species encode a wild type-like NC protein restoring full infectivity to the progeny virus particles. Additionally, the data suggest that this occurs only after infection. The deletion probably arises by intramolecular displacement of the replication complex between repeat sequences.

Relationships among virus spread, cytopathogenicity, and virulence as revealed by the noncytopathic mutants of Newcastle disease virus.

We have studied protein synthesis in cultured cells infected with the six noncytopathic (nc) mutants of the Australia-Victoria strain (AV-WT) of Newcastle disease virus and their plaque-forming revertants. Virus-specific polypeptides accumulated at 30 to 63% of wild-type levels in nc mutant-infected cells and between 66 and 175% of wild-type levels in revertant-infected cells. An exception was the L polypeptide, which accumulated in nc mutant-infected cells at only 5 to 20% of the levels found in wild-type infection. The reduced accumulation of the L polypeptide did not appear to be due to increased degradation of that polypeptide. A new polypeptide (X) accumulated instead of polypeptide P in cells infected with mutants nc4 or nc16 and in virions released from them. Peptide mapping identified X as an altered form of P. A revertant of mutant nc4 (nc4S1), which forms larger hemadsorbing spots, but still does not form plaques, accumulated P instead of the X polypeptide. Thus, a lesion in P can affect virus spread without affecting cytopathogenicity. Virions of mutant nc7 and two naturally occurring avirulent strains of Newcastle disease virus (NJ LaSota and B1-Hitchner) contained polypeptides (F(7) and F(A), respectively) related to, but migrating more rapidly than, F(0) in sodium dodecyl sulfate-polyacrylamide gels. As previously reported for avirulent strains, a brief treatment of nc7 virions with trypsin converted F(7) to F and increased infectivity. Similarly, culturing nc7-infected cells in the presence of trypsin facilitated fusion from within and viral spread from cell to cell. A plaque-forming revertant of nc7 still accumulated F(7) in virions, indicating that the lesions responsible for the F(7) and noncytopathic phenotypes are genetically separable. The virulent parental strain, AV-WT, exhibited a mean embryo death time of 42 h. Both the larger-spot-forming revertant of nc4 (nc4S1) and the small-plaque-forming revertant of nc7 exhibited a decrease in mean embryo death time (increase in virulence) from 74 to 63 h. A second-step, plaque-forming revertant derived from nc4S1 (nc4S1R1) exhibited a further decrease in mean embryo death time from 63 to 44 h. The results suggest that the F(A)-F(7) and X lesions affect the ability of virus to spread from cell to cell. In addition, these lesions appear to be genetically separable from those responsible for the noncytopathic phenotype. However, both types of lesions cause an extension of mean embryo death time and, thus, may be relevant to virulence in vivo.

Noncytopathic mutants of Newcastle disease virus are defective in virus-specific RNA synthesis.

We have studied virus-specific RNA synthesis in cells infected by six noncytopathic (nc) mutants of the Australia-Victoria wild-type strain (AV-WT) of Newcastle disease virus (NDV) (19). The rates of NDV-specific RNA synthesis in mutant infection were three to sevenfold lower than those observed in wild-type infection. Velocity sedimentation of this NDV-specific RNA revealed that the lower rates of synthesis in mutant infection correlated with reduced accumulation of 18S and 35S mRNA. Electrophoresis in polyacrylamide-urea gels showed that accumulation of all of the 18S mRNA species was reduced and no new species could be detected. Primary transcription appeared unaltered in mutant infection. Cells infected with two naturally occurring avirulent strains of NDV also showed less accumulation of 18S mRNA. Electrophoresis of this RNA resulted in patterns which differed from those obtained with RNA from either AV-WT or nc mutant infection. Complementation for RNA accumulation between the nc mutants and RNA- temperature-sensitive mutants of AV-WT (32) suggested a common defect in the nc mutants. Analysis of plaque-forming revertants of five of the nc mutants revealed that viral RNA synthetic capacity, cell killing, and plaque-forming ability correlated absolutely. These results suggest that viral RNA synthesis and cytopathogenicity may be causally related. In addition, several of the plaque-forming (and cell-killing) revertants were found to be unable to induce fusion from within in infected cell cultures. This result, coupled with the finding that several of the nc mutants are capable of wild-type levels of fusion from within, suggests that the ability to cause such fusion does not correlate with the ability to kill cells.

Tubulin and Microtubules in the Early Development of the Axolotl and Other Amphibia

SYNOPSIS. Unfertilized eggs of the axolotl, Ambystoma mexicanum , contain a pool of soluble tubulin accumulated during oogenesis. After initiation of cleavage the tubulin pool decreases somewhat and then remains constant through early development. Some properties of tubulin alter during development, but at least some of these changes are not due to changes in tubulin perse. However, the tubulin in axolotl oocytes, eggs, and embryos differs in some electrophoretic properties from tubulin in adult axolotl brain and testis. Equivalent differences were observed in Necturus maculosus tubulins. Heterogeneity of axolotl tubulins was confirmed by peptide mapping: Different patterns of peptides were formed by specific limited proteolysis of soluble tubulin from eggs and testis. The heterogeneity was more marked in the a than in the β subunit. Mobilization of soluble tubulin into the mitotic apparatus depends on the functioning of microtubule organizing centers after activation of the egg at fertilization. In eggs of the nc mutant axolotl there is a lesion in some step of activation, one effect of which is that even though the eggs contain an essentially normal pool of tubulin, microtubules fail to assemble, no mitotic apparatus forms, and embryonic development does not begin. These eggs can be partially corrected by injection of heterologous microtubule fragments, which elicit the mobilization of nc tubulin into arrays of microtubules, followed by initiation of cleavage and development to a partial blastula stage. The results of these experiments are discussed in comparison with other reports in the literature about the function of microtubule organizing centers during amphibian egg development.

Microtubule assembly mutant.

WE report here a new maternal effect mutation (nc, for no cleavage) in the Mexican axolotl, Ambystoma mexicanum. This mutation involves a defect in the assembly of microtubules in the fertilised egg. There is a large literature describing the molecular and biochemical events which accompany the in vitro self-assembly of microtubules; the nc mutant is the first known system with a genetic lesion in the regulation of these events in vivo. This mutant will provide a means to examine directly the control of microtubule assembly, and may also be useful in the identification of the events which trigger initiation of cleavage after activation of the egg at fertilisation.