Ability of the matrix protein of vesicular stomatitis virus to suppress beta interferon gene expression is genetically correlated with the inhibition of host RNA and protein synthesis

The vesicular stomatitis virus (VSV) matrix (M) protein plays a major role in the virus-induced inhibition of host gene expression. It has been proposed that the inhibition of host gene expression by M protein is responsible for suppressing activation of host interferon gene expression. Most wild-type (wt) strains of VSV induce little if any interferon gene expression. Interferon-inducing mutants of VSV have been isolated previously, many of which contain mutations in their M proteins. However, it was not known whether these M protein mutations were responsible for the interferon-inducing phenotype of these viruses. Alternatively, mutations in other genes besides the M gene may enhance the ability of VSV to induce interferons. These hypotheses were tested by transfecting cells with mRNA expressing wt and mutant M proteins in the absence of other viral components and determining their ability to inhibit interferon gene expression. The M protein mutations were the M51R mutation originally found in the tsO82 and T1026R1 mutant viruses, the double substitution V221F and S226R found in the TP3 mutant virus, and the triple substitution E213A, V221F, and S226R found in the TP2 mutant virus. wt M proteins suppressed expression of luciferase from the simian virus 40 promoter and from the beta interferon (IFN-beta) promoter, while M proteins of interferon-inducing viruses were unable to inhibit luciferase expression from either promoter. The M genes of the interferon-inducing mutants of VSV were incorporated into the wt background of a recombinant VSV infectious cDNA clone. The resulting recombinant viruses were tested for their ability to activate interferon gene expression and for their ability to inhibit host RNA and protein synthesis. Each of the recombinant viruses containing M protein mutations induced expression of a luciferase reporter gene driven by the IFN-beta promoter and induced production of interferon bioactivity more effectively than viruses containing wt M proteins. Furthermore, the M protein mutant viruses were defective in their ability to inhibit both host RNA synthesis and host protein synthesis. These data support the idea that wt M protein suppresses interferon gene expression through the general inhibition of host RNA and protein synthesis.     

Homology modeling of the structure of tobacco acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis

A reliable model of tobacco acetohydroxy acid synthase (AHAS) was obtained by homology modeling based on a yeast AHAS X-ray structure using the Swiss-Model server. Conserved residues at the dimer interface were identified, of which the functional roles of four residues, namely H142, E143, M489, and M542, were determined by site-directed mutagenesis. Eight mutants were successfully generated and purified, five of which (H142T, M489V, M542C, M542I, and M542V) were found to be inactive under various assay conditions. The H142K mutant was moderately altered in all kinetic parameters to a similar extent. In addition, the mutant was more thermo-labile than wild type enzyme. The E143A mutant increased the Km value more than 20-fold while other parameters were not significantly changed. All mutations carried out on residue M542 inactivated the enzyme. Though showing a single band on SDS-PAGE, the M542C mutant lost its native tertiary structure and was aggregated. Except M542C, each of the other mutants showed a secondary structure similar to that of wild type enzyme. Although all the inactive mutants were able to bind FAD, the mutants M489V and M542C showed a very low affinity for FAD. None of the active mutants constructed was strongly resistant to three tested herbicides. Taken together, the results suggest that the residues of H142, E143, M489, and M542 are essential for catalytic activity. Furthermore, it seems that H142 residue is involved in stabilizing the dimer interaction, while E143 residue may be involved in binding with substrate pyruvate. The data from the site-directed mutagenesis imply that the constructed homology model of tobacco AHAS is realistic.     

Multidrug resistance to HIV-1 protease inhibition requires cooperative coupling between distal mutations

The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/AIDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir. This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the K(m) of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than 1 order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggesting that the mutations at the dimer interface (L10I/L90M) play a major role in eliciting a cooperative response. These studies demonstrate that cooperative interactions contribute an average of 1.2 +/- 0.7 kcal/mol to the overall resistance, most of the cooperative effect (0.8 +/- 0.7 kcal/mol) being mediated by the mutations at the dimerization interface. Not all inhibitors in clinical use are affected the same by long-range cooperative interactions between mutations. These interactions can amplify the effects of individual mutations by factors ranging between 2 and 40 depending on the inhibitor. Dissection of the energetics of drug resistance into enthalpic and entropic components provides a quantitative account of the inhibitor response and a set of thermodynamic guidelines for the design of inhibitors with a lower susceptibility to this type of mutations.     

Cancer-related mutations in BRCA1-BRCT cause long-range structural changes in protein-protein binding sites: a molecular dynamics study

Cancer-associated mutations in the BRCT domain of BRCA1 (BRCA1-BRCT) abolish its tumor suppressor function by disrupting interactions with other proteins such as BACH1. Many cancer-related mutations do not cause sufficient destabilization to lead to global unfolding under physiological conditions, and thus abrogation of function probably is due to localized structural changes. To explore the reasons for mutation-induced loss of function, the authors performed molecular dynamics simulations on three cancer-associated mutants, A1708E, M1775R, and Y1853ter, and on the wild type and benign M1652I mutant, and compared the structures and fluctuations. Only the cancer-associated mutants exhibited significant backbone structure differences from the wild-type crystal structure in BACH1-binding regions, some of which are far from the mutation sites. Backbone differences of the A1708E mutant from the liganded wild type structure in these regions are much larger than those of the unliganded wild type X-ray or molecular dynamics structures. These BACH1-binding regions of the cancer-associated mutants also exhibited increases in their fluctuation magnitudes compared with the same regions in the wild type and M1562I mutant, as quantified by quasiharmonic analysis. Several of the regions of increased fluctuation magnitude correspond to correlated motions of residues in contact that provide a continuous path of fluctuating amino acids in contact from the A1708E and Y1853ter mutation sites to the BACH1-binding sites with altered structure and dynamics. The increased fluctuations in the disease-related mutants suggest an increase in vibrational entropy in the unliganded state that could result in a larger entropy loss in the disease-related mutants upon binding BACH1 than in the wild type. To investigate this possibility, vibrational entropies of the A1708E and wild type in the free state and bound to a BACH1-derived phosphopeptide were calculated using quasiharmonic analysis, to determine the binding entropy difference DeltaDeltaS between the A1708E mutant and the wild type. DeltaDeltaS was determined to be -4.0 cal mol(-1) K(-1), with an uncertainty of 2 cal mol(-1) K(-1); that is, the entropy loss upon binding the peptide is 4.0 cal mol(-1) K(-1) greater for the A1708E mutant, corresponding to an entropic contribution to the DeltaDeltaG of binding (-TDeltaDeltaS) 1.1 kcal mol(-1) more positive for the mutant. The observed differences in structure, flexibility, and entropy of binding likely are responsible for abolition of BACH1 binding, and illustrate that many disease- related mutations could have very long-range effects. The methods described here have potential for identifying correlated motions responsible for other long-range effects of deleterious mutations.     

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.     

The mutational specificity of DNA polymerases-alpha and -gamma during in vitro DNA synthesis

The frequency and specificity of mutations produced during in vitro DNA synthesis of the lacZ alpha gene in M13mp2 DNA by eucaryotic DNA polymerase-alpha (pol-alpha) and DNA polymerase-gamma (pol-gamma) have been determined. Pol-alpha, purified from five different sources, produces mutations resulting in loss of alpha-complementation at a frequency of 0.8-1.6%/single round of gap-filling DNA synthesis. DNA sequence analysis of 420 independent mutants produced by pol-alpha demonstrates three classes of errors. The majority of mutations result from single base substitutions, while single base frameshifts are detected at a lower but substantial frequency. Large deletions are also observed, with a frequency and specificity suggesting that they too are produced by pol-alpha in vitro. In contrast, pol-gamma is more accurate, producing mutants at a frequency of 0.3-0.5%. The specificity of pol-gamma errors is also different, since more than 90% of the mutants result from single base substitutions, while frameshift errors are not observed at a frequency significantly above background. The pol-gamma mutant spectrum also contains deletion mutations (10 of 179 mutants) presumably resulting from aberrant in vitro synthesis. When considered together with previous results using pol-beta (Kunkel, T. A. (1985) J. Biol. Chem. 260, 5787-5796) the relative accuracy of the three classes of purified vertebrate DNA polymerases for base substitutions, frameshifts, and deletions is in the order gamma greater than alpha greater than beta. These data demonstrate a correlation between the accuracy and processivity of DNA polymerization. Thus, the most accurate DNA polymerase (pol-gamma) also incorporates the most nucleotides per association with the primer-template, while the least accurate enzyme (pol-beta) is the least processive. This correlation exists both for base substitution mutations and for single base frameshifts, and is most obvious for minus-one-base frameshifts in runs of pyrimidines. In support of this correlation, increasing the processivity of pol-beta from 1 to 4-6 incorporations per association increases the accuracy of in vitro DNA synthesis by severalfold. The data imply that the processivity of DNA synthesis could be an important factor in controlling the levels of spontaneous and perhaps induced mutations.     

Construction and analysis of Sip1Aa insecticidal protein random recombination library

The Sip1Aa protein from Bacillus thuringiensis is highly toxic to Colaphellus bowringi Baly. In order to obtain mutant proteins with higher insecticidal activity, a random recombinant library of Sip1Aa protein was constructed using error-prone PCR. A total number of 100 positive transformants were randomly selected for sequence determination, and 25 mutants (M1 to M25) were selected and expressed the respective Sip1Aa mutants. These Sip1Aa variants had a total of 29 base mutations, with an average of 1.2 base mutations per mutant. Compared with that of the wild-type Sip1Aa protein, the insecticidal activity of the mutants M1 (A31G, Y118C, D227E), M5 (K168R) and M21 (I307T) was significantly decreased, with and LC₅₀ values 4 to 6 times higher than the Sip1Aa protein. The mutant M8 (R174S) showed increase in the insecticidal activity against the Colaphellus bowringi Baly was obtained, with an LC₅₀ value 4-fold less than the Sip1Aa protein. The results of this study provide reference for the molecular modification of Sip1Aa protein and the study of key sites of its insecticidal activity.     

UV- and X-ray-sensitive double mutants of mouse L5178Y cells are synergistically more sensitive to 4-nitroquinoline-1-oxide than is either of the single mutants

The X-ray-sensitive mutant M10 and the UV-sensitive mutant Q31 of mouse lymphoma L5178Y cells are both sensitive to killing by 4-nitroquinoline-1-oxide (4NQO). Since cell hybridization experiments showed that the 4NQO sensitivities in M10 and Q31 cells behaved as codominant traits (Shiomi et al., 1982c), it is not possible to determine by complementation test whether the M10 and the Q31 mutations responsible for 4NQO sensitivities are allelic. We have obviated this difficulty by selecting double mutants that are sensitive to both X-rays and UV. From X-ray-sensitive M10 cells, two UV-sensitive mutants (XU 1 and XU 2) were isolated by a cell-suspension spotting method. XU 1 and XU 2 were found to belong to the same complementation group as Q31 (group I). Double mutants XU 1 and XU 2 were 30-37-fold more sensitive to 4NQO than parental L5178Y cells, whereas the single mutants M10 and Q31 were only 6-8-fold more sensitive to 4NQO than L5178Y cells in terms of D10 values (dose required to reduce survival to 10%). These results show that the M10-Q31-double mutations enhance 4NQO sensitivity synergistically, indicating that the M10 and the Q31 mutations relevant to 4NQO sensitivities are non-allelic. The implications of this finding are discussed.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ethyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3--9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.     

Revertants and Secondary arom-2 Mutants Induced in Non-Complementing Mutants in the arom Gene Cluster of Neurospora Crassa

Extensive genetical and biochemical studies have been performed with revertants and secondary arom-2 mutants induced in two different primary non-complementing mutants which map within the arom gene cluster of Neurospora crassa. These studies indicate that mutant M54 but not M25 can revert by super-suppressor mutations in unlinked genes, thus confirming previous evidence that M54 contains a nonsense codon. At least three new super suppressors of M54 have been detected. All four super suppressors (including one previously detected) when combined with M54 result in high levels of all five of the arom enzymic activities in the form of arom multienzyme complexes very similar to (but not necessarily identical with) that in wild type (WT).-Evidence has also been obtained that the two non-complementing mutants can yield revertants which appear to result from true back mutations and produce arom aggregates essentially indistinguishable from that of WT. In addition, M25, but not M54, when plated on quinic acid yields revertants (secondary mutants) some of which are phenotypically indistinguishable from arom-2 primary mutants and others of which, although also mapping within the arom-2 gene, exhibit unusual properties. Genetic evidence indicates that the M25 secondary mutants are localized within the arom-2 gene, but that they arise from mutational events more complex than ones resulting in single base pair changes in the M25 codon.-The recovery of secondary arom-2 mutants as revertants of non-complementing arom mutants provides strong evidence, independent of earlier recombination data, that non-complementing arom mutants are located within the arom-2 structural gene of the arom gene cluster. In addition, the occurrence and characteristics of these secondary arom-2 mutants provide strong evidence, independent of the results with nonsense suppressors, that the arom gene cluster is transcribed, beginning with the arom-2 gene, as a single polycistronic messenger ribonucleic acid (mRNA) molecule which is subsequently translated into the arom multienzyme complex.     

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex.     

Mutations at alternative 5' splice sites of M1 mRNA negatively affect influenza A virus viability and growth rate

Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5' splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5' splice site), C51G (at the 3' end of the exon of the M2 mRNA 5' splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3' end of exon of the mRNA3 5' splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3' end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5' splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.     

Epistatic Interactions of Radiation-Sensitive (rad) Mutants of CAENORHABDITIS ELEGANS

Six double mutants and a quadruple mutant were derived from four UV radiation-hypersensitive single mutants (rad-1, rad-2, rad-3 and rad-7). Sensitivities of the 11 strains to UV, gamma-radiation and methyl methanesulfonate (MMS) were compared. Of the six double mutants, only the rad-1;rad-2 and rad-3;rad-7 doubles were no more hypersensitive than the most sensitive single mutant to UV-radiation. Thus, rad-1 and rad-2 define one epistasis group, whereas rad-3 and rad-7 define another. Consistent with this model was the observation that rad-1 and rad-2, but not rad-3 and rad-7, were hypersensitive to gamma-radiation. In addition, none of the multiple mutants was more hypersensitive to gamma-radiation than the most sensitive single rad mutant. No synergistic interactions of the rad mutations with respect to MMS sensitivities were observed.     

Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase

Methotrexate-resistant forms of human dihydrofolate reductase have the potential to protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained by combining two active-site mutations that independently confer weak MTX-resistance. In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR) variants for this application, we used a semi-rational approach to obtain combinatorial active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial mutant library encoding various amino acids at residues Phe31, Phe34 and Gln35. In vivo library selection was achieved in a bacterial system on medium containing high concentrations of MTX. We characterized ten novel MTX-resistant mutants with different amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters of the purified mutants revealed that higher MTX-resistance roughly correlated with a greater number of mutations, the most highly-resistant mutants containing three active site mutations (Ki(MTX)=59-180 nM; wild-type Ki(MTX)<0.03 nM). An inverse correlation was observed between resistance and catalytic efficiency, which decreased mostly as a result of increased KM toward the substrate dihydrofolate. We verified that the MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting the most resistant mutants into DHFR-knock-out CHO cells. The transfected variants conferred survival at concentrations of MTX between 100-fold and >4000-fold higher than the wild-type enzyme, the most resistant triple mutant offering protection beyond the maximal concentration of MTX that could be included in the medium. These highly resistant variants of hDHFR offer potential for myeloprotection during administration of MTX in cancer treatment.     

A major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein

The two major constituents of coronavirus virions are the membrane (M) and nucleocapsid (N) proteins. The M protein is anchored in the viral envelope by three transmembrane segments flanked by a short amino-terminal ectodomain and a large carboxy-terminal endodomain. The M endodomain interacts with the viral nucleocapsid, which consists of the positive-strand RNA genome helically encapsidated by N protein monomers. In previous work with the coronavirus mouse hepatitis virus (MHV), a highly defective M protein mutant, MDelta2, was constructed. This mutant contained a 2-amino-acid carboxy-terminal truncation of the M protein. Analysis of second-site revertants of MDelta2 revealed mutations in the carboxy-terminal region of the N protein that compensated for the defect in the M protein. To seek further genetic evidence corroborating this interaction, we generated a comprehensive set of clustered charged-to-alanine mutants in the carboxy-terminal domain 3 of N protein. One of these mutants, CCA4, had a highly defective phenotype similar to that of MDelta2. Transfer of the CCA4 mutation into a partially diploid MHV genome showed that CCA4 was a loss-of-function mutation rather than a dominant-negative mutation. Analysis of multiple second-site revertants of CCA4 revealed mutations in both the M protein and the N protein that could compensate for the original lesion in N. These data more precisely define the region of the N protein that interacts with the M protein. Further, we found that fusion of domain 3 of the N protein to the carboxy terminus of a heterologous protein caused it to be incorporated into MHV virions.